Methanol Fixation Research Articles

In-cell processing enables rapid and in-depth proteome analysis of low-input Caenorhabditis elegans.

Caenorhabditis elegans is a widely used genetic model organism, however, the worm cuticle complicates extraction of intracellular proteins, a prerequisite for typical bottom-up proteomics. Conventional physical disruption procedures are not only time-consuming, but can also cause significant sample loss, making it difficult to perform proteomics with low-input samples. Here, for the first time, we present an on-filter in-cell (OFIC) processing approach, which can digest C. elegans proteins directly in the cells of the organism after methanol fixation. With OFIC processing and single-shot LCMS analysis, we identified over 9,400 proteins from a sample of only 200 worms, the largest C. elegans proteome reported to date that did not require fractionation or enrichment. We systematically evaluated the performance of the OFIC approach by comparing it with conventional lysis-based methods. Our data suggest equivalent and unbiased performance of OFIC processing for C. elegans proteome identification and quantitation. We further evaluated the OFIC approach with even lower input samples, then used this method to determine how the proteome is impacted by loss of superoxide dismutase sod-1, the ortholog of human SOD-1, a gene associated with amyotrophic lateral sclerosis (ALS). Analysis of 8,800 proteins from only 50 worms as the initial input showed that loss of sod-1 affects the abundance of proteins required for stress response, ribosome biogenesis, and metabolism. In conclusion, our streamlined OFIC approach, which can be broadly applied to other systems, minimizes sample loss while offering the simplest workflow reported to date for C. elegans proteomics analysis.

A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue.

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.

Echeveria Leaf Morpho-Anatomical Analysis and Its Implications for Environmental Stress Conditions

Echeveria, classified in the Crassulaceae family, possesses unique adaptive strategies with xeromorphic features to withstand semi-arid environments. The diversity and ecological adaptation of succulent plants offer valuable insights into addressing climate change challenges. In particular, the epidermis, hypodermis, vascular bundles arrangement, and stomata characteristics are commonly used to investigate light, humidity, temperature, and water availability adaptations. While leaf anatomical analysis is a common approach, limited studies have been conducted on Echeveria, especially among cultivars. To understand how succulents cope with environmental stress, leaf morpho-anatomical features were analyzed using the free-hand sectioning method with methanol fixation of fifteen Echeveria cultivars. The finding revealed a robust correlation between epidermis and hypodermis size (r = 0.362–0.729), and a positive association between leaf thickness and the epidermis (r = 0.362–0.536), suggesting implications for water storage. Most cultivars displayed a 3D vascular arrangement, with minor vascular bundles surrounding the main vascular bundle at the center, along with small stomata size, and low stomata frequency in the adaxial surface. Moreover, these cultivars grown under controlled conditions maintain their xeromorphic characteristics with the presence of epicuticular wax and thick and fully expanded small leaves. Likewise, the features of cultivars ultimately suggest that these succulents are tolerant to high temperatures and limited water supply. This study provides a fundamental understanding of Echeveria plants’ leaf anatomy and the correlation of their leaf structures toward environmental stress. Likewise, the methods and results of this study will serve as a benchmark for other research in related species.

Effects of Chemical Fixatives on Kinetic Measurements of Biomolecular Interaction on Cell Membrane.

Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared the effects of five fixatives, i.e., formaldehyde vapor (FV), paraformaldehyde (PFA), acetone, methanol, and ethanol, on kinetic measurements via the LigandTracer method. We found that all five fixatives exerted insignificant effects on lectin-glycan interaction. However, antibody-receptor interaction is markedly perturbed by coagulant fixatives. The acetone fixation changed the binding of the anti-human epidermal growth factor receptor 2 (HER2) antibody to HER2 on the cell membrane from a 1:2 to a 1:1 binding model, while methanol and ethanol abolished the antibody binding possibly by removal of the HER2 receptors on the cell membrane. The capability of binding was retained when methanol fixation was performed at lower temperatures, albeit with a binding model of 1:1 instead. Moreover, whereas cell morphology does not exert a substantial impact on lectin-glycan interaction, it can indeed modify the binding model of antibody-receptor interaction. Our results provided insights into the selection of fixatives for cell-based kinetic studies.

Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation.

The application of single-cell RNA sequencing has greatly improved our understanding of various cellular and molecular mechanisms involved in physiological and pathophysiological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. In this study, we selected two AML samples that had been fixed in methanol for 12-18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effectively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus.

Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and library construction, raising the need for efficient sample preservation methods which are compatible with scRNA-seq downstream analysis. Several studies evaluated the effect of methanol fixation as preservation method, yet none of them deeply assessed its effect on adult primary dissociated brain tissue. Here, we evaluated its effect on murine dentate gyrus (DG) single cell suspensions and on subsequent scRNA-seq downstream analysis by performing SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CEL-seq2 protocol. Our results show that MeOH fixation preserves RNA integrity and has no apparent effects on cDNA library construction. They also suggest that fixation protects from sorting-induced cell stress and increases the proportion of high-quality cells. Despite evidence of mRNA leakage in fixed cells, their relative gene expression levels correlate well with those of fresh cells and fixation does not significantly affect the variance of the dataset. Moreover, it allows the identification of all major DG cell populations, including neural precursors, granule neurons and different glial cell types, with a tendency to preserve more neurons that are underrepresented in fresh samples. Overall, our data show that MeOH fixation is suitable for preserving primary neural cells for subsequent single-cell RNA profiling, helping to overcome challenges arising from complex workflows, improve experimental flexibility and facilitate scientific collaboration.

Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells

The main critical step in single-cell transcriptomics is sample preparation. Several methods have been developed to preserve cells after dissociation to uncouple sample handling from library preparation. Yet, the suitability of these methods depends on the cell types to be processed. In this project, we perform a systematic comparison of preservation methods for droplet-based single-cell RNA-seq on neural and glial cells derived from induced pluripotent stem cells. Our results show that while DMSO provides the highest cell quality in terms of RNA molecules and genes detected per cell, it strongly affects the cellular composition and induces the expression of stress and apoptosis genes. In contrast, methanol fixed samples display a cellular composition similar to fresh samples and provide a good cell quality and little expression biases. Taken together, our results show that methanol fixation is the method of choice for performing droplet-based single-cell transcriptomics experiments on neural cell populations.

Comparison of methanol fixation versus cryopreservation of the placenta for metabolomics analysis

Methods for collection of placental tissue at room temperature for metabolic profiling are described. Specimens were excised from the maternal side of the placenta and immediately flash frozen or fixed and stored for 1, 6, 12, 24, or 48 h in 80% methanol. Untargeted metabolic profiling was performed on both the methanol-fixed tissue and the methanol extract. Data were analyzed using Gaussian generalized estimating equations, two sample t-tests with false discovery rate (FDR) corrections, and principal components analysis. Methanol-fixed tissue samples and methanol extracts had a similar number of metabolites (p = 0.45, p = 0.21 in positive vs. negative ion mode). In positive ion mode, when compared to flash frozen tissue, both the methanol extract and methanol-fixed tissue (6 h) had a higher number of metabolites detected (146 additional metabolites, pFDR = 0.020; 149 additional metabolites, pFDR = 0.017; respectively), but these associations were not found in negative ion mode (all pFDR ≥ 0.05). Principle components analysis demonstrated separation of the metabolite features in the methanol extract, but similarity between methanol-fixed tissue and flash frozen tissue. These results show that placental tissue samples collected in 80% methanol at room temperature can yield similar metabolic data to flash frozen specimens.

Assessment of stained direct cytology smears of breast cancer for whole transcriptome and targeted messenger RNA sequencing.

