Cell Homogenates Research Articles

Neutrophil heterogeneity and emergence of a distinct population of CD11b/CD18-activated low-density neutrophils after trauma.

Multiple large clinical trauma trials have documented an increased susceptibility to infection after injury. Although neutrophils (polymorphonuclear leukocytes [PMNs]) were historically considered a homogeneous cell type, we hypothesized that injury could alter neutrophil heterogeneity and predispose to dysfunction. To explore whether trauma modifies PMN heterogeneity, we performed an observational mass-spectrometry-based cytometry study on total leukocytes and low-density PMNs found in the peripheral blood mononuclear cell fraction of leukocytes from healthy controls and trauma patients. A total of 74 samples from 12 trauma patients, each sampled at 1 or more time points, and matched controls were fractionated and profiled by mass-spectrometry-based cytometry using a panel of 44 distinct markers. After deconvolution and conservative gating on neutrophils, data were analyzed using Seurat, followed by clustering of principal components. Eleven distinct neutrophil populations were resolved in control and trauma neutrophils based on differential protein surface marker expression. Trauma markedly altered the basal heterogeneity of neutrophil subgroups seen in the control samples, with loss of a dominant population of resting neutrophils marked by high expression of C3AR and low levels of CD63, CD64, and CD177 (cluster 1), and expansion of two alternative neutrophil populations, one of which is marked by high expression of CD177 with suppression of CD10, CD16, C3AR, CD63, and CD64 (cluster 6). Remarkably, following trauma, a substantially larger percentage of neutrophils sediment in the monocyte fraction. These low-density neutrophils bear markers of functional exhaustion and form a unique trauma-induced population (cluster 9) with markedly upregulated expression of active surface adhesion molecules (activated CD11b/CD18), with suppression of nearly all other surface markers, including receptors for formyl peptides, leukotrienes, chemokines, and complement. Circulating neutrophils demonstrate considerable evidence of functional heterogeneity that is markedly altered by trauma. Trauma induces evolution of a novel, exhausted, low-density neutrophil population with immunosuppressive features.

Wildtype heterogeneity contributes to clonal variability in genome edited cells

Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype–phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.

A Biomimetic Nonwoven-Reinforced Hydrogel for Spinal Cord Injury Repair.

In clinical trials, new scaffolds for regeneration after spinal cord injury (SCI) should reflect the importance of a mechanically optimised, hydrated environment. Composite scaffolds of nonwovens, self-assembling peptides (SAPs) and hydrogels offer the ability to mimic native spinal cord tissue, promote aligned tissue regeneration and tailor mechanical properties. This work studies the effects of an aligned electrospun nonwoven of P11-8—enriched poly(ε-caprolactone) (PCL) fibres, integrated with a photo-crosslinked hydrogel of glycidylmethacrylated collagen (collagen-GMA), on neurite extension. Mechanical properties of collagen-GMA hydrogel in compression and shear were recorded, along with cell viability. Collagen-GMA hydrogels showed J-shaped stress–strain curves in compression, mimicking native spinal cord tissue. For hydrogels prepared with a 0.8-1.1 wt.% collagen-GMA concentration, strain at break values were 68 ± 1–81 ± 1% (±SE); maximum stress values were 128 ± 9–311 ± 18 kPa (±SE); and maximum force values were 1.0 ± 0.1–2.5 ± 0.1 N (±SE). These values closely mimicked the compression values for feline and porcine tissue in the literature, especially those for 0.8 wt.%. Complex shear modulus values fell in the range 345–2588 Pa, with the lower modulus hydrogels in the range optimal for neural cell survival and growth. Collagen-GMA hydrogel provided an environment for homogenous and three-dimensional cell encapsulation, and high cell viability of 84 ± 2%. Combination of the aligned PCL/P11-8 electrospun nonwoven and collagen-GMA hydrogel retained fibre alignment and pore structure, respectively, and promoted aligned neurite extension of PC12 cells. Thus, it is possible to conclude that scaffolds with mechanical properties that both closely mimic native spinal cord tissue and are optimal for neural cells can be produced, which also promote aligned tissue regeneration when the benefits of hydrogels and electrospun nonwovens are combined.

