Multiple Centrifugation Research Articles

One-step on-chip preparation of nanoparticle-conjugated red blood cell carriers

Red blood cell (RBC)-based carriers have emerged as promising vehicles for drug delivery due to their inherent biocompatibility and biodegradability. Traditional methods for loading nanoparticles (NPs) onto RBC surfaces often involve labor-intensive processes like incubation and multiple centrifugation steps, limiting their practicality and controllability. In this study, we introduce a fully integrated acoustofluidic platform that enables one-step preparation of NP-loaded RBC carriers with controlled modification and on-site purification. By incorporating a high-frequency bulk acoustic wave (BAW) resonator into a microfluidic chip, we utilize acoustic streaming effects to manipulate the movement and interaction of RBCs and NPs within the microchannel. This design allows for precise control over NP loading efficiency by adjusting the input power to the resonator. Experimental results using 200 nm positively charged fluorescent NPs demonstrate that our platform significantly enhances the interaction between RBCs and NPs, achieving efficient and controllable surface loading of NPs onto RBCs. Furthermore, the platform simplifies post-processing by directing excess NPs to waste outlets, eliminating the need for repetitive washing and centrifugation. This acoustofluidics approach not only automates the loading process but also offers high controllability, highlighting its potential for various applications in particle and cell surface modification.

An acoustofluidic device for the automated separation of platelet-reduced plasma from whole blood

Separating plasma from whole blood is an important sample processing technique required for fundamental biomedical research, medical diagnostics, and therapeutic applications. Traditional protocols for plasma isolation require multiple centrifugation steps or multiunit microfluidic processing to sequentially remove large red blood cells (RBCs) and white blood cells (WBCs), followed by the removal of small platelets. Here, we present an acoustofluidic platform capable of efficiently removing RBCs, WBCs, and platelets from whole blood in a single step. By leveraging differences in the acoustic impedances of fluids, our device generates significantly greater forces on suspended particles than conventional microfluidic approaches, enabling the removal of both large blood cells and smaller platelets in a single unit. As a result, undiluted human whole blood can be processed by our device to remove both blood cells and platelets (>90%) at low voltages (25 Vpp). The ability to successfully remove blood cells and platelets from plasma without altering the properties of the proteins and antibodies present creates numerous potential applications for our platform in biomedical research, as well as plasma-based diagnostics and therapeutics. Furthermore, the microfluidic nature of our device offers advantages such as portability, cost efficiency, and the ability to process small-volume samples.

Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

Cell Membrane-Coated Nanoparticles for Precision Medicine: A Comprehensive Review of Coating Techniques for Tissue-Specific Therapeutics.

Nanoencapsulation has become a recent advancement in drug delivery, enhancing stability, bioavailability, and enabling controlled, targeted substance delivery to specific cells or tissues. However, traditional nanoparticle delivery faces challenges such as a short circulation time and immune recognition. To tackle these issues, cell membrane-coated nanoparticles have been suggested as a practical alternative. The production process involves three main stages: cell lysis and membrane fragmentation, membrane isolation, and nanoparticle coating. Cell membranes are typically fragmented using hypotonic lysis with homogenization or sonication. Subsequent membrane fragments are isolated through multiple centrifugation steps. Coating nanoparticles can be achieved through extrusion, sonication, or a combination of both methods. Notably, this analysis reveals the absence of a universally applicable method for nanoparticle coating, as the three stages differ significantly in their procedures. This review explores current developments and approaches to cell membrane-coated nanoparticles, highlighting their potential as an effective alternative for targeted drug delivery and various therapeutic applications.