Rather than surgical resection, cytologic specimens are often used as first-line clinical diagnostic procedures due to higher safety, speed, and cost-effectiveness. Archival diagnostic cytology slides containing cancer can be equivalent to tissue biopsies for DNA mutation testing, but the accuracy of transcriptomic profiling by RNA sequencing (RNA-seq) is less understood. This study compares the results from whole transcriptome RNA-seq and a targeted RNA-seq assay of stained cytology smears (CS) versus matched tumor tissue samples preserved fresh-frozen (FF) and processed as formalin-fixed paraffin-embedded (FFPE) sections. Cellular cytology scrapes from all 11 breast cancers were fixed and stained using three common protocols: Carnoy's (CS_C) or 95% ethanol (CS_E) fixation and then Papanicolaou stain or air-dried then methanol fixation and DiffQuik stain (CS_DQ). Agreement between samples was assessed using Lin's concordance correlation coefficient. Library yield for CS_DQ was too low, therefore it was not sequenced. The distributions of concordance correlation coefficient of gene expression levels in comparison to FF were comparable between CS_C and CS_E, but expression of genes enriched in stroma was lower in cytosmear samples than in FF or FFPE. Six signatures showed similar concordance to FF for all methods and two were slightly worse in CS_C and CS_E. Genomic signatures were highly concordant using targeted RNA-seq. The allele fraction of selected mutations calculated on cytosmear specimens was highly correlated with FF tissues using both RNA-seq methods. RNA can be reliably extracted from cytology smears and is suitable for transcriptome profiling or mutation detection, except for signatures of tumor stroma.

Saline dry fixation for improved cell composition analysis using Raman spectroscopy.

Raman spectroscopy enables the label-free assessment of cellular composition. While live cell analysis is the most accurate approach for cellular Raman spectroscopy, the analysis of fixed cells has proved to be very useful, particularly in collaborative projects where samples need to be serially examined by different laboratories or stored and reanalyzed at a later date. However, many chemicals that are widely used for cell fixation directly affect cellular biomolecules, yielding Raman spectra with missing or altered information. In this article, we compared the suitability of dry-fixation with saline versus chemical fixatives. We compared the Raman spectroscopy of saline dry-fixed cells with the more commonly used formaldehyde and methanol fixation and found that dry-fixed cell spectra preserved more cellular information than either chemical fixative. We also assessed the stability of dry-fixed cells over time and found that they were stable for at least 5 months. Finally, a comparison of dry-fixed and live cell spectra revealed effects due to the hydration state of the cells since they were recovered upon rehydrating dry-fixed samples. Thus, for fixed cell Raman spectroscopy, we recommend dry-fixation with unbuffered saline as a superior method to formaldehyde or methanol fixation.

Confocal Raman microscopy for assessing effects of preservation methods on symbiotic deep-sea mussel gills

Confocal Raman microscopy (CRM) is a powerful tool for biological research, which can provide information regarding the composition and distribution of biomolecules in an in situ, label-free, non-destructive manner and with high spatial resolution. Sample preservation is often an unavoidable step, especially for symbiotic deep-sea samples. Moreover, protocols for the preservation of samples for CRM have not been established and specific effects of different preservation methods on biomolecules have not been studied for relevant samples. In this study, we used deep-sea mussel Gigantidas platifrons, an ideal model in the study of deep-sea symbiosis and investigated the effect of four common preservation methods on the results of CRM imaging and signals. The methods included snap-freeze (SF), SF followed by rapid fixation in methanol (SF-MeOH), 2.5% glutaraldehyde and 2% paraformaldehyde fixation (SF-GP), and 4% paraformaldehyde and alcohol fixation (PS-PA). The results of this study indicate that SF was the most effective method for the comprehensive analysis of the biomolecular composition although the sectioning success rate was relatively low. Moreover, SF-MeOH was found to be effective when SF is not sufficient in obtaining good morphology in sections, or when the effect of chemical bonding on the composition of biomolecules upon SF-MeOH can be neglected. Finally, SF-GP and PS-PA were found to be the most effective methods considering the overall morphological observation. However, they were less suitable for metabolic studies. We believe our results can provide guidance for further studies of Raman on symbiotic deep-sea biological samples. It is of great importance for the wide application of Raman technique.