Histopathological Features of Primary Cutaneous Diffuse Large B-Cell Lymphoma Leg Type: A Case Report

Background: Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT), is a rare and aggressive lymphoma with poor prognostic. It is difficult to diagnose PCDLBCL-LT at an early stage due to its nonspecific manifestations that overlap with other lymphomas. A histopathological examination can be performed to establish the diagnosis.
 Case Presentation: A 74-year-old female presented with itchy red, swollen patches accompanied by a burning sensation on both her legs. Clinical examination revealed a hard ping pong ball-sized lump palpable on her left inner thigh. Haematoxylin eosin staining demonstrated the proliferation of diffused neoplastic cells, homogenous cells with atypical nuclei, and mitoses. Immunohistochemical examination revealed positive for CD 45 as well as CD 20 and negative for CD3. These findings were consistent with a diagnosis of PCDLBCL-LT.
 Conclusion: The presented case demonstrates that skin lesions in patients with diagnosed PCDLBCL-LT may have a variable clinical presentation. Histologically is characterized by a diffuse dermal infiltrate mostly consisting of activated B-cell (centroblast) and activated lymphocyte (immunoblast), with high mitotic activity and a minimal T-reactive component. Immunohistochemical analysis is necessary for establishing the diagnosis of PCDLBCL-LT which shows positive markers for CD 45, CD 20, CD79a, Bcl-2, MUM-1, FOX-P1, CD10, IgM, CD 138, Ki-67, CD 30 and MYC.

Impact of Non-Muscle Cells on Excitation-Contraction Coupling in the Heart and the Importance of In Vitro Models.

Excitation-coupling (ECC) is paramount for coordinated contraction to maintain sufficient cardiac output. The study of ECC regulation has primarily been limited to cardiomyocytes (CMs), which conduct voltage waves via calcium fluxes from one cell to another, eliciting contraction of the atria followed by the ventricles. CMs rapidly transmit ionic flux via gap junction proteins, predominantly connexin 43. While the expression of connexin isoforms has been identified in each of the individual cell populations comprising the heart, the formation of gap junctions with nonmuscle cells (i.e., macrophages and Schwann cells) has gained new attention. Evaluating nonmuscle contributions to ECC in vivo or in situ remains difficult and necessitates the development of simple, yet biomimetic in vitro models to better understand and prevent physiological dysfunction. Standard 2D cell culture often consists of homogenous cell populations and lacks the dynamic mechanical environment of native tissue, confounding the phenotypic and proteomic makeup of these highly mechanosensitive cell populations in prolonged culture conditions. This review will highlight the recent developments and the importance of new microphysiological systems to better understand the complex regulation of ECC in cardiac tissue.

Neutrophils in autoimmunity: when the hero becomes the villain.

Neutrophils were long considered to be a short-lived homogenous cell population, limited to their role as first responders in anti-bacterial and -fungal immunity. While it is true that neutrophils are first to infiltrate the site of infection to eliminate pathogens, growing evidence suggests their functions could extend beyond those of basic innate immune cells. Along with their well-established role in pathogen elimination, utilizing effector functions such as phagocytosis, degranulation, and the deployment of neutrophil extracellular traps (NETs), neutrophils have recently been shown to possess antigen-presenting capabilities. Moreover, the identification of different subtypes of neutrophils points to a multifactorial heterogeneous cell population with great plasticity in which some subsets have enhanced pro-inflammatory characteristics, while others seem to behave as immunosuppressors. Interestingly, the aberrant presence of activated neutrophils with a pro-inflammatory profile in several systemic and organ-specific autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), multiple sclerosis (MS), and type 1 diabetes (T1D) could potentially be exploited in novel therapeutic strategies. The full extent of the involvement of neutrophils, and more specifically that of their various subtypes, in the pathophysiology of autoimmune diseases is yet to be elucidated.

"SIT" with Emx1-NuTRAP Mice: Simultaneous INTACT and TRAP for Paired Transcriptomic and Epigenetic Sequencing.

