Lysis Process Research Articles

Comparison of standard and global hemostasis assays in cord and peripheral blood of newborns.

Umbilical cord blood is used for the testing of various parameters in newborns. However, data on its applicability for hemostasis assays is insufficient. To evaluate whether umbilical cord blood can be used for standard tests, thromboelastometry and thrombodynamics for preterm and term newborns. 187 newborns were included in the study. Blood was taken from the umbilical cord and by venipuncture of the newborn. Clotting times, fibrinogen, D-dimer, thromboelastometry and thrombodynamics were measured. Clotting times and fibrinogen indicated a hypocoagulable shift, while thromboelastometry and thrombodynamics showed a hypercoagulable shift in hemostasis in umbilical cord blood compared to newborn blood. D-dimer indicated an enhanced process of thrombus lysis in newborn blood compared to cord blood. Collecting blood into a tube with the addition of a contact pathway inhibitor did not significantly change the global assay parameters in either umbilical cord blood or newborn blood. In the thrombodynamics assay, spontaneous clotting was detected but suppressed by the addition of a tissue factor inhibitor. Hemostasis in cord and newborn blood differs for both global and standard tests. Hypercoagulability in newborns registered with the global assay thrombodynamics is associated with the presence of tissue factor in the blood. 1. We found a hypercoagulation shift in newborns compared with the adult references, possibly due to the presence of tissue factor in blood. 2. Blood coagulation is enhanced in cord blood compared with blood sampled from the vein of a newborn according to thromboelastometry and thrombodynamics assays. 3. Clotting times and fibrinogen concentrations in cord blood differ from these parameters in newborn blood. 4. Studying of the (patho)physiological features of hemostasis in newborns should consider differences in cord blood and vein sampled blood.

DMF-DM-seq: Digital-Microfluidics Enabled Dual-Modality Sequencing of Single-Cell mRNA and microRNA with High Integration, Sensitivity, and Automation.

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

Application of direct PCR technique using specified mitochondrial DNAencoded ND4 and D-Loop genes on detecting pork DNA in processed meat products

Adulteration or counterfeiting of food products raises public concerns about the halal issue. Many issues related to food products such as meatballs, sausages, and corned beef use pork for counterfeiting. This study aimed to determine a direct polymerase chain reaction (dPCR) technique for pork DNA detection in processed meat products such as meatballs, sausages, and corned beef in a short time and at a low cost using specific primers, ND4 and D-Loop. The study used a direct PCR technique with variations in the incubation time and temperature of the lysis process on the sample. The concentration of DNA obtained from the lysis results with variations in incubation time and temperature in fresh meat and processed meat products was 66.17-469.23 ng/µL, and the purity of DNA in fresh and processed meat products was 1.70-2.25. ND4 primer was more sensitive to DNA amplification than D-Loop in processed meat products using direct PCR. Meatballs, sausages, and corned beef products written on the packaging using beef did not detect any mixing or addition of pork. Direct PCR can detect pork DNA in processed meat products such as meatballs, sausages and corned beef.

Histomorphological assessment of the germanium-doped calcium phosphate ceramics on reparative osteogenesis in rabbits with systemic osteoporosis