O-174 The association of chromosomal abnormalities with embryo ultrastructure, spindle anomalies and metabolic profiling

Abstract Study question Can metabolic profiling predict embryos at risk of chromosomal abnormalities and how are these reflected in the ultrustructure and cytoskeleton? Summary answer Different metabolic profiles are observed between normal and aneuploid/chaotic embryos which are linked to altered mitochondrial and other organelles’ structure/function and spindle and nuclear abnormalities. What is known already One of the greatest challenges in IVF is the selection of the best ‘fit’ embryo for implantation in a non-invasive way. Down’s syndrome embryos and Monosomy 21 embryos have previously been shown to have differential expression of metabolites compared to normal embryos, but limited studies, have investigated in detail the metabolic profiling of embryos with other abnormalities in comparison to chromosomally normal embryos or their reflection in the ultrustructure and the cytoskeleton. Study design, size, duration Culture media collected on day 3 from 200 embryos which underwent PGT-A, were analysed by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS). The chromosomally normal embryos were transferred to the uterus (1–2 embryos/ET) or still remain vitrified for clinical use and 120 of the embryos that were diagnosed with chromosomal abnormalities were either processed for spindle/chromosome configurations analysis (n = 60) by confocal laser scanning microscopy(CLSM) or for ultrastructural analysis (n = 60) by Transmission Electron Microscopy(TEM). Participants/materials, setting, methods Metabolic profiling was conducted in a Forensic Toxicology Laboratory by HILIC-MS/MS (100 metabolites). Spindle Chromosome Configuration analysis was conducted in an academic hospital after methanol fixation and immunostaining with α-tubulin, γ-tubulin, acetylated-tubulin antibodies and DAPI or/ PI to visualise DNA. Ultrastructure analysis by TEM was carried out in a Histology/Embryology Laboratory following embryo fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance This study provides screening for >100 primary metabolites using HILIC-MS/MS in a single run of 40 minutes. Characteristic patient specific metabolic profiles were observed which differed between normal embryos that had resulted in a viable pregnancy and aneuploid and chaotic embryos. Logistic regression analysis revealed a number of metabolites that had a high predictive value including Isoleucine, lysine and glucose and models were created in combination with embryo score which in the future could serve as non-invasive markers for the detection of chromosomal abnormalities before embryo transfer. TEM analysis revealed differences in the quality of cells and organelle activity which were reflected in the embryo metabolic profiles. Chaotic poor quality embryos showed a lower number of mitochondria, often with no cisternae, increased number of vacuoles, and frequently problems in junctions between cells. Aneuploid but well developed hatching blastocysts had mainly cells with good mitochondrial morphology/ activity, nice Golgi apparatus and well developed rough and smooth endoplasmic reticulum but depending on the aneuploidy involved, inner cell mass cells with limited organelles and occasionally lipofuscin droplets in the trophectoderm were evident. Nuclear and chromosomal abnormalities were interrelated through abnormalities in cytokinesis and spindle formation and reflected in the embryo metabolic profiles. Limitations, reasons for caution Although metabolic profiles were compared between normal and chromosomally abnormal embryos identified by PGT-A, all the normal embryos were transferred to the uterus or remain vitrified for clinical purposes and therefore the ultrastructure analysis and the spindle chromosome configuration analysis are based only on chromosomally abnormal embryos. Wider implications of the findings This study identified distinct differences in the metabolic profiles of normal and chromosomally abnormal embryos and provides unique metabolites which in the future could serve as non-invasive biomarkers for the detection of chromosomal abnormalities before embryo transfer. Trial registration number not applicable

Immunofluorescence Detection of Plasma Membranous PTEN in Cultured Cells.