Optimization and validation of RP-HPLC method for evaluation of pyrrole -containing hydrazones in isolated rat synaptosomes

In the current study, RP-HPLC method for evaluation of 2 pyrrol-based hydrazones in isolated rat synaptosomes was optimized and validated according to ICH guidelines. The synaptosomes were obtained by multiple centrifugations with Percoll reagent and the selected 2 N-pyrollyl hydrazide-hydrazones were incubated for 2 hours at 37 °C. Subsequently, the purified fraction through protein precipitation was analyzed by an UltiMateDionex 3000 DAD system with Purospher STAR C18 (4.6 x 12.5 cm, 5 µm) column. The mobile phase, consisting of acetonitrile: phosphate buffer pH 3.5: methanol in ratio 42/36/22 (v/v/v), was eluted isocratically with 0.8 mL/min flow rate. Afterwards, the novel and rapid method was applied effectively for identification of biotransformation in isolated rat brain synaptosomes. The analysis results indicated an absence of new peaks and persistent sample concentration which determined the stability of the analyzed ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2- oxoethyl)-2-methyl- 1H-pyrrole-3-carboxylate (11b) and ethyl 5-(4-bromophenyl)-1-(3-(2-(2-hydroxybenzylidene)hydrazinyl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (12b) and pointed these structures as promising.

Newly Synthesized Creatine Derivatives as Potential Neuroprotective and Antioxidant Agents on In Vitro Models of Parkinson's Disease.

Oxidative stress is one of the key factors responsible for many diseases-neurodegenerative (Parkinson and Alzheimer) diseases, diabetes, atherosclerosis, etc. Creatine, a natural amino acid derivative, is capable of exerting mild, direct antioxidant activity in cultured mammalian cells acutely injured with an array of different reactive oxygen species (ROS) generating compounds. The aim of the study was in vitro (on isolated rat brain sub-cellular fractions-synaptosomes, mitochondria and microsomes) evaluation of newly synthetized creatine derivatives for possible antioxidant and neuroprotective activity. The synaptosomes and mitochondria were obtained by multiple centrifugations with Percoll, while microsomes-only by multiple centrifugations. Varying models of oxidative stress were used to study the possible antioxidant and neuroprotective effects of the respective compounds: on synaptosomes-6-hydroxydopamine; on mitochondria-tert-butyl hydroperoxide; and on microsomes-iron/ascorbate (non-enzyme-induced lipid peroxidation). Administered alone, creatine derivatives and creatine (at concentration 38 µM) revealed neurotoxic and pro-oxidant effects on isolated rat brain subcellular fractions (synaptosomes, mitochondria and microsomes). In models of 6-hydroxydopamine (on synaptosomes), tert-butyl hydroperoxide (on mitochondria) and iron/ascorbate (on microsomes)-induced oxidative stress, the derivatives showed neuroprotective and antioxidant effects. These effects may be due to the preservation of the reduced glutathione level, ROS scavenging and membranes' stabilizers against free radicals. Thus, they play a role in the antioxidative defense system and have a promising potential as therapeutic neuroprotective agents for the treatment of neurodegenerative disorders, connected with oxidative stress, such as Parkinson's disease.

Immunohistochemistry (IHC) staining of in-vitro cancer cell-generated tumoroids

Targeting different pathways in combinational therapy may lead to synergistic effects with higher drug efficiency. Due to a large number of candidate drugs and the variability in the genomic landscape of the disease, conventional cell culture models have limited success. Three-dimensional (3D) cell culture platforms such as tumoroids not only provide a pathophysiological relevant condition but also allow for low-cost and high-throughput drug screening strategies. Immunostaining of targeted proteins within a tumoroid is challenging as the interior cells are difficult to access via a non-destructive method. Immunohistochemistry (IHC) is an important technique in clinical research to explore the expression of various biomarkers. IHC staining of tumoroids allows non-destructive detection of unstable proteins by direct fixation of cells at the state of tumor microenvironment (TME) context, providing two main advantages. First, the target protein can be fixed without dissociating cells and disintegration of tumoroids into a single-cell suspension. Second, staining the preserved structure of tumoroids helps identify the location of the target proteins as well as the spatial distribution throughout the tumoroid geometry. In this protocol, we describe the detailed methodology of a non-destructive IHC staining of cancer biomarkers which minimizes the manipulation of tumoroids prior to fixation by eliminating multiple centrifugations and shaking steps typically required for removing excess hydrogel and collecting tumoroids. The protocol can be used in studies involving prognostic and predictive biomarker investigations in new anti-tumor drug development strategies.