Bone fractures are mostly urgent in nature, complex in pathogenetic, diagnostic and therapeutic aspects, and reparative osteogenesis is multimodal and depends on the balanced and reciprocal interaction of many factors. The results of histological studies for osteoreplacement of bone defects in rabbits with systemic osteoporosis are presented. The purpose of the work is histomorphological evaluation of bone regenerates after osteoreplacement with germanium-doped hydroxyapatite ceramics in rabbits with secondary osteoporosis. Experimental osteoporosis in rabbits (n=18) was induced by administration of 0.4% dexamethasone solution. In animals of the experimental group, bone defects were replaced with granules of hydroxyapatite ceramics doped with germanium, and in animals of the control group, they healed under a blood clot. Histological sections were made on a rotary microtome with a thickness of 5 to 10 μm and stained with Weigert's iron hematoxylin and 1% alcohol-based eosin solution (manufactured by Diapath, Italy). On the 60th day of reparative osteogenesis in the animals of the experimental group, the site of the bone defect was flled with compact bone tissue with minor remnants of spongy bone tissue. Slightly expanded Haversian canals were noted. In the control group, the defect site was flled with coarse and spongy bone tissue. Bone beams of various thicknesses with a small number of osteoblasts and single walled osteocytes were visualized. A signifcant number of osteocyte lacunae were empty due to the process of osteocyte lysis. Haversian canals are signifcantly dilated with a small number of vessels. Also, lacunae of bone tissue resorption and acellular areas were observed on histological specimens. Histomorphological assessment of bone regenerates confrms the realization of osteoconductive, osteointegrative and osteoinductive properties of calcium-phosphate ceramics doped with germanium in conditions of osteoporotic fractures of tubular bones. Keywords: systemic osteoporosis, bone fractures, cancellous and compact bone tissue, histological sections, histomorphological changes, rabbits.

Reduction of selenite to selenium nanoparticles by highly selenite-tolerant bacteria isolated from seleniferous soil

The microbial reduction of selenite to elemental selenium nanoparticles (SeNPs) is thought to be an effective detoxification process of selenite for many bacteria. In this study, Metasolibacillus sp. ES129 and Oceanobacillus sp. ES111 with high selenite reduction efficiency or tolerance were selected for systematic and comparative studies on their performance in selenite removal and valuable SeNPs recovery. The kinetic monitoring of selenite reduction showed that the highest transformation efficiency of selenite to SeNPs was achieved at a concentration of 4.24 mM for ES129 and 4.88 mM for ES111. Ultramicroscopic analysis suggested that the SeNPs produced by ES111 and ES129 had been formed in cytoplasm and subsequently released to extracellular space through cell lysis process. Furthermore, the transcriptome analysis indicated that the expression of genes involved in bacillithiol biosynthesis, selenocompound metabolism and proline metabolism were significantly up-regulated during selenite reduction, suggesting that the transformation of selenite to Se0 may involve multiple pathways. Besides, the up-regulation of genes associated with nucleotide excision repair and antioxidation-related enzymes may enhance the tolerance of bacteria to selenite. Generally, the exploration of selenite reduction and tolerance mechanisms of the highly selenite-tolerant bacteria is of great significance for the effective utilization of microorganisms for environmental remediation.

Fully integrated and automated centrifugal microfluidic chip for point-of-care multiplexed molecular diagnostics

Public health events caused by pathogens have imposed significant economic and societal burdens. However, conventional methods still face challenges including complex operations, the need for trained operators, and sophisticated instruments. Here, we proposed a fully integrated and automated centrifugal microfluidic chip, also termed IACMC, for point-of-care multiplexed molecular diagnostics by harnessing the advantages of active and passive valves. The IACMC incorporates multiple essential components including a pneumatic balance module for sequential release of multiple reagents, a pneumatic centrifugation-assisted module for on-demand solution release, an on-chip silicon membrane module for nucleic acid extraction, a Coriolis force-mediated fluid switching module, and an amplification module. Numerical simulation and visual validation were employed to iterate and optimize the chip's structure. Upon sample loading, the chip automatically executes the entire process of bacterial sample lysis, nucleic acid capture, elution quantification, and isothermal LAMP amplification. By optimizing crucial parameters including centrifugation speed, direction of rotation, and silicone membrane thickness, the chip achieves exceptional sensitivity (twenty-five Salmonella or forty Escherichia coli) and specificity in detecting Escherichia coli and Salmonella within 40 min. The development of IACMC will drive advancements in centrifugal microfluidics for point-of-care testing and holds potential for broader applications in precision medicine including high-throughput biochemical analysis immune diagnostics, and drug susceptibility testing.

