Removal Of Cell Debris Research Articles

High hydrostatic pressure treatment for advanced tissue grafts in reconstructive head and neck surgery.

The increasing importance of regenerative medicine has resulted in a growing need for advanced tissue replacement materials in head and neck surgery. Allo- and xenogenic graft processing is often time-consuming and can deteriorate the extracellular matrix (ECM). High hydrostatic pressure (HHP)-treatment could allow specific devitalization while retaining the essential properties of the ECM. Porcine connective tissue and cartilage were HHP-treated at 100-400 MPa for 10 min. Structural modifications following HHP-exposure were examined using electron microscopy, while devitalization was assessed through metabolism and cell death analyses. Furthermore, ECM alterations and decellularization were evaluated by histology, biomechanical testing, and DNA content analysis. Additionally, the inflammatory potential of HHP-treated tissue was evaluated in vivo using a dorsal skinfold chamber in a mouse model. The devitalization effects of HHP were dose-dependent, with a threshold identified at 200 MPa for fibroblasts and chondrocytes. At this pressure level, HHP induced structural alterations in cells, with a shift toward late-stage apoptosis. HHP-treatment preserved ECM structure and biomechanical properties, but did not remove cell debris from the tissue. This study observed a pressure-dependent increase of markers suggesting the occurrence of immunogenic cell death. In vivo investigations revealed an absence of inflammatory responses to HHP-treated tissue, indicating a favorable biological response to HHP. In conclusion, application of HHP devitalizes fibroblasts and chondrocytes at 200 MPa while retaining the essential properties of the ECM. Prospectively, HHP may simplify the preparation of allo- and xenogenic tissue replacement materials and increase the availability of grafts in head and neck surgery.

Effects of blood-processing protocols on cell-free DNA fragmentomics in plasma: Comparisons of one- and two-step centrifugations

BackgroundCell-free DNA (cfDNA) fragmentomic characteristics are promising analytes with abundant physiological signals for non-invasive disease diagnosis and monitoring. Previous studies on plasma cfDNA fragmentomics commonly employed a two-step centrifugation process for removing cell debris, involving a low-speed centrifugation followed by a high-speed centrifugation. However, the effects of centrifugation conditions on the analysis of cfDNA fragmentome remain uncertain. MethodsWe collected blood samples from 10 healthy individuals and divided each sample into two aliquots for plasma preparation with one- and two-step centrifugation processes. We performed whole genome sequencing (WGS) of the plasma cfDNA in the two groups and comprehensively compared the cfDNA fragmentomic features. Additionally, we reanalyzed the fragmentomic features of cfDNA from 16 healthy individuals and 16 COVID-19 patients, processed through one- and two-step centrifugation in our previous study, to investigate the impact of centrifugation on disease signals. ResultsOur results showed that there were no significant differences observed in the characteristics of nuclear cfDNA, including size, motif diversity score (MDS) of end motifs, and genome distribution, between plasma samples treated with one- and two-step centrifugation. The cfDNA size shortening in COVID-19 patients was observed in plasma samples with one- and two-step centrifugation methods. However, we observed a significantly higher relative abundance and longer size of cell-free mitochondrial DNA (mtDNA) in the one-step samples compared to the two-step samples. This difference in mtDNA caused by the one- and two-step centrifugation methods surpasses the pathological difference between COVID-19 patients and healthy individuals. ConclusionsOur findings indicate that one-step low-speed centrifugation is a simple and potentially suitable method for analyzing nuclear cfDNA fragmentation characteristics. These results offer valuable guidance for cfDNA research in various clinical scenarios.

Regeneration and reuse of transwell-type culture inserts for blood-brain barrier modelling

Culture inserts of Transwell type are widely used in laboratories worldwide to model non-cerebral vessels, the blood-brain barrier (BBB), and other blood-tissue barriers, and also for the study of chemotaxis and transmigration. However, the use of inserts can generate plastic waste that has an environmental impact due to their one-off design and massive consumption. Thus, it is important to develop a method that can reduce the utilization of inserts but not affect research efficiency. In this study, we propose using a 1:1 (v:v) mix of 30% hydrogen peroxide and 99% sulfuric acid (“piranha solution’’) to completely remove cell debris and matrix from culture inserts. BBB models with inserts regenerated using piranha solution have barrier properties comparable to those of fresh inserts. We show that piranha solution is an effective reagent and allows for the reuse of Transwell-type inserts for BBB modeling up to 5 times. Therefore, the use of this method greatly reduces the production of laboratory waste and benefits numerous laboratories worldwide.

