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Microalgae are a promising feedstock with proven biostimulant activity that is enhanced by their biochemical components (e.g., amino acids and phytohormones), which turns them into an appealing feedstock to reduce the use of fertilisers in agriculture and improve crop productivity and resilience. Thus, this work aimed to isolate protein-rich microalgal mutants with increased biostimulant activity. Random mutagenesis was performed with Chlorella vulgaris, and a selection of protein-rich mutants were sorted through fluorescence-activated cell sorting (FACS), resulting in the isolation of 17 protein-rich mutant strains with protein contents 19–34% higher than that of the wildtype (WT). Furthermore, mutant F4 displayed a 38%, 22% and 62% higher biomass productivity, growth rate and chlorophyll content, respectively. This mutant was then scaled up to a 7 L benchtop reactor to produce biomass and evaluate the biostimulant potential of this novel strain towards garden cress seeds. Compared to water (control), the germination index and the relative total growth increased by 7% and 19%, respectively, after the application of 0.1 g L−1 of this bioproduct, which highlights its biostimulant potential. [ABSTRACT FROM AUTHOR]

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools. [ABSTRACT FROM AUTHOR]

Abstract A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.

Multilineage-differentiating stress-enduring (Muse) cells are a type of pluripotent cell with unique characteristics such as non-tumorigenic and pluripotent differentiation ability. After homing, Muse cells spontaneously differentiate into tissue component cells and supplement damaged/lost cells to participate in tissue repair. Importantly, Muse cells can survive in injured tissue for an extended period, stabilizing and promoting tissue repair. In addition, it has been confirmed that injection of exogenous Muse cells exerts anti-inflammatory, anti-apoptosis, anti-fibrosis, immunomodulatory, and paracrine protective effects in vivo. The discovery of Muse cells is an important breakthrough in the field of regenerative medicine. The article provides a comprehensive review of the characteristics, sources, and potential mechanisms of Muse cells for tissue repair and regeneration. This review serves as a foundation for the further utilization of Muse cells as a key clinical tool in regenerative medicine. [ABSTRACT FROM AUTHOR]

Neurogenesis involves the precisely coordinated action of genetic programs controlling large-scale neuronal fate specification down to terminal events of neuronal differentiation. The Q neuroblasts in Caenorhabditis elegans , QL on the left and QR on the right, divide, differentiate, and migrate in a similar pattern to produce three neurons each. However, QL on the left migrates posteriorly, and QR on the right migrates anteriorly. The MAB-5 /Hox transcription factor is necessary and sufficient for posterior Q lineage migration and is normally expressed only in the QL lineage. To define genes controlled by MAB-5 in the Q cells, fluorescence-activated cell sorting was utilized to isolate populations of Q cells at a time in early L1 larvae when MAB-5 first becomes active. Sorted Q cells from wild-type, mab-5 loss-of-function (lof), and mab-5 gain-of-function (gof) mutants were subject to RNA-seq and differential expression analysis. Genes enriched in Q cells included those involved in cell division, DNA replication, and DNA repair, consist with the neuroblast stem cell identity of the Q cells at this stage. Genes affected by mab-5 included those involved in neurogenesis, neural development, and interaction with the extracellular matrix. cwn-1 , which encodes a Wnt signaling molecule, showed a paired response to mab-5 in the Q cells: cwn-1 expression was reduced in mab-5 (lof) and increased in mab-5 (gof) , suggesting that MAB-5 is required for cwn-1 expression in Q cells. MAB-5 is required to prevent anterior migration of the Q lineage while it transcriptionally reprograms the Q lineage for posterior migration. Functional genetic analysis revealed that CWN-1 is required downstream of MAB-5 to inhibit anterior migration of the QL lineage, likely in parallel to EGL-20 /Wnt in a noncanonical Wnt pathway. In sum, work here describes a Q cell transcriptome, and a set of genes regulated by MAB-5 in the QL lineage. One of these genes, cwn-1 , acts downstream of mab-5 in QL migration, indicating that this gene set includes other genes utilized by MAB-5 to facilitate posterior neuroblast migration. [ABSTRACT FROM AUTHOR]

A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

The ability to link a DNA profile to a source of biological material has significant implications in forensic investigations. Epithelial cells are the main constituent of many of the samples collected as part of forensic casework. However, it is currently not possible to distinguish between or isolate epithelial cells from different anatomical regions. Flow cytometry represents a potential method that could be used in this context. This study was conducted to optimize procedures for the collection and processing of epidermal, buccal, vaginal and penile epithelial cells prior to analysis by flow cytometry. [ABSTRACT FROM AUTHOR]