Epigenetic regulation of transcription is gaining increasing importance in the study of neurobiology. The advent of sequencing technology has enabled the study of this regulation across the entire genome and transcriptome. However, modern methods that allow the correlation of transcriptomic data with epigenomic regulation have had several key limitations, including use of separate tissue sources and detection of low-expression genes. This article describes a method combining isolation of nuclei tagged in specific cell types (INTACT) with translating ribosome affinity purification (TRAP) in the same cell homogenate, referred to as Simultaneous INTACT and TRAP (SIT). We used this technical approach to directly couple transcriptomic sequencing with epigenomic data in neurons derived from the mouse hippocampus. We demonstrate this method with an Emx1-NuTRAP transgenic mouse model. Here, we present protocols for SIT and for the generation and validation of the Emx1-NuTRAP mouse model that we used to demonstrate SIT. These methods enable cell type-specific comparison of translating mRNA and chromatin data from the same set of cells. Using SIT and the Emx1-NuTRAP transgenic mouse model, researchers can compare epigenomic data to transcriptomic data in the same set of hippocampal excitatory neurons. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Emx1-NuTRAP transgenic mouse line for labeling excitatory neurons in the hippocampus Basic Protocol 2: SIT: Simultaneous Isolation of nuclei tagged in specific cell types (INTACT) and Translating ribosome affinity purification (TRAP).

Evaluation of Poly-3-Hydroxybutyrate (P3HB) Scaffolds Used for Epidermal Cells Growth as Potential Biomatrix.

Advances in tissue engineering have made possible the construction of organs and tissues with the use of biomaterials and cells. Three important elements are considered: a specific cell culture, an adequate environment, and a scaffold. The present study aimed to develop P3HB scaffolds by 3D printing and evaluate their biocompatibility with HaCaT epidermal cells, as a potential model that allows the formation of functional tissue. By using a method of extraction and purification with ethanol and acetone, a biopolymer having suitable properties for use as a tissue support was obtained. This polymer exhibited a higher molecular weight (1500 kDa) and lower contact angle (less than 90°) compared to the material obtained using the conventional method. The biocompatibility analysis reveals that the scaffold obtained using the ethanol–acetone method and produced by 3D printing without pores was not cytotoxic, did not self-degrade, and allowed high homogenous cell proliferation of HaCaT cells. In summary, it is possible to conclude that the P3HB scaffold obtained by 3D printing and a simplified extraction method is a suitable support for the homogeneous development of HaCaT keratinocyte cell lineage, which would allow the evaluation of this material to be used as a biomatrix for tissue engineering.

Crop phenotype prediction using biclustering to explain genotype-by-environment interactions.

Phenotypic variation in plants is attributed to genotype (G), environment (E), and genotype-by-environment interaction (GEI). Although the main effects of G and E are typically larger and easier to model, the GEI interaction effects are important and a critical factor when considering such issues as to why some genotypes perform consistently well across a range of environments. In plant breeding, a major challenge is limited information, including a single genotype is tested in only a small subset of all possible test environments. The two-way table of phenotype responses will therefore commonly contain missing data. In this paper, we propose a new model of GEI effects that only requires an input of a two-way table of phenotype observations, with genotypes as rows and environments as columns that do not assume the completeness of data. Our analysis can deal with this scenario as it utilizes a novel biclustering algorithm that can handle missing values, resulting in an output of homogeneous cells with no interactions between G and E. In other words, we identify subsets of genotypes and environments where phenotype can be modeled simply. Based on this, we fit no-interaction models to predict phenotypes of a given crop and draw insights into how a particular cultivar will perform in the unused test environments. Our new methodology is validated on data from different plant species and phenotypes and shows superior performance compared to well-studied statistical approaches.

A modern automated patch-clamp approach for high throughput electrophysiology recordings in native cardiomyocytes

Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp (APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases.

Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Given that HCC is an extraordinarily heterogeneous malignant disease, finding an effective therapeutic strategy for treating it has been difficult. Because of the importance of angiogenesis in tumorigenesis, targeting the more homogenous HCC endothelial cells may be a better therapeutic strategy. In a unpublished manuscript, we found that the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase 2/9 (MMP2/9) were reduced in human HCC tissues that overexpressed DNA damage repair gene general transcription factor II subunit H2 (GTF2H2). This suggested that GTF2H2 may have an inhibitory effect on angiogenesis. Therefore, we hypothesized that GTF2H2 acts as an anti-angiogenesis gene. However, our results showed that GTF2H2 overexpression had no effect on endothelial cell viability, migration, or permeability. To our surprise, treating human umbilical vein endothelial cells (HUVECs) with the culture medium of Huh 7 cells overexpressing GTF2H2 could inhibit their viability, migration, and permeability. We then isolated the culture medium into exosomes and other components from the culture medium. Only GTF2H2-enriched exosomes could inhibit the viability, migration, tube formation, and permeability of HUVECs. Our results suggest that overexpressing GTF2H2 had no effect on HUVECs, while GTF2H2 enriched exosomes from Huh7 cells could inhibit HUVEC phenotypes such as proliferation and migration. Therefore, GTF2H2-enriched exosomes can possibly be utilized as a novel drug for treating HCC and also serve as a potential molecular target for inhibiting tumor angiogenesis.