Research on enhancing sewage sludge lysis and bioreactor initiation through composite thermophilic strains

Returning lysed sludge to biochemical tanks to achieve in-situ sludge reduction is promising. A novel approach for regulating and initiating the sludge lysis process utilizing thermophilic strains is proposed. In this study, three strains were isolated from sludge, and sequencing of the 16 S rDNA amplicon revealed that the three thermophilic strains were the closest relatives to the genus Geobacillus, and they were designated as Geobacillus sp. DX5, Geobacillus sp. DX8 and Geobacillus sp. DX11. Sludge lysis experiments demonstrated that composite thermophilic strains (CTS) were dosed at 65°C, and the sludge VSS removal efficiency after 24 h was 37.8%, significantly surpassing individual strains. Moreover, 65.8% of proteins and 52.0% of polysaccharides in Extracellular polymeric substances (EPS) of sludge were degraded. Furthermore, Three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy results revealed a substantial release of dissolved organic matter during sludge lysis. Inoculation of CTS under optimal conditions (65°C, pH 7, 4 × 1010cfu/L) resulted in VSS removal efficiency and protease activity of 42.7% and 2584 U/L, respectively. After 20 days of inoculation with CTS to initiate sludge lysis reactor, VSS removal efficiency reached 46%. Additionally, it stimulated the growth of the thermophilic bacterial community, engaging indigenous thermophilic strains in the sludge lysis process.

Reusable magnetically-modified Enteromorpha prolifera-based biochar hydrogels: competitive removal mechanism for metal-organic dye composite contaminants

Dyes and heavy metals pollute the environment. Biochar-based hydrogel is an excellent adsorbent, but the competitive adsorption mechanism associated with the removal of pollutants using biochar is yet to be understood in detail. Biochar was prepared following the process of high-temperature lysis of marine green tide (Enteromorpha prolifera). The prepared biochar was cross-linked with water-soluble chitosan and compounded with nano-Fe3O4 to synthesize magnetically-modified Enteromorpha prolifera-based biochar hydrogel (MM-EBC-HD). The competitive removal performance of the hydrogel was studied, and the pollutant removal mechanism was analyzed against a binary system consisting of common environmental pollutants (methyl orange [MO] and hexavalent chromium [Cr (VI)]. The physical and chemical properties of the composites were studied before and after contaminant removal, and the associated pollutant removal mechanisms were analyzed by SEM, EDS, FTIR, XRD, and XPS techniques. The effects of pH, temperature and initial pollutant concentration on the adsorption performance of the materials were examined. The maximum adsorption of MO on MM-EBC-HD was 71.18 mg g−1, and adsorption equilibrium was attained at approximately 60 min. Electrostatic forces, hydrophobic bonds, and hydrogen bonds were exploited for MO adsorption. And the maximum adsorption amount of Cr (VI) was recorded to be 115.41 mg g−1, and equilibrium was attained in approximately 10 min. Electrostatic and ion exchange effects were exploited to adsorb Cr (VI) efficiently. The MO and Cr (VI) adsorption processes could be explained by the second-order kinetic model and Langmuir adsorption isotherms, respectively. The adsorption performance recorded for the binary adsorption system was poorer than that recorded for the single adsorption system for both pollutants. MO and Cr (VI) adsorption decreased from 74.88% to 47.65% and from 62.33% to 42.4%, respectively. Competition between MO and Cr (VI) in the dual system can be attributed to the presence of amino and hydroxyl groups. The MO–Cr complex, which was more compact in structure than a single contaminant, was formed, allowing few reactive groups to be exposed to the surface of the hydrogel. Additionally, MM-EBC-HD nanocomposites presented a recovery rate of 87% after 5 cycles and thus could be used to avoid adsorbents-caused environmental hazards.Graphical

Molecular Machinery of the Triad Holin, Endolysin, and Spanin: Key Players Orchestrating Bacteriophage-Induced Cell Lysis and their Therapeutic Applications.