There is a strong relationship between the dysregulation of epidermal growth factor receptor (EGFR) and the development of epithelial-derived cancers. Therefore, EGFR has usually been considered the desired target for gene therapy. Here, we propose an approach for targeting EGFR-expressing cells by phage particles capable of displaying EGF and GFP as tumor-targeting and reporting elements, respectively. For this purpose, the superfolder GFP-EGF (sfGFP-EGF) coding sequence was inserted at the N-terminus of the pIII gene in the pIT2 phagemid. The capability of the constructed phage to recognize EGFR-overexpressing cells was monitored by fluorescence microscopy, fluorescence-activated cell sorting (FACS), and cell-based ELISA experiments. FACS analysis showed a significant shift in the mean fluorescence intensity (MFI) of the cells treated with phage displaying sfGFP-EGF compared to phage displaying only sfGFP. The binding of phage displaying sfGFP-EGF to A-431 cells, monitored by fluorescence microscopy, indicated the formation of the sfGFP-EGF-EGFR complex on the surface of the treated cells. Cell-based ELISA experiments showed that phages displaying either EGF or sfGFP-EGF can specifically bind EGFR-expressing cells. The vector constructed in the current study has the potential to be engineered for gene delivery purposes as well as cell-based imaging for tumor detection. [ABSTRACT FROM AUTHOR]

Background: Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) play a pivotal role in maintaining lifelong hematopoiesis. The distinction between stem cells and other progenitors, as well as the assessment of their functions, has long been a central focus in stem cell research. In recent years, deep learning has emerged as a powerful tool for cell image analysis and classification/prediction. Methods: In this study, we explored the feasibility of employing deep learning techniques to differentiate murine HSCs and MPPs based solely on their morphology, as observed through light microscopy (DIC) images. Results: After rigorous training and validation using extensive image datasets, we successfully developed a three-class classifier, referred to as the LSM model, capable of reliably distinguishing long-term HSCs, short-term HSCs, and MPPs. The LSM model extracts intrinsic morphological features unique to different cell types, irrespective of the methods used for cell identification and isolation, such as surface markers or intracellular GFP markers. Furthermore, employing the same deep learning framework, we created a two-class classifier that effectively discriminates between aged HSCs and young HSCs. This discovery is particularly significant as both cell types share identical surface markers yet serve distinct functions. This classifier holds the potential to offer a novel, rapid, and efficient means of assessing the functional states of HSCs, thus obviating the need for time-consuming transplantation experiments. Conclusion: Our study represents the pioneering use of deep learning to differentiate HSCs and MPPs under steady-state conditions. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into the molecular mechanisms underlying stem cell self-renewal. [ABSTRACT FROM AUTHOR]

Fluorescence-activated cell sorting (FACS) holds great promise for the separation of single cells or cell populations according to specific light scattering and fluorescent characteristics. Here, we present a new perspective on microfluidic FACS (μFACS) with predictable geometry, which meets the requirements of high-throughput analysis and sorting. Instead of the widely applied elastic polydimethylsiloxane (PDMS), a rigid epoxy resin chip was rapidly fabricated and irreversibly encapsulated to eliminate channel deformation (tenfold reduction) and enhance performance while meeting high pressure (>600 kPa) and high flow rate application scenarios. Fluorescence discrimination and particle differentiation were additionally validated in a self-contained μFACS system using calibration microspheres and mammalian cells. The μFACS chip and system were integrally optimized to achieve a minimum interval (0.58 ms) with a mean flow rate of 1.5 m/s. Ultimately, event recording and automated sorting were accomplished in real time while achieving a sorting efficiency of 87% at cell throughput of 8,000 events/s. This rigid chip for high-throughput μFACS, which is independent of the physical properties of cells could pave the way for cell screening in plasma samples for personalized medicine.