Single-cell phenotypic profiling to identify a set of immune cell protein biomarkers for relapsed and refractory diffuse large B cell lymphoma: A single-center study.

Diffuse large B-cell lymphoma (DLBCL) is the most common invasive type of non-Hodgkin lymphoma. Cell-of-origin (COO) classification is related to patients' prognoses. Primary drug resistance in treatment for DLBCL has been observed. The specific serum biomarkers in these patients who suffer from relapsed and refractory (R/R)-DLBCL remains unclear. In the current study, using single-cell RNA sequencing (scRNA-seq) and mass cytometry (CyTOF), we determined and verified immune cell biomarkers at the mRNA and protein levels in single-cell resolution from 18 diagnostic PBMC specimens collected from patients with R/R DLBCL. As controls, 5 PBMC specimens from healthy volunteers were obtained. We identified a panel of 35 surface marker genes for the features of R/R DLBCL unique cell cluster by scRNA-seq of 8 R/R DLBCL patient samples and validated its efficiency in an external cohort consisting of 10 R/R DLBCL patients by CyTOF. The cell clustering and dimension reduction were compared among R/R DLBCL samples in CyTOF Space with COO as well as the C-MYC expression designation. Immune cells from each patient occupied unique regions in the 32-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Significant heterogeneity observed in subgroups was mainly attributed to individual differences among samples and not to expression differences in a single, homogeneous immune cell subpopulation. The marker panel showed reliability in labeling R/R DLBCL without any influence from COO stratification and C-MYC expression designation. Furthermore, we compared all the markers between R/R DLBCL and normal samples. A total of 12 biomarkers were significantly overexpressed in R/R DLBCL relative to the normal samples. Therefore, we further optimized the diagnostic biomarker panel of R/R DLBCL comprising CD82, CD55, CD36, CD63, CD59, IKZF1, CD69, CD163, CD14, CD226, CD84, and CD31. In summary, we developed a novel set of biomarkers for the diagnoses of patients with R/R DLBCL. Detections procedures at single-cell resolution provide precise biomarkers, which may substantially overcome intertumoral and intratumoral heterogeneity among primary samples. The findings confirmed that each case was unique and may comprise multiple, genetically distinct subclones.

Cell Membrane and V2C MXene-Based Electrochemical Immunosensor with Enhanced Antifouling Capability for Detection of CD44.

The inactive adsorption and interference of biomolecules in electrochemical biosensors is a topic of intense interest. Directly utilizing native cell membranes to endow electrochemical surfaces with antifouling and biocompatible features is a promising strategy, rather than attempting to synthetically replicate complex biological interface properties. In this study, we present a facial and sensitive sandwich-type antifouling immunoassay through platelet membrane/Au nanoparticle/delaminated V2C nanosheet (PM/AuNPs/d-V2C)-modified electrode as the substrate of sensing interface and methylene blue/aminated metal organic framework (MB@NH2-Fe-MOF-Zn) as an electrochemical signal probe. The biosensor perfectly integrates the high conductivity of AuNPs-loaded V2C MXene with the excellent loading property of NH2-Fe-MOF-Zn to improve the electrochemical sensing performance. In addition, the excellent antifouling properties of the homogeneous cell membrane can effectively prevent the non-specific adsorption of model proteins. The obtained antifouling biosensor possesses the capability of ultrasensitive detection of CD44 and CD44-positive cancer cell in complex liquids and exhibits good analytical performance for the analysis of CD44 with a linear range from 0.5 ng/mL to 500 ng/mL. This strategy of developing cell membrane-based biosensing systems with enhanced antifouling capability can be easily expanded to the construction of other complex biosensors, and the advanced biological probes and analytical methods provide a favorable means to accurately quantify biomarkers associated with tumor progression.

Multilayer Electrospun-Aligned Fibroin/Gelatin Implant for Annulus Fibrosus Repair: An In Vitro and In Vivo Evaluation.