PTEN is a well-known tumor suppressor with various functions that depend on its intracellular localization. Green fluorescent protein (GFP)-tagged live-cell images clarified the crucial amino acids needed to regulate the localization of PTEN in cells. However, it currently remains unknown whether GFP itself affects the intracellular localization of PTEN and its mutants, and the establishment of fixed-cell imaging is important for identifying the exact location of PTEN in cells. I herein investigated a number of immunofluorescence strategies for cell fixation, membrane permeabilization, and antigen retrieval. Permeabilization by detergents was necessary to observe nuclear and cytosolic PTEN in paraformaldehyde (PFA)-fixed cells; however, this permeabilization was not always valid. On the other hand, antigen retrieval by the pre-boiled EDTA treatment was useful for detecting plasma membranous PTEN in PFA-fixed cells in the same manner as in in vivo studies. Furthermore, methanol-fixed images of PTEN were consistent with GFP-tagged live-cell images. Two immunofluorescence methods (the PFA-fixed/pre-boiled EDTA treatment and methanol fixation) are applicable to investigations of the intracellular localization of PTEN without a GFP tag in cultured cells. In conclusion, live-cell imaging and appropriate immunofluorescence including a novel antigen retrieval treatment were both useful for detecting the cellular localization of PTEN, particularly at the plasma membrane.

Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms

Objective and designDrug delivery to inflammatory cells is dependent upon poorly understood, complex endocytic processes. Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules in mast cells (MC), but the mechanism of BBR internalization is unknown.MethodsBMMC were treated with various concentrations of BBR for different amounts of time and BBR internalization was assessed by flow cytometry and fluorescence microscopy. BMMC were pretreated with endocytic inhibitors or a growth factor (IL-3) prior to BBR exposure to access mechanisms of its internalization.ResultsAfter 24 h, 48 ± 0.8% of BMMC internalized BBR and this process was dependent upon temperature and the presence of glucose in the medium. Methanol fixation reduced BBR internalization, suggesting the involvement of an energy-dependent active transport mechanism. To determine mode of internalization, BBR was encapsulated into Lipofectamine TM lipoplexes since these are known to circumvent classical endocytic pathways. Incorporating BBR into lipoplexes decreased BBR internalization by 26% and 10% (10 μg/ml and 100 μg/ml Lipo-BBR respectively) by BMMC. BBR endocytosis was significantly reduced by Latrunculin B (88%), Cytochalasin B (87%), Chloroquine (86.5%) and 3-methyladenine (91%), indicating that actin polymerization, lysosomal pH and lysosomal self-degradation via the autophagy pathway was involved. In contrast, IL-3 treatment significantly enhanced BBR endocytosis (54% by 40 ng/ml IL-3) suggesting that IL-3 signaling pathways play a role in internalization.ConclusionsOur data suggests that internalization of BBR by resting and IL-3-activated BMMC utilizes an energy-dependent pathway that is dependent upon glucose metabolism and temperature. Furthermore, this process requires actin polymerization and lysosomal trafficking. These data suggest internalization of benzylisoquinoline compounds is an active and complex process.

High-throughput RNA sequencing of paraformaldehyde-fixed single cells

Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.

The effect of methanol fixation on single-cell RNA sequencing data

BackgroundSingle-cell RNA sequencing (scRNA-seq) has led to remarkable progress in our understanding of tissue heterogeneity in health and disease. Recently, the need for scRNA-seq sample fixation has emerged in many scenarios, such as when samples need long-term transportation, or when experiments need to be temporally synchronized. Methanol fixation is a simple and gentle method that has been routinely applied in scRNA-sEq. Yet, concerns remain that fixation may result in biases which may change the RNA-seq outcome.ResultsWe adapted an existing methanol fixation protocol and performed scRNA-seq on both live and methanol fixed cells. Analyses of the results show methanol fixation can faithfully preserve biological related signals, while the discrepancy caused by fixation is subtle and relevant to library construction methods. By grouping transcripts based on their lengths and GC content, we find that transcripts with different features are affected by fixation to different degrees in full-length sequencing data, while the effect is alleviated in Drop-seq result.ConclusionsOur deep analysis reveals the effects of methanol fixation on sample RNA integrity and elucidates the potential consequences of using fixation in various scRNA-seq experiment designs.

Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms

Abstract Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules of myeloid-derived mast cells, but, the mechanism of berberine’s internalization is poorly understood. Our data showed that 84±3.3% of highly granulated bone marrow-derived mast cells (BMMC) internalized BBR (10 ug/mL) after 45 hr. Methanol fixation completely blocked this process, suggesting an energy-dependent active transport mechanism, and 3-mtheyladenine (600 uM) inhibited internalization by ~43%, indicating that lysosomal self-degradation via the autophagy pathway was involved. BBR incorporation into lipoplexes did not improve BBR internalization, but, rather decreased it by ~30% (1 ug/mL Lipo/BBR; n=3, p<0.05), suggesting that BBR internalization may utilize clathrin-dependent endosomal endocytosis. Interleukin-3 (IL-3)-mediated activation of BMMC augmented BBR internalization (~54% by 40 ng/mL IL-3) in a concentration-dependent manner, further suggesting that clathrin-mediated endocytosis as well as active cell division, have a profound role in BBR internalization. Altogether, our data suggest that internalization of BBR in resting and IL-3 activated BMMC, utilizes clathrin-dependent pathway. Our findings provide important new mechanistic insights into the internalization of benzylsoquinoline compounds by resting and activated mast cells and open new avenues for designing organelle-targeted nanotherapeutics.

Antimicrobial Surface Functionality of PEG Coated and AgNPs Immobilized Extracorporeal Biomaterials

Nanotechnology is a promising field for generating new medicine applications like colloidal silver to prevent nosocomial infections. In this study, the objection of the presented manuscript was to test the antimicrobial activity of PEG/silver nanoparticles (AgNPs) coated blood tubing used in hemodialysis. The modification of plastic tubing was first performed by PEG coating, which was followed by immobilization of AgNPs synthesized with a green chemical approach from the leaves of Liquidambar Orientalis Miller. The stabilization of each surface modification stage was assured by methanol fixation. The characterization of AgNPs, surface modification steps, and final tubing surface appearances was performed by spectrophotometric measurements, DLS analysis, FTIR spectrums, and SEM images. The results indicated that the surfaces of hemodialysis tubing were successfully coated with PEG/AgNPs processed sequentially. Antimicrobial activities were evaluated against pathogens (Escherichia coli, Pseudomonas aeruginosa. Klebsiella pneumonia, Staphylococcus aureus, and Candida albicans), which are commonly involved in catheter-related infections. In conclusion, a new AgNPs immobilization method to polymeric catheter surfaces after PEG layering was developed. The antimicrobial function against five nosocomial microorganisms would have potential in biomedical applications.

An optimised GAS-pharyngeal cell biofilm model

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

Effects of fixation on bacterial cellular dimensions and integrity

SummaryFixation facilitates imaging of subcellular localization and cell morphology, yet it remains unknown how fixation affects cellular dimensions and intracellular fluorescence patterns, particularly during long-term storage. Here, we characterized the effects of multiple fixatives on several bacterial species. Fixation generally reduced cell length by 5–15%; single-cell tracking in microfluidics revealed that the length decrease was an aggregate effect of many steps in the fixation protocol and that fluorescence of cytoplasmic GFP but not membrane-bound MreB-msfGFP was rapidly lost with formaldehyde-based fixatives. Cellular dimensions were preserved in formaldehyde-based fixatives for ≥4 days, but methanol caused length to decrease. Although methanol preserved cytoplasmic fluorescence better than formaldehyde-based fixatives, some Escherichia coli cells were able to grow directly after fixation. Moreover, methanol fixation caused lysis in a subpopulation of cells, with virtually all Bacillus subtilis cells lysing after one day. These findings highlight tradeoffs between maintenance of fluorescence and membrane integrity for future applications of fixation.