Microalgae are a promising feedstock with proven biostimulant activity that is enhanced by their biochemical components (e.g., amino acids and phytohormones), which turns them into an appealing feedstock to reduce the use of fertilisers in agriculture and improve crop productivity and resilience. Thus, this work aimed to isolate protein-rich microalgal mutants with increased biostimulant activity. Random mutagenesis was performed with Chlorella vulgaris, and a selection of protein-rich mutants were sorted through fluorescence-activated cell sorting (FACS), resulting in the isolation of 17 protein-rich mutant strains with protein contents 19–34% higher than that of the wildtype (WT). Furthermore, mutant F4 displayed a 38%, 22% and 62% higher biomass productivity, growth rate and chlorophyll content, respectively. This mutant was then scaled up to a 7 L benchtop reactor to produce biomass and evaluate the biostimulant potential of this novel strain towards garden cress seeds. Compared to water (control), the germination index and the relative total growth increased by 7% and 19%, respectively, after the application of 0.1 g L−1 of this bioproduct, which highlights its biostimulant potential.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.

Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence‐activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into x‐ray‐irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts, which can proliferate in the x‐ray‐irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes such as Hoechst 33342, DRAQ5, and DyeCycle, showed, more or less, toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the x‐ray‐sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% → ca. 89%), the sorted cells could actively proliferate when they were injected into x‐ray‐irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes. [ABSTRACT FROM AUTHOR]

Flow cytometry(FCM)is a rapid and relatively quantitative multi-parameter analytical technique for analyzing a large number of cells at the single-cell level. The constructed fluorescence activated cell sorting(FACS)based on FCM can physically separate fluorescent-labeled single cells and has been widely used in the field of directed evolution of enzyme. Directed evolution has been proven to be an effective method for obtaining industrial enzymes with excellent properties, such as high enzyme activity, strong thermal stability and solvent resistance. To conduct directed evolution, mutant library firstly needs to be constructed by random mutagenesis or DNA recombination, and then be screened under manual selection pressure. Traditional screening methods such as plate and microplate methods have limitations of low throughput, high cost, large errors, and poor accuracy, and cannot achieve high-throughput screening of large mutant library. Compared with traditional screening methods, FACS has the advantages of high throughput, low cost, small error and high precision. In this article, we first summarized the development process of FCM and FACS, as well as the composition and classification of related instruments derived from them. Then, we discussed the application status and limitations of FACS in the directed evolution of enzyme. Finally, we summarized and prospected the development direction of FACS and its application in the field of directed evolution of enzyme. [ABSTRACT FROM AUTHOR]

l-Lysine, an essential amino acid for humans and mammals, is widely used in the food, feed, medicine, and cosmetics industries. In this study, a lysine over-producing Escherichia coli mutant was isolated using a fluorescence-based screen and an E. coli strain lacking five of the six L-lysine tRNA-UUU genes. Firstly, an l-lysine codon-rich protein was fused with a green fluorescent protein (all AAG codons were replaced with AAA), yielding a rare codon-rich screening marker positively correlated with l-lysine content. After association and room temperature plasma (ARTP) mutagenesis and induced fluorescent protein expression culture, mutant strains with strong fluorescence were sorted using flow cytometry. The fermentation performance of the high-yielding l-lysine strains were evaluated, which resulted in 16 of the 29 mutant strains showing increased L-lysine yields compared with those of the wild-type strains and a screening efficiency of up to 55.2%. Following a 48 h fermentation, the production of l-lysine (14.8 g/L) and biomass by E. coli QD01ΔtRNA L2 were 12.1 and 4.5% higher than those of the wild-type strain. The screening strategy for high-yielding strains based on the artificial rare cryptosystem established in this study will provide an efficient, accurate, and simple method for screening other amino-acid-producing microorganisms. [ABSTRACT FROM AUTHOR]

Abstract Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood–brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

Abstract Background In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum l-histidine producer. Since all known genetic targets contributing to l-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to l-histidine biosynthesis. Results In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and l-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10–80% more l-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled l-histidine titer (29 mM) and product yield (0.13 C-mol C-mol−1) in comparison to the starting variant. Conclusions This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood–brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke. [ABSTRACT FROM AUTHOR]

Background: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicuml-histidine producer. Since all known genetic targets contributing to l-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to l-histidine biosynthesis. Results: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and l-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10–80% more l-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled l-histidine titer (29 mM) and product yield (0.13 C-mol C-mol−1) in comparison to the starting variant. Conclusions: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance. [ABSTRACT FROM AUTHOR]