Annulus fibrosus (AF) damage is proven to prompt intervertebral disc (IVD) degeneration, and unrepaired AF lesions after surgical discectomy may boost herniation of the nucleus pulposus (NP) which may lead to further compression of neural structures. Moreover, vascular and neural ingrowth may occur within the defect which is known as a possible reason for discogenic pain. Due to a limited healing capacity, an effective strategy to repair and close the AF defect is necessary. In this study, using electrospinning technology, two nature polymers, silk fibroin and gelatin, were linked to imitate the unique lamellae structure of native AF. Our findings revealed that a multilayer electrospun-aligned fibroin/gelatin scaffold with mechanical and morphological properties mimicking those of native AF lamellae have been developed. The average diameter of the nanofiber is 162.9 ± 38.8 nm. The young’s modulus is around 6.70 MPa with an ultimate tensile strength of around 1.81 MP along preferred orientation. The in vitro test confirmed its biocompatibility and ability to maintain cell viability and colonization. Using a porcine model, we demonstrated that the multilayer-aligned scaffold offered a crucial microenvironment to induce collagen fibrous tissue production within native AF defect. In the implant-repaired AF, H&E staining showed homogeneous fibroblast-like cell infiltration at the repaired defect with very little vascular ingrowth, which was confirmed by magnetic resonance imaging findings. Picrosirius red staining and immunohistochemical staining against type I collagen revealed positively stained fibrous tissue in an aligned pattern within the implant-integrated site. Relative to the intact control group, the disc height index of the serial X-ray decreased significantly in both the injury control and implant group at 4 weeks and 8 weeks (p < 0.05) which indicated this scaffold may not reverse the degenerative process. However, the results of the discography showed that the effectiveness of annulus repair of the implant group is much superior to that of the untreated group. The scaffold, composed with nature fibroin/gelatin polymers, could potentially enhance AF healing that could prevent IVD recurrent herniation, as well as neural and neovascular ingrowth after discectomy surgeries.

Heterogeneity of the NIH3T3 Fibroblast Cell Line.

The embryonic mouse fibroblast cell line NIH3T3 is widely used in life science research, including the study of cell cycle control and primary cilia. Fibroblasts are the most important cell type in connective tissue, as they produce components of the extracellular matrix and determine tissue architecture. However, they are very heterogeneous and consist of subtypes specific to their organ of residence, among others. The NIH3T3 cell line was derived from whole mouse embryos that developed to pre-birth and is therefore most likely composed of different fibroblast subtypes. Furthermore, prolonged proliferation may have influenced their cellular composition. A heterogeneous cell population is unsuitable for any sophisticated research project. We found that the proportion of ciliated cells in the total NIH3T3 cell population was highly variable and asked whether this was a consequence of cellular heterogeneity and what molecular signatures were associated with it. We have established sub-cell lines by clonal expansion of single cells and characterized them morphologically and molecularly. Eventually, a myofibroblast-like and a fibroblast-like cell line were generated that differ in ciliation and proliferation. These homogeneous cell lines are valuable for a more detailed study of their molecular signatures, not least to uncover further the molecular pathways that contribute to the formation of the primary cilium.

3D numerical simulations and microstructural modeling of anisotropic and tension–compression asymmetric ductile materials

In the present work, we have analyzed the effect of anisotropy on void growth and stress–strain behavior for materials that exhibit remarkable tension–compression asymmetry (i.e., zirconium alloys). For that purpose, we have performed finite element simulations using a cubic 3D cell with a spherical void inside and subjected to periodic boundary conditions. Nonlinear kinematic constraints are also imposed as boundary conditions in order to maintain the values of macroscopic ratios constant during the whole loading history of the cell and account for a general (3D) stress state. The behavior of the matrix material is described by the CPB06 anisotropic criterion developed by Cazacu et al. (2006). The numerical results are compared to those considering 3D homogeneous (without void) cell with the same initial porosity as the voided one and governed by the anisotropic porous yield criterion developed by Stewart and Cazacu (2011). To investigate the influence of prescribed stress, strength differential parameter and strain hardening exponent on stress–strain behavior and void growth in the non-homogeneous (with void) and the homogeneous (without void) cells, we have used several stress ratios, three strength differential parameters and three strain hardening exponents. Finite element results obtained from different stress ratios show the strength differential parameter significantly affect void growth in both homogeneous and non-homogeneous cells. Moreover, comparison of two cells proves that both stress–strain behavior and porosity evolution are in good qualitative agreement for all three values of strength differential parameter. In contrast, as the value of strain hardening exponent increases, the agreement between results obtained from homogeneous and non-homogeneous cells is worse. An heuristic extension of the Stewart and Cazacu (2011)’s model is proposed in this work in an attempt to improve the accuracy of the model.