Piezocatalytic Schottky Junction Treats Atherosclerosis by a Biomimetic Trojan Horse Strategy.

The atherosclerotic vulnerable plaque is characterized by thefoamy macrophage burden, involving impaired cholesterol efflux and deficient efferocytosis. Correspondingly, piezocatalytic therapy is an emerging solution for eliminating the foamy macrophage burden with satisfactory spatiotemporal controllability and deep penetration depth. Herein, a biomimetic Trojan horse (Au-ZnO@MM) is engineered by coating the macrophage membrane (MM) onto the surface of a rod-like Au-ZnO Schottky Junction to effectively relieve the atherosclerotic progression. These Trojan horses with the coating of MM are actively transported into subsistent foamy macrophages and generate abundant reactive oxygen species (ROS) via ultrasound-activated piezocatalysis. ROS-initiated autophagy and mitochondrial dysfunction induce substantial cell apoptosis, alleviating the burden of subsistent foamy macrophages. The resulting apoptotic fragments further significantly facilitate cholesterol excretion and trigger efferocytosis of intraplaque fresh macrophages. Ultimately, the biomimetic Au-ZnO@MM piezocatalyst not only inhibits the foaming capacity of macrophages, but also improves the function of removing cell debris, which can stabilize atherosclerotic vulnerable plaque. Meanwhile, the plasmon resonance effect of integrated gold nanoparticles enables favorable photoacoustic molecular imaging for real-time image-guided atherosclerotic therapy. This proposed biomimetic Trojan horse strategy provides the paradigm of employing ultrasound-activated piezocatalytic methodology for enhanced atherosclerotic theranostics.

Critical Factors in Sample Collection and Preparation for Clinical Metabolomics of Underexplored Biological Specimens.

This review article compiles critical pre-analytical factors for sample collection and extraction of eight uncommon or underexplored biological specimens (human breast milk, ocular fluids, sebum, seminal plasma, sweat, hair, saliva, and cerebrospinal fluid) under the perspective of clinical metabolomics. These samples are interesting for metabolomics studies as they reflect the status of living organisms and can be applied for diagnostic purposes and biomarker discovery. Pre-collection and collection procedures are critical, requiring protocols to be standardized to avoid contamination and bias. Such procedures must consider cleaning the collection area, sample stimulation, diet, and food and drug intake, among other factors that impact the lack of homogeneity of the sample group. Precipitation of proteins and removal of salts and cell debris are the most used sample preparation procedures. This review intends to provide a global view of the practical aspects that most impact results, serving as a starting point for the designing of metabolomic experiments.

A Biomimetic Polymer for the Extraction and Purification of Superior Analogues of Amphotericin B.

Amphotericin B has been an essential drug in the fight against leishmaniasis and fungal pathogens for decades, and has more recently gained attention for the very limited microbial resistance displayed against it. However, its toxicity has restricted its use to only the most severe cases of disease, and attempts to reduce these ill effects via formulation have had only minor success. Genetic engineering has allowed the development of superior amphotericin analogues, notably 16-descarboxyl-16-methyl amphotericin B (MeAmB), which shows a ten-fold reduction in toxicity in addition to a slight improvement in therapeutic activity. However, MeAmB is difficult to extract from its bacterial source and purify. Presented here is an alternative method of MeAmB purification. A biomimetic polymer with a high affinity for MeAmB was designed via computational modelling and synthesised. Prepared as a separation column, the polymer was able to retain the target MeAmB whilst allowing the removal of cell debris from the bacterial extract. Starting with a simple bacterial extract, the relatively simple process allowed the purification of an MeAmB salt complex at approximately 70% MeAmB, and likely higher purification from further extraction. The mean MeAmB recovery between the pre-purification extract sample and the final product was 81%. This is the first successful demonstration of extraction or purification of any amphotericin molecule with any polymeric material. The biomimetic polymer was additionally reusable and simple to fabricate, giving this technique significant advantages over traditional methods of extraction and purification of valuable compounds.