Simple Summary: The protein, mouse atonal homolog 6 (Math6), is required for correct placental development in mice and causes loss of pregnancy, leading to reproductive insufficiency in females, when it is missing. It is currently unclear which cell types of the placenta predominantly express this protein. With the help of transgenic Math6 Flag-tag mice, we established two methods to characterize the Math6-positive cells: immunohistochemical staining and cell sorting. Using these methods, we discovered that Math6 protein is expressed in multiple layers of the murine placenta in diverse cell types: decidua cells, uterine natural killer cells, macrophages, trophoblast giant cells, and endothelial cells. The transcription factor Math6, mouse atonal homolog 6, belongs to the family of highly conserved basic helix–loop–helix transcription factors. It plays an important role in embryonic development and shows a wide expression pattern in murine tissues. The placenta, as a life-sustaining transient organ for the fetus, also depends on the expression of Math6. The adverse effects of deleting Math6 in mice, leading to deficient placental development and pregnancy loss, have already been demonstrated by us. Until now, detailed investigations regarding the specific mechanisms underlying the improper placental development in these murine mutants have failed, as the Math6 expression could not be confined to a specific cell type due to the lack of a highly specific Math6 antibody. To circumvent this problem, we used transgenic mice, where Math6 is marked with a Flag sequence that functions as a specific epitope. Tissues from these transgenic mice were used to establish immunohistochemical staining and fluorescence-activated cell sorting (FACS). The establishment of these methods yielded initial findings pertaining to the identification of Math6-expressing cell types and their localization. Our results reveal that Math6 shows a wide expression pattern in both maternal and fetal components of the murine placenta. It shows expression in various cell types, but predominantly in trophoblast giant cells, endothelial cells and macrophages. The largest subpopulation that we detected in the group of Math6-positive cells were identified as DBA+ uterine natural killer cells. These findings reveal information and a chance for further investigation on the involvement of Math6 in placental development and the molecular pathomechanisms of spontaneous abortion. [ABSTRACT FROM AUTHOR]

Simple Summary: There is a lack of biomarkers for predicting the response to chemotherapy in gastric cancer (GC). This study investigates the prognostic significance of PD-1 expression in CD8+ T cells in the blood of 68 patients with advanced GC receiving platinum-based chemotherapy. Patients were divided based on baseline frequencies of PD-1+CD8+ T cells and whether these frequencies increased or decreased on day 7 after treatment. While baseline frequencies of PD-1+CD8+ T cells had no impact on survival, the patients whose frequencies of PD-1+CD8+ T cells increased on day 7 from the treatment initiation showed significantly longer survival than those whose frequencies of PD-1+CD8+ T cells decreased. This difference was also reflected in the mean duration of response between the groups. These findings suggest that an early increase in frequencies of PD-1+CD8+ T cells could predict favorable prognoses and durable responses in patients with advanced GC receiving chemotherapy. The clinical significance of PD-1 expression in circulating CD8+ T cells in patients with gastric cancer (GC) receiving chemotherapy remains unelucidated. Therefore, we aimed to examine its prognostic significance in blood samples of 68 patients with advanced GC who received platinum-based chemotherapy. The correlation between peripheral blood mononuclear cells, measured using fluorescence-activated cell sorting, was evaluated. Patients were divided into two groups according to the changes in PD-1+CD8+ T-cell frequencies between day 0 and 7. They were categorized as increased or decreased PD-1+CD8+ T-cell groups. The increased PD-1+CD8+ T-cell group showed longer progression-free survival (PFS) and overall survival (OS) than the decreased PD-1+CD8+ T-cell group (PFS: 8.7 months vs. 6.1 months, p = 0.007; OS: 20.7 months vs. 10.8 months, p = 0.003). The mean duration of response was significantly different between the groups (5.7 months vs. 2.5 months, p = 0.041). Multivariate analysis revealed that an increase in PD-1+CD8+ T-cell frequency was an independent prognostic factor. We concluded that the early increase in PD-1+CD8+ T-cell frequency is a potential predictor of favorable prognoses and durable responses in patients with advanced GC receiving chemotherapy. [ABSTRACT FROM AUTHOR]

Bioprinting is a powerful technology with the potential to transform medical device manufacturing, organ replacement, and the treatment of diseases and physiologic malformations. However, current bioprinters are unable to reliably print the fundamental unit of all living things, single cells. A high-definition single-cell printing, a novel microfluidic technology, is presented here that can accurately print single cells from a mixture of multiple candidates. The bioprinter employs a highly miniaturized microfluidic sorter to deterministically select single cells of interest for printing, achieving an accuracy of ≈10 µm and speed of ≈100 Hz. This approach is demonstrated by fabricating intricate cell patterns with pre-defined features through selective single-cell printing. The approach is used to synthesize well-defined spheroids with controlled composition and morphology. The speed, accuracy, and flexibility of the approach will advance bioprinting to enable new studies in organoid science, tissue engineering, and spatially targeted cell therapies.