Screening of the active Ingredients in Huanglian Jiedu decoction through amide bond-Immobilized magnetic nanoparticle-assisted cell membrane chromatography.

Introduction: The Huanglian Jiedu decoction (HLJDD) is a Chinese herbal formula that exerts neuroprotective effects by alleviating oxidative stress injuries and may potentially be prescribed for treating Alzheimer's disease; however, its active ingredients have not yet been identified. Cell membrane chromatography is a high-throughput method for screening active ingredients, but traditional cell membrane chromatography requires multiple centrifugation steps, which affects its separation efficiency. Magnetic nanoparticles are unparalleled in solid-liquid separation and can overcome the shortcomings of traditional cell membrane chromatography. Methods: In this study, the neuroprotective effects of the components of HLJDD were screened through a novel magnetic nanoparticle-assisted cell membrane chromatography method. Magnetic nanoparticles and cell membranes were stably immobilized by amide bonds. Magnetic bead (MB)-immobilized cell membranes of HT-22 cells were incubated with the HLJDD extract to isolate specific binding components. The specific binding components were then identified by ultraperformance liquid chromatography (UPLC)-Orbitrap Fusion Tribrid MS after solid-phase extraction. The bioactivity of these components was analyzed in an HT-22 cellular model of glutamate-induced injury. Results and Discussion: The preparation method of the composite of cell membrane and MBs has the advantages of simple preparation and no introduction of toxic organic reagents. MBs not only provide support for cell membranes, but also greatly improve the separation efficiency compared with traditional cell membrane chromatography. Fifteen of these components were found to specifically bind to the cell membranes, and seven of them were confirmed to reduce varying degrees of glutamate-induced toxicity in HT-22 cells. In conclusion, our findings suggest that the amide bond-based immobilization of magnetic nanoparticles on cell membranes, along with solid-phase extraction and UPLC, is an effective method for isolating and discovering the bioactive components of traditional Chinese medicines.

Comparison of methods to isolate peripheral blood mononuclear cells from cattle blood

Peripheral blood mononuclear cells (PBMCs) are critical for assessment of host immune responses to infectious disease. The isolation of PBMCs from whole blood is a laborious process involving density gradients and multiple centrifugation steps. In the present study we compared a more traditional method of PBMC isolation used in our laboratory to two novel methods of cell isolation for efficiency, cell viability, and enumeration of cell subsets. Our laboratory method uses Histopaque-1077 density gradient in standard conical tubes and this was compared with isolation of cells using SepMate™ tubes, a novel conical tube containing an insert to separate the density gradient. Multiple experiments were performed to optimize the SepMate™ tubes for use with cattle blood. A final experiment was conducted to compare traditional methodology, the optimized SepMate™ method with a more novel method using cell preparation tubes (CPT-10 vacutainers containing density gradient). Results demonstrated that optimization of the SepMate™ tube methodology was necessary, including dilution of blood and addition of centrifugation steps to reduce platelet contamination. The CPT-10 tubes worked well but cell recovery was lower compared to other methods. Both of the newer methods were comparable to a modified version of our traditional laboratory method of PBMC isolation in terms of numbers of recovered viable cells and the frequency of immune cell subsets. Additionally, efficiency was improved, particularly with the SepMate™ tube method, resulting in reduced time in the laboratory as well as reduced usage of plasticware.

Separation and Extraction of Mixed Grinding Chips of Artificial Joints with Different Densities by Multiple Centrifugal Separations

Aseptic loosening caused by the wear and tear of the artificial joint prosthesis after implantation is one of the main causes of artificial joint failure. Therefore, it is important to investigate the wear debris generated due to wear when developing new artificial joint materials. Aseptic loosening is related to the size, number, and morphology of wear debris, and this study proposed the separation and extraction of mixed wear debris with different density ratios of artificial joints by centrifugation to study the characteristics of different artificial joint wear and wear debris extraction rates. The results showed that multiple centrifugations to separate the mixed wear debris were able to reintroduce the wear debris on the wall of the centrifuge tube into the solution and that the wear debris extraction rate was increased. Suspensions with different density ratios of artificially jointed mixed wear debris were effectively separated by this method. The total extraction rate of the three repeated extractions compared to the first extraction rate, the extraction rate of CoCrMo wear debris increased by 6.7%, ultra-high molecular weight polyethylene (UHMWPE) wear debris increased by 15.1–23.44%, ZrO2 wear debris increased by 10.91%, and that of polyether ether ketone (PEEK) wear debris increased by 9.95%. This method for separating and extracting wear debris from artificial joints can realize the separation of mixed wear debris from artificial joints and obtain a high extraction rate and high-quality wear debris images, investigate the wear mechanism of artificial joint implants, and provide valuable information on the wear performance of new artificial joint implants under investigation.