Microglial tissue surveillance: The never-resting gardener in the developing and adult CNS.

Immunosurveillance by microglia is a dynamic process in the central nervous system (CNS) with versatile functions to maintain tissue homeostasis and provide immune defense. A tightly controlled microglia network throughout the CNS parenchyma facilitates efficient immunosurveillance, where each cell guards a certain tissue territory. Each cell is constantly surveilling its environment and the surrounding cells, screening for pathogens but also removing cell debris and metabolites, grooming neighboring cells and facilitating cellular crosstalk. In the absence of inflammation, this "tissue surveillance" by microglia presents an essential process for CNS homeostasis and development. In this review, we provide a summary on different tissue surveillance functions mediated by microglia, the underlying molecular machineries, and how defects, such as genetic mutations, can alter these surveillance mechanisms and cause disease onset.

Separating and Purifying Mycosporine-like Amino Acids from Cyanobacteria for Application in Commercial Sunscreen Formulations.

Using algal-derived mycosporine-like amino acids (MAAs) in sunscreen formulations is constrained by low cellular concentrations of MAAs and by the high costs associated with harvesting algal cells and extracting the MAAs. Here, we report an industrial scalable method using a membrane filtration approach to purify and concentrate aqueous extracts of MAAs. The method includes an additional biorefinery step enabling purification of phycocyanin, an established valuable natural product. Cultivated cells of the cyanobacterium Chlorogloeopsis fritschii (PCC 6912) were concentrated and homogenised to produce a feed for sequential processing through three membranes of decreasing pore size to obtain a retentate and permeate for each step. Microfiltration (0.2 µm) was used to remove cell debris. Ultrafiltration (10,000 Da) was used to remove large molecules and recover phycocyanin. Finally, nanofiltration (300-400 Da) was used to remove water and other small molecules. Permeate and retentate were analysed using UV-visible spectrophotometry and HPLC. The initial homogenised feed had a shinorine concentration of 5.6 ± 07 mg L-1. The final nanofiltered retentate resulted in a 3.3 times-purified concentrate (shinorine concentration of 18.71 ± 0.29 mg L-1). Significant process losses (35%) highlight scope for improvement. Results confirm the potential of membrane filtration to purify and concentrate aqueous solutions of MAAs with simultaneous separation of phycocyanin highlighting a biorefinery approach.

Neuroglia Cells Transcriptomic in Brain Development, Aging and Neurodegenerative Diseases.

Glia cells are essential for brain functioning during development, aging and disease. However, the role of astroglia plays during brain development is quite different from the role played in the adult lesioned brain. Therefore, a deeper understanding of pathomechanisms underlying astroglia activity in the aging brain and cerebrovascular diseases is essential to guide the development of new therapeutic strategies. To this end, this review provides a comparison between the transcriptomic activity of astroglia cells during development, aging and neurodegenerative diseases, including cerebral ischemia. During fetal brain development, astrocytes and microglia often affect the same developmental processes such as neuro-/gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis, and synaptic pruning. In the adult brain astrocytes are a critical player in the synapse remodeling by mediating synapse elimination while microglia activity has been associated with changes in synaptic plasticity and remove cell debris by constantly sensing the environment. However, in the lesioned brain astrocytes proliferate and play essential functions with regard to energy supply to the neurons, neurotransmission and buildup of a protective scar isolating the lesion site from the surroundings. Inflammation, neurodegeneration, or loss of brain homeostasis induce changes in microglia gene expression, morphology, and function, generally referred to as "primed" microglia. These changes in gene expression are characterized by an enrichment of phagosome, lysosome, and antigen presentation signaling pathways and is associated with an up-regulation of genes encoding cell surface receptors. In addition, primed microglia are characterized by upregulation of a network of genes in response to interferon gamma. Conclusion. A comparison of astroglia cells transcriptomic activity during brain development, aging and neurodegenerative disorders might provide us with new therapeutic strategies with which to protect the aging brain and improve clinical outcome.