Abstract The possible role of somatic copy number variations (CNVs) in Alzheimer’s disease (AD) aetiology has been controversial. Although cytogenetic studies suggested increased CNV loads in AD brains, a recent single-cell whole-genome sequencing (scWGS) experiment, studying frontal cortex brain samples, found no such evidence. Here we readdressed this issue using low-coverage scWGS on pyramidal neurons dissected via both laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) across five brain regions: entorhinal cortex, temporal cortex, hippocampal CA1, hippocampal CA3, and the cerebellum. Among reliably detected somatic CNVs identified in 1301 cells obtained from the brains of 13 AD patients and 7 healthy controls, deletions were more frequent compared to duplications. Interestingly, we observed slightly higher frequencies of CNV events in cells from AD compared to similar numbers of cells from controls (4.1% vs. 1.4%, or 0.9% vs. 0.7%, using different filtering approaches), although the differences were not statistically significant. On the technical aspects, we observed that LCM-isolated cells show higher within-cell read depth variation compared to cells isolated with FACS. To reduce within-cell read depth variation, we proposed a principal component analysis-based denoising approach that significantly improves signal-to-noise ratios. Lastly, we showed that LCM-isolated neurons in AD harbour slightly more read depth variability than neurons of controls, which might be related to the reported hyperploid profiles of some AD-affected neurons.

Identifying and interrogating cell type–specific populations within the heterogeneous milieu of the human brain is paramount to resolving the processes of normal brain homeostasis and the pathogenesis of neurological disorders. While brain cell type–specific markers are well established, most are localized on cellular membranes or within the cytoplasm, with limited literature describing those found in the nucleus. Due to the complex cytoarchitecture of the human brain, immunohistochemical studies require well-defined cell-specific nuclear markers for more precise and efficient quantification of the cellular populations. Furthermore, efficient nuclear markers are required for cell type–specific purification and transcriptomic interrogation of archived human brain tissue through nuclei isolation–based RNA sequencing. To sate the growing demand for robust cell type–specific nuclear markers, we thought it prudent to comprehensively review the current literature to identify and consolidate a novel series of robust cell type–specific nuclear markers that can assist researchers across a range of neuroscientific disciplines. The following review article collates and discusses several key and prospective cell type–specific nuclei markers for each of the major human brain cell types; it then concludes by discussing the potential applications of cell type–specific nuclear workflows and the power of nuclear-based neuroscientific research. [ABSTRACT FROM AUTHOR]

l-Lysine, an essential amino acid for humans and mammals, is widely used in the food, feed, medicine, and cosmetics industries. In this study, a lysine over-producing Escherichia coli mutant was isolated using a fluorescence-based screen and an E. coli strain lacking five of the six L-lysine tRNA-UUU genes. Firstly, an l-lysine codon-rich protein was fused with a green fluorescent protein (all AAG codons were replaced with AAA), yielding a rare codon-rich screening marker positively correlated with l-lysine content. After association and room temperature plasma (ARTP) mutagenesis and induced fluorescent protein expression culture, mutant strains with strong fluorescence were sorted using flow cytometry. The fermentation performance of the high-yielding l-lysine strains were evaluated, which resulted in 16 of the 29 mutant strains showing increased L-lysine yields compared with those of the wild-type strains and a screening efficiency of up to 55.2%. Following a 48 h fermentation, the production of l-lysine (14.8 g/L) and biomass by E. coli QD01ΔtRNA L2 were 12.1 and 4.5% higher than those of the wild-type strain. The screening strategy for high-yielding strains based on the artificial rare cryptosystem established in this study will provide an efficient, accurate, and simple method for screening other amino-acid-producing microorganisms.