Phage therapy, a promising alternative to combat multidrug-resistant bacterial infections, harnesses the lytic cycle of bacteriophages to target and eliminate bacteria. Key players in this process are the phage lysis proteins, including holin, endolysin, and spanin, which work synergistically to disrupt the bacterial cell wall and induce lysis. Understanding the structure and function of these proteins is crucial for the development of effective therapies. Recombinant versions of these proteins have been engineered to enhance their stability and efficacy. Recent progress in the field has led to the approval of bacteriophage-based therapeutics as drugs, paving the way for their clinical use. These proteins can be combined in phage cocktails or combined with antibiotics to enhance their activity against bacterial biofilms, a common cause of treatment failure. Animal studies and clinical trials are being conducted to evaluate the safety and efficacy of phage therapy in humans. Overall, phage therapy holds great potential as a valuable tool in the fight against multidrug- resistant bacteria, offering hope for the future of infectious disease treatment.

Application of Oncolytic Poxviruses: An Emerging Paradigm in Cancer Therapy.

Despite the significant advancement of new tools and technology in the field of medical biology and molecular biology, the challenges in the treatment of most cancer types remain constant with the problem of developing resistance toward drugs and no substantial enhancement in the overall survival rate of cancer patients. Immunotherapy has shown the most promising results in different clinical and preclinical trials in the treatment of various cancer due to its higher efficacy and minimum collateral damage in many cancer patients as compared to conventional chemotherapy and radiotherapy. An oncolytic virus is a new class of immunotherapy that can selectively replicate in tumor cells and destroy them by the process of cell lysis while exerting minimum or no effect on a normal cell. Besides this, it can also activate the host's innate immune system, which generates an anti-tumor immune response to eliminate the tumor cells. Several wild types and genetically modified viruses have been investigated to show oncolytic behavior. Vaccinia virus has been studied extensively and tested for its promising oncolytic nature on various model systems and clinical trials. Recently, several engineered vaccinia viruses have been developed that express the desired genes encoded for selective penetration in tumor cells and enhanced activation of the immune system for generating anti-tumor immunity. However, further investigation is required to prove their potential and enhance their therapeutic efficacy.

Cytopathic effects in Mimivirus infection: understanding the kinetics of virus-cell interaction.

Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.

Construction of an autolytic system in Escherichia coli Nissle 1917 for efficient release of intracellular recombinant proteins

BackgroundEscherichia coli has emerged as the preferred option in the field of recombinant protein production. However, this necessitates a cell lysis process to extract substantial quantities of recombinant protein. The E. coli Nissle 1917 (EcN) autolysis system was engineered to enhance convenience in the recovery process. This concept would simplify the procedure for retrieving recombinant proteins, streamlining the overall production process. MethodsPrevious studies showed that the fusion of cell-penetrating peptides (Pa) with T4 lysozyme (T4L) to generate 2PaT4L enables E. coli to undergo autolysis, releasing intracellular proteins into the cultivation medium. In this study, the expression plasmid of 2PaT4L was constructed and transformed into ENL6P, the ECN T7 expression system host cell. The induction of this autolysis process is achieved through the induction of IPTG. Significant FindingsAfter IPTG induction, the autolysis of cells gradually reached a dynamic equilibrium with the growth at a low cell concentration. After 24 h of cell autolysis induction, the yields of green fluorescent protein and trehalose synthase could reach 177.27 ± 7.40 and 84.59 ± 4.39 mg/L, respectively. The results show that the ECN autolytic expression system can overexpress recombinant proteins and simplify the recovery process.

Modelling biofilm development: The importance of considering the link between EPS distribution, detachment mechanisms and physical properties