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets

Digital droplet PCR (ddPCR) is classified as the third-generation PCR technology that enables absolute quantitative detection of nucleic acid molecules and has become an increasingly powerful tool for clinic diagnosis. We previously established a CLEAR-dPCR technique based on the combination of CLEAR droplets generated by micro-centrifuge-based microtubule arrays (MiCA) and insitu 3D readout by light-sheet fluorescence imaging. This CLEAR-dPCR technique attains very high readout speed and dynamic range. Meanwhile, it is free from sample loss and contamination, showing its advantages over commercial d-PCR technologies. However, a conventional orthogonal light-sheet imaging setup in CLEAR d-PCR cannot image multiple centrifuge tubes, thereby limiting its widespread application to large-scale, high-speed dd-PCR assays. Herein, we propose an in-parallel 3D dd-PCR readout technique based on an open-top light-sheet microscopy setup. This approach can continuously scan multiple centrifuge tubes which contain CLEAR emulsions with highly diverse concentrations, and thus further boost the scale and throughput of our 3D dd-PCR technique.

Comparison of sperm preparation methods to improve the recovery of mature spermatozoa in sub-fertile males.

The integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.

Robust Dual-Biomimetic Titanium Dioxide-Cellulose Monolith for Enrichment of Phosphopeptides.

Metal oxide affinity chromatography (MOAC) is considered to be one of the most effective methods for phosphopeptide enrichment. However, most of the materials used in the method are powder; frequent centrifugation is necessitated during the enrichment process, and potential risks of loss of peptides and materials and clogging of the column employed for liquid chromatography-mass spectrometry (LC-MS) arise. Moreover, the reusability of these materials to achieve sustainability was hardly investigated. To overcome these limitations, herein, inorganic titanium dioxide (TiO2) was coated onto the skeletal surface of the organic cellulose monolith (CM) material with a coral-like structure via a sol-gel method. This produced an organic-inorganic hybrid TiO2-CM material, which contained a combination of organic and inorganic substances, making it mimic the mollusk shell in terms of composition. The prepared TiO2-CM material as monolith exhibited excellent mechanical strength and did not break during the enrichment process; thus, the tedious implementation of multiple centrifugation cycles was prevented, thereby streamlining the experimental procedure and avoiding the loss of peptides and materials. Moreover, a large amount of TiO2 was introduced onto the surface of the CM material, and thus, the resultant TiO2-CM material exhibited a large surface area. As a result, the fabricated TiO2-CM material was successfully applied to the enrichment of phosphopeptides obtained from the tryptic digests of a BSA/β-casein (mass ratio, 500/1) mixture. The results were superior to those achieved for commercial TiO2 beads, confirming that TiO2-CM has excellent selectivity for phosphopeptides and reusability. Furthermore, 9287 unique phosphopeptides derived from the 2661 phosphoproteins were successfully identified from two milligrams of tryptic digests of Hela cell exosomes obtained through five independent replications after enriching using the TiO2-CM material. The results indicated that the material has good application prospects in the analysis of protein phosphorylation. Furthermore, TiO2-CM consists of green and cheap cellulose as the skeleton, and its synthesis process is environment-friendly, simple, and inexpensive.

Silica nanoparticle-loaded thermoresponsive block copolymer vesicles: a new post-polymerization encapsulation strategy and thermally triggered release.