The Innate Immune System in Cardiovascular Diseases and Its Role in Doxorubicin-Induced Cardiotoxicity

Innate immune cells are the early responders to infection and tissue damage. They play a critical role in the initiation and resolution of inflammation in response to insult as well as tissue repair. Following ischemic or non-ischemic cardiac injury, a strong inflammatory response plays a critical role in the removal of cell debris and tissue remodeling. However, persistent inflammation could be detrimental to the heart. Studies suggest that cardiac inflammation and tissue repair needs to be tightly regulated such that the timely resolution of the inflammation may prevent adverse cardiac damage. This involves the recognition of damage; activation and release of soluble mediators such as cytokines, chemokines, and proteases; and immune cells such as monocytes, macrophages, and neutrophils. This is important in the context of doxorubicin-induced cardiotoxicity as well. Doxorubicin (Dox) is an effective chemotherapy against multiple cancers but at the cost of cardiotoxicity. The innate immune system has emerged as a contributor to exacerbate the disease. In this review, we discuss the current understanding of the role of innate immunity in the pathogenesis of cardiovascular disease and dox-induced cardiotoxicity and provide potential therapeutic targets to alleviate the damage.

Decellularization of Articular Cartilage: A Hydrochloric Acid-Based Strategy.

Removing cellular material from a tissue, a process called decellularization, reduces the risk of adverse host reactions, allows for efficient decontamination, and extends the shelf-life of the matrix. It facilitates the use of cartilage tissue as human-derived allograft, thus providing the field of cartilage regeneration with a biomaterial unmatched in its similarity to native cartilage in terms of structure, composition, and mechanical properties.The dense extracellular matrix of articular cartilage requires a particularly thorough process to achieve the removal of cells, cell debris, and reagents used in the process. In our studies (Nürnberger et al., EBioMedicine 64:103196, 2021; Schneider et al., Tissue Eng Part C Methods 22(12):1095-1107, 2016), we have successfully developed a protocol for achieving decellularization via physical, chemical, and enzymatic steps. Combining freeze-thaw cycles for devitalization, hydrochloric acid as decellularization agent and the enzymatic removal of glycosaminoglycans, results in an acellular scaffold that is fully biocompatible and promotes cellular attachment. The structure and sophisticated architecture of collagen type II is left intact.This chapter provides a comprehensive guide to the steps and reagents needed to decellularize articular cartilage. In addition to the standard decell-deGAG protocol, a fast option is given which is suitable for thin specimen. Histological evaluation is presented to illustrate treatment success.

Roadmap to success in AAV purification. In-process control, high throughput & novel column modalities as necessary means for control over scalable AAV process

Manufacture and purification of recombinant adeno-associated viruses (rAAV) require development and optimization of processes to ensure the best possible quality of the final rAAV product. To do so, different strategies in upstream can be used to achieve the highest possible viral titer and lowest amount of impurities, both of which further influence downstream. Second challenge involves removal of cell debris where different pre-treatments can be utilized. In the next step, optimized capture of rAAV on a cation-exchange chromatography should be developed to remove impurities and achieve a high recovery of rAAV. In the end, several chromatographic options are available to remove empty and defected capsids, so only functional viruses can be isolated. Here, the process of manufacturing and purification of rAAV has been designed using monolithic columns to achieve this important goal of preparing rAAV for the use in gene therapy. 