In industry, treatments against biofilms need to be optimized and, in the wastewater treatment field, biofilm composition needs to be controlled. Therefore, describing the biochemical and physical structures of biofilms is now required to better understand the influence of operating parameters and treatment on biofilms. The present study aims to investigate how growth conditions influence EPS composition, biofilm physical properties and volume detachment using a 1D biofilm model. Two types of EPS are considered in the present model, proteins and polysaccharides. The main hypotheses are that: (i) the production of polysaccharides occurs mainly under strong nutrient limitation(s) while the production of proteins is coupled to both the substrate uptake rate and the lysis process; (ii) the local biofilm porosity depends on the local biofilm composition. Both volume and surface detachment occur in biofilms and volume detachment extent depends on the biofilm local cohesion and thus on the local composition of biofilms for a given shear stress.The model is based on experimental trends and aims to represent these observations on the basis of biochemical and physical processes. Four case studies covering a wide range of contrasting growth conditions such as different COD/N ratios, applied SOLR and shear stresses are investigated. The model predicts how the biochemical and physical biofilm structures change as a result of contrasting growth conditions. More precisely simulation results are in good agreement with the main experimental observations reported in the literature, such as: (i) a strong nitrogen limitation of growth induces an important accumulation of polysaccharides leading to a more porous and homogenous biofilm, (ii) a high applied surface organic loading load allows to obtain a high biofilm thickness, (iii) a strong shear stress applied during the biofilm growth leads to a reduction of the biofilm thickness and to a consolidation of the biofilm structure. Overall, this model represents a relevant decision tool for the selection of appropriate enzymatic treatments in the context of negative biofilm control. From our results, it appears that protease based treatments should be more appropriate for biofilms developed under low COD/N ratios (about 20 gCOD/gN) whereas both glucosidases and proteases based treatments should be more appropriate for biofilms developed under high COD/N ratio (about 70 gCOD/gN). In addition, the model could be useful for other applications such as resource recovery in biofilms or granules, and help to better understand biological membrane fouling.

Investigation of lysing excess sludge slurry using hydrolase secreting thermophilic bacterial communities

Sludge reduction is a critical challenge in biological wastewater treatment. Combining excess sludge slurry lysis technology with traditional activated sludge processes is a promising approach for in-situ sludge reduction. Here, a strategy for excess sludge slurry lysis based on thermophilic bacterial communities (LTBC) was proposed. This investigation focused on the process of excess sludge slurry lysis dominated by thermophilic bacterial communities domesticated at different temperatures (55–75 °C). The evolution of sludge lysate was analyzed, and the mechanism of excess sludge slurry lysis under the action of thermophilic bacterial communities was elucidated through amplicon sequencing analysis. The results demonstrated that the aerobic thermophilic bacterial communities adapted to 75 °C exhibit the highest efficiency in sludge slurry lysis. During LTBC process, the removal efficiency of volatile suspended solids reached 53.9 ± 1.8% within 2 h, and 97.0 ± 1.0% of the protein and 96.0 ± 1.0% of the polysaccharide in the extracellular polymers was solubilized, and bacterial cell walls in sludge were disrupted. Fourier transform infrared spectroscopy and excitation-emission matrix spectroscopy of the sludge lysate demonstrated that the LTBC process was accompanied by humification process. The accumulation of humic acid primarily occurred at 55 °C and 65 °C, while fulvic acid occurred at 75 °C. The thermophilic bacterial communities adapted to 75 °C were dominated by Thermus and Thermaerobacter. Phylogenetic studies showed that the LTBC hydrolase system comprises enzymes related to protein hydrolysis, carbohydrate hydrolysis, and peptidoglycan hydrolysis, including metalopeptidase MepB, neutral α-glucosidase C, N-acetyl Muramyl-L-alanine amidase, and others enzymes. These results provide a theoretical basis for the application of LTBC technology in the reduction of sludge which generated in traditional waste water activated sludge processes.