A thermoresponsive amphiphilic diblock copolymer that can form spheres, worms or vesicles in aqueous media at neutral pH by simply raising the dispersion temperature from 1 °C (spheres) to 25 °C (worms) to 50 °C (vesicles) is prepared via polymerization-induced self-assembly (PISA). Heating such an aqueous copolymer dispersion from 1 °C up to 50 °C in the presence of 19 nm glycerol-functionalized silica nanoparticles enables this remarkable ‘shape-shifting’ behavior to be exploited as a new post-polymerization encapsulation strategy. The silica-loaded vesicles formed at 50 °C are then crosslinked using a disulfide-based dihydrazide reagent. Such covalent stabilization enables the dispersion to be cooled to room temperature without loss of the vesicle morphology, thus aiding characterization and enabling the loading efficiency to be determined as a function of both copolymer and silica concentration. Small-angle X-ray scattering (SAXS) analysis indicated a mean vesicle membrane thickness of approximately 20 ± 2 nm for the linear vesicles and TEM studies confirmed encapsulation of the silica nanoparticles within these nano-objects. After removal of the non-encapsulated silica nanoparticles via multiple centrifugation–redispersion cycles, thermogravimetric analysis indicated that vesicle loading efficiencies of up to 86% can be achieved under optimized conditions. Thermally-triggered release of the silica nanoparticles is achieved by cleaving the disulfide bonds at 50 °C using tris(2-carboxyethyl)phosphine (TCEP), followed by cooling to 20 °C to induce vesicle dissociation. SAXS is also used to confirm the release of silica nanoparticles by monitoring the disappearance of the structure factor peak arising from silica–silica interactions.

Strategies for Engineering Affordable Technologies for Point-of-Care Diagnostics of Infectious Diseases.

Disease prevalence is highest in low-resource settings (LRS) due to the lack of funds, infrastructure, and personnel required to carry out laboratory-based molecular tests. In high-resource settings, gold-standard molecular tests for diseases consist of nucleic acid amplification tests (NAATs) due to their excellent sensitivity and specificity. These tests require the extraction, amplification, and detection of nucleic acids from clinical samples. In high-resource settings, all three of these steps require highly specialized, costly, and onerous equipment that cannot be used in LRS. Nucleic acid extraction involves multiple centrifugation steps. Amplification consists of the polymerase chain reaction (PCR), which requires thermal cyclers. The detection of amplified DNA is typically done with specialized thermal cyclers that are capable of fluorescence detection. Traditional methods used to extract, amplify, and detect nucleic acids cannot be used outside of a laboratory in LRS. Thus, there is a need for affordable point-of-care devices to ease the high burden of disease in LRS.The past decade of work on paper-based fluidic devices has resulted in the invention of many paper-based biosensors for disease detection as well as isothermal amplification techniques that replace PCR. However, a challenge still remains in detecting pathogenic biomarkers from complex human samples without specialized laboratory equipment. Our research has focused on the development of affordable technologies to extract and detect nucleic acids in clinical samples with minimal equipment. Here we describe methods for the paper-based extraction, amplification, and detection of nucleic acids. This Account provides an overview of our latest technologies developed to detect an array of diseases in low-resource settings. We focus on detecting nucleic acids of H1N1, human papillomavirus (HPV), Neisseria gonorrheae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), and malaria from a variety of clinical sample types. H1N1 RNA was extracted from nasopharyngeal swabs; HPV, NG, and CT DNA were extracted from either cervical, urethral, or vaginal swabs; TV DNA was extracted from urine; and malaria DNA was extracted from whole blood. Different sample types necessitate different nucleic extraction protocols; we provide guidelines for assay design based on the clinical sample type used. We compare the pros and cons of different isothermal amplification techniques, namely, helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), and a novel isothermal amplification technique that we developed: isothermal-identical multirepeat sequences (iso-IMRS). Finally, we compare various detection mechanisms, including lateral-flow and electrochemical readouts. Electrochemical readouts frequently employ gold electrodes due to strong gold-thiol coupling. However, the high cost of gold precludes their use in LRS. We discuss our development of novel gold leaf electrodes that can be made without specialized equipment for a fraction of the cost of commercially available gold electrodes.