Sepax-2 cell processing device: a study assessing reproducibility of concentrating thawed hematopoietic progenitor cells

BackgroundAutologous hematopoietic progenitor cell (HPC) transplantation is currently the standard of care for a fraction of patients with newly diagnosed myelomas and relapsed or refractory lymphomas. After high-dose chemotherapy, cryopreserved HPC are either infused directly after bedside thawing or washed and concentrated before infusion. We previously reported on the comparability of washing/concentrating HPC post-thaw vs. infusion without manipulation in terms of hematopoietic engraftment, yet settled for the prior favoring cell debris and DMSO removal. For almost two decades, automation of this critical step of washing/concentrating cells has been feasible. As part of continuous process verification, we aim to evaluate reproducibility of this procedure by assessing intra-batch and inter-batch variability upon concentration of thawed HPC products using the Sepax 2 S-100 cell separation system.MethodsAutologous HPC collected from the same patient were thawed and washed either in two batches processed within a 3-4 h interval and immediately infused on the same day (intra-batch, n = 45), or in two batches on different days (inter-batch, n = 49) for those patients requiring 2 or more high-dose chemotherapy cycles. Quality attributes assessed were CD34+ cell recovery, viability and CD45+ viability; CFU assay was only performed for allogeneic grafts.ResultsIntra-batch and inter-batch median CD34+ cell recovery was comparable (75% vs. 73% and 77% vs. 77%, respectively). Similarly, intra-batch and inter-batch median CD45+ cell viability was comparable (79% vs. 80% and 79% vs. 78%, respectively). Bland-Altman analysis describing agreement between batches per patient revealed a bias close to 0%. Additionally, lower HPC recoveries noted in batch 1 were noted as well in batch 2, regardless of the CD34+ cell dose before cryopreservation, both intra- and inter-batch, suggesting that the quality of the collected product plays an important role in downstream recovery. Intrinsic (high mature and immature granulocyte content) and extrinsic (delay between apheresis and cryopreservation) variables of the collected product resulted in a significantly lower CD45+ viability and CD34+ cell recovery upon thawing/washing.ConclusionsAutomated post-thaw HPC concentration provides reproducible cell recoveries and viabilities between different batches. Implications of this work go beyond HPC to concentrate cell suspension/products during manufacturing of cell and gene therapy products.

Multiplexed Single-Nucleus RNA Sequencing Using Lipid-Oligo Barcodes.

This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled with the same lipid-coupled oligonucleotide barcode (10X Genomics). Nuclei from as many as 12 individual samples can be pooled together and simultaneously processed for cDNA library construction and subsequent DNA sequencing. While previous protocols using lipid-coupled oligonucleotide barcodes were optimized for analysis of samples consisting of viable cells, this protocol is optimized for analyses of quick-frozen cell samples. The protocol ensures efficient recovery of nuclei both by incorporating high sucrose buffered solutions and by including a tracking dye (trypan blue) during nuclei isolation. The protocol also describes a procedure for removing single nuclei 'artifacts' by removing cell debris prior to single nuclear fractionation. This protocol informs the use of computational tools for filtering poorly labeled nuclei and assigning sample identity using barcode unique molecular identifier (UMI) read counts percentages. The computational pipeline is applicable to either cultured or primary, fresh or frozen cells, regardless of their cell types and species. Overall, this protocol reduces batch effects and experimental costs while enhancing sample comparison. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling cells with lipid oligo barcodes and generating multiplexed single-nucleus RNA-seq libraries Basic Protocol 2: Bioinformatic deconvolution of the multiplexed snRNAseq libraries.

Aqueous two-phase extraction to separate alpha-ketoglutaric acid from fermentation broth by combining crystallization

Alpha-ketoglutaric acid (α-KG) is a crucial intermediate with broad application prospects. However, the downstream separation of α-KG from fermentation broth with low concentration and complex components has become the bottleneck of large-scale production. In this study, α-KG was separated from the actual fermentation broth by aqueous two-phase system (ATPS) and crystallization. The optimal ATPS composed of acetone/(NH4)2SO4 was selected by studying the phase diagram and α-KG distribution behavior in different organic solvents and salts/sugars. Under the optimized conditions, the purity of α-KG products from crude fermentation broth reached 98.08%, and the one-pass extraction efficiency was 90.52%. The removal of cell debris and soluble proteins increased with the increase of acetone and (NH4)2SO4 concentrations, with the removal ratios reaching up to 99.44% and 86.54%. In addition, 92.56% of the glucose was distributed in the aqueous phase. Although the α-KG fermentation broth is more complex than the whole-cell biotransformation, impurities in the crude fermentation broth could be efficiently removed and a higher α-KG distribution coefficient was obtained. Meanwhile, the recovered (NH4)2SO4 could be reused for fermentation medium. This strategy provides an economical and effective method combined with upstream biosynthesis to recover α-KG and other bioproducts from crude fermentation broth.