GFP fusion promotes the soluble and active expression of a pea actin isoform (PEAc1) in Escherichia coli

In the present study, we report that a GFP fusion tag facilitated the soluble expression of a pea actin isoform (PEAc1) in E. coli. To investigate the influence of a GFP fusion tag on PEAc1 structure and activity, PEAc1, His-tagged PEAc1 (His-PEAc1), His-tagged GFP (His-GFP), and His-tagged PEAc1 fusion with GFP (His-PEAc1-GFP) were expressed in E. coli. SDS-PAGE and western blot analyses reveal that the solubility of His-PEAc1-GFP was higher than that of PEAc1 and His-PEAc1. The His-PEAc1-GFP and His-GFP fusion proteins were purified from the supernatant of cell homogenate on a Ni-affinity column, and PEAc1 and His-PEAc1 were purified from inclusion bodies. CD spectrum analysis of the four purified proteins indicated that the proportion of α-helix and β-sheet in PEAc1 was closest to the predicted data in His-PEAc1-GFP (compared with His-PEAc1 or PEAc1). In addition, the actin-associated activities of His-PEAc1-GFP, including polymerization to microfilaments under specific ionic conditions and DNase I inhibition by monomers, were more similar to those of muscle actin (compared with PEAc1 and His-PEAc1). These improvements in PEAc1 solubility and activity are likely the result of correct PEAc1 folding mediated by GFP fusion.

Biomimetic Keratin-Coated Gold Nanoparticles for Photo-Thermal Therapy in a 3D Bioprinted Glioblastoma Tumor Model.

Before entering human clinical studies to evaluate their safety and effectiveness, new drugs and novel medical treatments are subject to extensive animal testing that are expensive and time-consuming. By contrast, advanced technologies enable the development of animal-free models that allow the efficacy of innovative therapies to be studied without sacrificing animals, while providing helpful information and details. We report on the powerful combination of 3D bioprinting (3DB) and photo-thermal therapy (PTT) applications. To this end, we realize a 3DB construct consisting of glioblastoma U87-MG cells in a 3D geometry, incorporating biomimetic keratin-coated gold nanoparticles (Ker-AuNPs) as a photo-thermal agent. The resulting plasmonic 3DB structures exhibit a homogeneous cell distribution throughout the entire volume while promoting the localization of Ker-AuNPs within the cells. A 3D immunofluorescence assay and transmission electron microscopy (TEM) confirm the uniform distribution of fluorescent-labeled Ker-AuNPs in the volume and their capability to enter the cells. Laser-assisted (λ = 532 nm) PTT experiments demonstrate the extraordinary ability of Ker-AuNPs to generate heating, producing the highest temperature rise of about 16 °C in less than 2 min.

Dynamic crystallization kinetics and morphology of carbonaceous nanofillers-reinforced poly (lactic acid) foams

Carbon black (CB), multi-walled carbon nanotubes (CNT), and graphene oxide (GO)-reinforced poly (lactic acid) (PLA) foams were prepared. Morphological studies indicated that incorporating nanoparticles into foamed PLA (F-PLA) changed the sphericity degree, diameter, and homogeneity of cells. Among all samples, foamed PLA containing 0.25 phr GO exhibited the highest void content (+46%) compared to foamed neat PLA, while its cell density diminished by 21%. In contrast, despite a lower improvement in void content (+18%), foamed PLA containing 0.25 phr CNT experienced a +3% enhancement in cell density compared to foamed neat PLA. Also, the nanoparticles were well dispersed in un-foamed PLA without any obvious aggregation. The crystallization kinetics of foams were studied using DSC. The results revealed that PLA foams reinforced with 0.25 phr CNT and GO had a significant effect on crystallization promotion. The crystallization kinetics of foamed samples were studied using different models such as Jeziorny, Avrami, Ozawa, Kissinger, and Doyle-Ozawa. The results demonstrated that adding nanofillers did not affect the crystallization mechanism of foamed PLA. Also, incorporating up to 0.5 phr CNT and GO decreased the apparent crystallization activation energy compared to neat F-PLA. An almost similar trend was observed for the local crystallization activation energy.

Isolation of Stably Transfected Melanoma Cell Clones.

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.