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

Peritoneal mucosa of mesothelial cells line the abdominal cavity, surround intestinal organs and the female reproductive organs and are responsible for immunological integrity, organ functionality and regeneration. Peritoneal diseases range from inflammation, adhesions, endometriosis, and cancer. Efficient technologies to isolate and cultivate healthy patient-derived mesothelial cells with maximal purity enable the generation of capable 2D and 3D as well as in vivo-like microfluidic cell culture models to investigate pathomechanisms and treatment strategies. Here, we describe a new and easily reproducible technique for the isolation and culture of primary human mesothelial cells from laparoscopic peritoneal wash cytology. We established a protocol containing multiple washing and centrifugation steps, followed by cell culture at the highest purity and over multiple passages. Isolated peritoneal mesothelial cells were characterized in detail, utilizing brightfield and immunofluorescence microscopy, flow cytometry as well as Raman microspectroscopy and multivariate data analysis. Thereby, cytokeratin expression enabled specific discrimination from primary peritoneal human fibroblasts. Raman microspectroscopy and imaging were used to study morphology and biochemical properties of primary mesothelial cell culture compared to cryo-fixed and cryo-sectioned peritoneal tissue.

Development of a multi-channel magnetic bead micro-probe assay for high-throughput detection of zearalenone in edible and medicinal Coix seed

A multi-channel magnetic bead micro-probes assay (MBPA) based on indirect competitive principle was developed for high-throughput detection of zearalenone (ZEA) in edible and medicinal Coix seed. This strategy introduced magnetic beads as the carriers, the specific primary antibodies as the capture probes for targets and the secondary antibodies functionalized goat anti-mouse immunoglobulin G labeled fluorescein isothiocyanate as the fluorescence signal probes. Through the competitive reaction of ZEA in Coix seed samples and that covalently coupled on the surface of MBs with their specific antibodies, as well as fast magnetic separation and sensitive fluorescence detection, the developed MBPA strategy allowed low limit of detection (2.03 ng/mL) with broad dynamic range (2.03–440.67 ng/mL), as well as excellent accuracy with the average recovery rate of 96.39% and relative standard deviation (RSD) of 5.48% for ZEA. 36 samples could realize simultaneous analysis in one operation within less than 20 min only needing 50 μL of solution and 30 s of sampling, avoiding large consumption of time and organic solvents. Multiple centrifugation and cleanup steps were omitted because of magnetic separation, avoiding the loss of targets. Diverse capture and fluorescent probes can be randomly bound onto the surface of MBs, making the MBPA strategy a promising tool for on-site high-throughput monitoring of various trace hazard factors in food safety, and environmental monitoring.

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z’ values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Standardized bacteriophage purification for personalized phage therapy.

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward-by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 1011 plaque-forming units (PFUs) per milliliter for Pseudomonas, Klebsiella, and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 109 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 106-fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.

Gene selection of non-small cell lung cancer data for adjuvant chemotherapy decision using cell separation algorithm

Since recommended treatment for Non-small cell lung cancer (NSCLC) after surgery is chemotherapy, the prediction of effectiveness or futileness of adjuvant chemotherapy (ACT) in early stage is important for future decision. Classification of NSCLC in gene expression data is performed to predict effectiveness or futileness of ACT. Selection of genes highly correlated with the class attribute, affects the classification accuracy. In this paper, a new cell separation algorithm is proposed which it imitates the action of cell separation using differential centrifugation process involving multiple centrifugation steps and increasing the rotor speed in each step. The CSA uses the application of centrifugal force to separate the solutions based on their objective function in different steps while the velocity is increased in each step. The CSA contributes to automatic trade-off between exploration and exploitation by control of selection rate during the search process. To examine the CSA, 25 test functions were used first and then the CSA was applied to predict effectiveness or futileness of ACT. The number of genes in candidate subsets is handled by increasing the subset size if after a certain number of iterations there is no improvement in fitness of the subset. This contributes to less time consideration and memory usage. In this experiment, the NSCLC data contain 280 samples collected from four institutes are used. As results, the minimum number of five genes with dependency degree equal to one and classification accuracy of higher than 94% for SVM, KNN and MLP classifiers is obtained.