Perivascular Mesenchymal Stem/Stromal Cells, an Immune Privileged Niche for Viruses?

Mesenchymal stem cells (MSCs) play a critical role in response to stress such as infection. They initiate the removal of cell debris, exert major immunoregulatory activities, control pathogens, and lead to a remodeling/scarring phase. Thus, host-derived ‘danger’ factors released from damaged/infected cells (called alarmins, e.g., HMGB1, ATP, DNA) as well as pathogen-associated molecular patterns (LPS, single strand RNA) can activate MSCs located in the parenchyma and around vessels to upregulate the expression of growth factors and chemoattractant molecules that influence immune cell recruitment and stem cell mobilization. MSC, in an ultimate contribution to tissue repair, may also directly trans- or de-differentiate into specific cellular phenotypes such as osteoblasts, chondrocytes, lipofibroblasts, myofibroblasts, Schwann cells, and they may somehow recapitulate their neural crest embryonic origin. Failure to terminate such repair processes induces pathological scarring, termed fibrosis, or vascular calcification. Interestingly, many viruses and particularly those associated to chronic infection and inflammation may hijack and polarize MSC’s immune regulatory activities. Several reports argue that MSC may constitute immune privileged sanctuaries for viruses and contributing to long-lasting effects posing infectious challenges, such as viruses rebounding in immunocompromised patients or following regenerative medicine therapies using MSC. We will herein review the capacity of several viruses not only to infect but also to polarize directly or indirectly the functions of MSC (immunoregulation, differentiation potential, and tissue repair) in clinical settings.

Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination by Murine Bone Marrow-Derived Macrophages (BMDMs)

ObjectivesTo develop BMEs that evade elimination by BMDMs. MethodsBovine mammary alveolar MAC-T cells secrete MEs and were used to engineer MEs that express proprietary protein features suspected to decrease elimination by BMDMs, denoted UNL1 and UNL2. MAC-T cells were transformed by using lentiviral vectors. BMEs featuring UNL1 or UNL2 were isolated from MAC-T cell culture media supernatant using polyethylene glycol (PEG) precipitation after removing cell debris by using a 0.22-μm filter. The BMEs were labeled using a carbonyl-reactive fluorescent dye and purified by ultracentrifugation. Primary BMDMs was isolated from mouse hind legs (C57BL/6J, aged 8–10 weeks) and seeded in 96-well plates for assessing BME uptake at a physiological concentration (1010 BMEs/mL). Uptake was compared to unmodified BMEs and normalized for BMDM density. Time points were compared pairwise by using t-test, and P < 0.05 was considered significant. ResultsThe uptake of BME UNL1 by BMDMs was reduced by 37%, 42%, 48% and 47% as compared to unmodified BMEs after 12 h, 24 h, 36 h and 48h, respectively in culture dishes (P < .05; n = 5). Data are preliminary (n = 3), yet encouraging, for BME UNL2: The uptake of BME UNL2 was reduced by 41%, 44%, 46% and 46% compared to unmodified BMEs after 12 h, 24 h, 36 h and 48 h, respectively. ConclusionsThe elimination of BMEs UNL1 and UNL2 is significantly reduced compared unmodified BMEs in BMDM cultures. This is of great importance when using BMEs for delivering therapeutics. Funding SourcesNIH P20GM104320, NIFA2016-67,001-25,301 and 2022–67,021-36,407), USDA Hatch and W-4002, and the SynGAP Research Fund (all to J. Z.). J.Z. serves as consultant for PureTech Health, Inc.

Multiparametric analysis in GBM plasma extracellular vesicles (Evs) and surface marker expression profile.