Optimization of intracellular L-Asparaginase production by Streptomyces paulus CA01 isolated from wheat bran using the response surface methodology

This study aimed to enhance the production of the intracellular L-asparaginase bacterial enzyme, which is known to impede the growth of cancer cells, by optimizing the culture conditions. In the present study, we exploited the ability of a new Actinomycete strain isolated from a food byproduct, wheat bran, to produce the desired enzyme. Streptomyces paulus CA01 was identified following morphological, biochemical, and molecular testing, and its total L-asparaginase activity was evaluated using ADS agar. Intracellular L-asparaginase activity was measured on the same medium using the well-diffusion method and was quantified by Nesslerization. A Box-Behnken design was employed to optimize the production of the enzyme by analyzing the effect of three factors: temperature, L-Asparagine concentration and glucose concentration. The ADS base medium, which was previously optimized and consisted of starch, K2HPO4, and MgSO4, was also included in the analysis. The optimal conditions for intracellular L-asparaginase production were temperature of 27.5 °C, L-Asparagine concentration of 1.19%, glucose concentration of 0.26% and ADS base medium composition, with which we achieved an incrase in enzymatic activity from 3.20 IU/mL to 8.39 IU/mL. This study indicates that Algerian wheat bran is a potential source of Streptomyces paulus CA01, which produces the bacterial intracellular L-asparaginase enzyme and also opens up prospects for the exploitation of agricultural by-products as sources of interesting micro-organisms. Despite the complex process of cell lysis required for its production, intracellular L-asparaginase is important and depends on culture conditions, making its optimization and increased yield feasible.

Rapid colorimetric and fluorescence identification of Pinelliae Rhizoma and adulterate Rhizoma Typhonii Flagelliformis using direct-LAMP assay

Pinelliae Rhizoma, renowned for its medicinal benefits against cough, insomnia and inflammation, is frequently utilized in botanical dietary supplements and herbal products. However, due to high demand, there is a risk of adulteration. This study introduced Direct-Loop-Mediated Isothermal Amplification (Direct-LAMP), a rapid colorimetric technique, for distinguishing Pinelliae Rhizoma from its primary adulterant, Rhizoma Typhonii Flagelliformis. This method comprises three parts: DNA extraction-free direct lysis, isothermal amplification and prompt color development. The direct lysis process allows for release of genomic DNA within 2 min. The isothermal amplification reaction exhibits excellent analytical performance, detecting Pinelliae Rhizoma at levels as low as 0.1 pg and Rhizoma Typhonii Flagelliformis at 0.08 ng, while also demonstrating good linearity across a wide range, making it suitable for quantitative analysis. Two different color reagents are employed for these two different plant materials, resulting in dual-color changes for captivating visual detection. The developed direct-LAMP was also successfully validated across 48 actual samples.

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.

A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis.

Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.

Polyaniline-based 3D network structure promotes entrapment and detection of drug-resistant bacteria

Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin.Graphical

Bacillus cereus strain L7 lyses Cylindrospermopsis raciborskii through intercellular contact

Cylindrospermopsis raciborskii is a new type of toxic filamentous cyanobacteria commonly found in tropical regions but has recently spread to temperate areas. It is considered an invasive species that poses a serious threat to drinking water sources and human health. Bacillus cereus strain L7, previously isolated and identified by our group, was found to have efficient algicidal activity against C. raciborskii FACHB-1503 in this study. The growth period and initial concentration of L7 had a significant impact on algicidal activity, with L7 culture in the decline phase exhibiting the highest algicidal activity (I = 99.62 %). The ideal environmental conditions for the coculture system were 30 °C, pH = 8, and a light-dark cycle (12 h light: 12 h dark). In experiments to confirm the algicidal mode of L7, the pellet resuspension with R2A medium had the highest algicidal efficiency (I = 98.54 %, t = 6 d), even higher than that of the L7 culture. In contrast, sterile filtrate and pellet resuspension with sterile water had no discernible impact on the algal growth, indicating that L7 performed algicidal activity through direct contact with algal cells. Following 6 days of coculture of L7 culture with C. raciborskii solution, the activity of the PSII system and the photosynthetic efficiency of algal cells were both zero. Following SEM and TEM results, it was discovered that the process of L7 lysis of algal cells begins with the disruption of their multicellular filamentous structure, rupture of the cell wall, efflux of the cell contents, and complete disappearance of the algal cell structure. This is the first research to demonstrate that L7 lyses C. raciborskii directly, which serves as an excellent strategic direction for launching innovative freshwater bloom control strategies.