2038 Background: Glioblastoma (GBM) is the most common malignant brain tumor with poor clinical prognosis. Management of GBM is hampered by the lack of an accurate test that can be used for differential diagnosis of tumor progression from inflammatory pseudoprogression. Plasma extracellular vesicles (EVs) has been shown to be a promising source for biomarker identification. In this project, we aimed to identify GBM plasma EV markers that could serve as the basis of a liquid biopsy. Methods: Sample preparation, assay controls and instrument calibration were performed following MIFlowCyt-EV guideline. Plasma samples were subjected to 2-step centrifugations to remove cell debris and platelets. 10ul of plasma sample was diluted with 90ul filtered PBS, then stained for EV surface markers including CD9, CD31, CD45, CD41a and CD11b, as well as actin phalloidin. Stained plasma samples were purified using IZON qEV1/70nm column, then EV fractions were analyzed using full spectrum Cytek Aurora flow cytometer. Clustering analysis was performed on EV events (CD9 +/ actin phalloidin -) using t-SNE and FlowSOM extensions from FlowJo plugins. Results: Compared to normal donors, GBM plasma EVs were bigger in size (higher SSC value) and expressed higher levels of CD9, CD31, CD45 and CD11b while ND plasma EVs had higher CD41a expression. t-SNE and FlowSOM analysis demonstrated that GBM plasma EVs had a unique surface marker expression profile compared to ND EVs. It also showed 10 EV sub-populations that differed in size as well as various surface marker expression levels. Four of these subpopulations were enriched in GBM EVs, while three of these were enriched in ND EVs. Conclusions: This multiparametric analysis revealed that GBM plasma EVs had a unique surface marker expression profile compared to ND plasma EVs. Further separation and molecular profiling analysis based on each sub population could reveal EV biomarkers that are unique to each sample population.

Inside Front Cover: A simple and sensitive liquid chromatography–tandem mass spectrometry method for quantification of multi‐residual pesticides in blood

DOI: 10.1002/sscp.202200067 The cover picture shows the extraction of pesticides from blood samples and a developed LC-MS/MS method to determine the levels of 33 different pesticides in human blood. The developed method used a simple extraction method that started with blood collection from farmers in an EDTA tube. A 100 μL of blood was added to a centrifuge tube and then adding cold acetonitrile to remove cell debris and then subjected to cold incubation for pesticides in blood to solubilize into the solvent. Finally, the extracted sample was filtered through 0.2-micron filters to get a clear sample. A 10 μL of the extracted sample was injected into an LCMS/MS system, and the results were recorded. An Agilent zorbax C18 reverse-phase chromatographic column was used in this study. This developed method efficiently separated the 33 pesticides of our interest. The chromatographic run involved a 20 min run with different retention times for each analyte. The developed method was checked by employing validation parameters such as robustness, ruggedness, recovery, and found that the method has passed all the parameters as per the European standard guidelines.

Collagen matrices are preserved following decellularization of a bovine bone scaffold.

The decellularization of bovine bone has emerged as a strategy for the repair, replacement, and regeneration of bone defects. To evaluate the effects of a new protocol of bone decellularization and its impact on the structure and collagen scaffold. Cancellous bone from bovine femur was dissected in fragments and decellularized based on protocol of multiple steps. The residual protein levels, histological, morphometric, and scanning electronic microscopy analyses were carried out to evaluate the effects of decellularization and the impact on the structure and collagen scaffold. A cytotoxicity assay was performed. Residual protein analysis showed an important removal of bone marrow components and cell debris from the bone. Sections revealed that collagen fibers presented integrity and absence of cells in the decellularized bone. Sirius Red-stained sections of collagen fiber collagen matrix were maintained after decellularization. Scanning electron microscopy revealed that the main bone structure, despite being irregular, was maintained in both groups, with no significant visual differences between the surface characteristics according to the groups. Decellularized bovine bone demonstrated a degree of toxicity of 3, indicating moderate reactivity. The present data demonstrate that the main bone structure was maintained. Additionally, the chemical and physical treatments were able to remove cellular debris, and extracellular matrix architecture and collagen were preserved. However, the tissue showed moderate toxicity.