The possible role of somatic copy number variations (CNVs) in Alzheimer's disease (AD) aetiology has been controversial. Although cytogenetic studies suggested increased CNV loads in AD brains, a recent single-cell whole-genome sequencing (scWGS) experiment, studying frontal cortex brain samples, found no such evidence. Here we readdressed this issue using low-coverage scWGS on pyramidal neurons dissected via both laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) across five brain regions: entorhinal cortex, temporal cortex, hippocampal CA1, hippocampal CA3, and the cerebellum. Among reliably detected somatic CNVs identified in 1301 cells obtained from the brains of 13 AD patients and 7 healthy controls, deletions were more frequent compared to duplications. Interestingly, we observed slightly higher frequencies of CNV events in cells from AD compared to similar numbers of cells from controls (4.1% vs. 1.4%, or 0.9% vs. 0.7%, using different filtering approaches), although the differences were not statistically significant. On the technical aspects, we observed that LCM-isolated cells show higher within-cell read depth variation compared to cells isolated with FACS. To reduce within-cell read depth variation, we proposed a principal component analysis-based denoising approach that significantly improves signal-to-noise ratios. Lastly, we showed that LCM-isolated neurons in AD harbour slightly more read depth variability than neurons of controls, which might be related to the reported hyperploid profiles of some AD-affected neurons. [ABSTRACT FROM AUTHOR]

Since the discovery of striatal neurons expressing dopamine-synthesizing enzymes, researchers have attempted to identify their phenotype and functional significance. In this study, it was shown that in transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) gene promoter, (i) there are striatal neurons expressing only TH, only aromatic L-amino acid decarboxylase (AADC), or both enzymes of dopamine synthesis; (ii) striatal neurons expressing dopamine-synthesizing enzymes are not dopaminergic since they lack a dopamine transporter; (iii) monoenzymatic neurons expressing individual complementary dopamine-synthesizing enzymes produce this neurotransmitter in cooperation; (iv) striatal nerve fibers containing only TH, only AADC, or both enzymes project into the lateral ventricles, providing delivery pathways for L-3,4-dihydroxyphenylalanine and dopamine to the cerebrospinal fluid; and (v) striatal GFP neurons express receptor genes for various signaling molecules, i.e., classical neurotransmitters, neuropeptides, and steroids, indicating fine regulation of these neurons. Based on our data, it is assumed that the synthesis of dopamine by striatal neurons is a compensatory response to the death of nigral dopaminergic neurons in Parkinson's disease, which opens broad prospects for the development of a fundamentally novel antiparkinsonian therapy. [ABSTRACT FROM AUTHOR]

Exposure to sublethal stresses related to food-processing may induce a heterogenous mixture of cells that co-exist, comprising healthy, sublethally injured, dormant and dead cells. Heterogeneity in survival capacity and dormancy of single cells may impede the detection of foodborne pathogens. In this study, we exposed Listeria monocytogenes Scott A strain, to peracetic acid (PAA; 20–40 ppm) and to acidic conditions (hydrochloric (HCl) and acetic (AA) acid, adjusted to pH 2.7–3.0, to evaluate the resuscitation capacity and outgrowth kinetics of metabolically active cells in two different media. Injury and the viable-but-non-culturable (VBNC) status of cells were assessed by flow cytometry using CFDA (metabolically active) and PI (dead) staining. Stressed CFDA+PI− cells were sorted on Tryptic Soy (TS) Agar or in TS broth, both supplemented with 0.6 % Yeast Extract (TSAYE or TSBYE), to evaluate culturability. Resuscitation capacity of CFDA+PI−sorted cells (10 events/well) was monitored by visual inspection on TSAYE and by optical density measurement in TSBYE for 5 days. Sorting of L. monocytogenes viable cells (CFDA+PI−) in Ringer's solution on TSAYE and TSBYE showed 100 % recovery in both media (control condition), while the mean lag time in TSBYE was 9.6 h. Treatment with 20 ppm PAA for 90 and 180 min resulted in 74.79 % and 85.82 % of non-culturable cells in TSBYE and increased the average lag time to 41.7 h and 43.8 h, respectively, compared to the control (9.6 h). The longest average lag time (79.5 h) was detected after treatment with 30 ppm PAA for 90 min, while at the same condition sorting of CFDA+PI− cells resulted in 95.05 % and 93.94 % non-culturable cells on TSAYE and TSBYE, respectively. The highest percentage of wells with non-culturable cells (96.17 %) was detected on TSAYE after treatment with 40 ppm PAA for 30 min. Fractions of VBNC cells were detected in TSBYE after treatment with HCl pH 3.0 for 60 and 240 min, and in TSAYE and TSBYE after exposure to AA pH 2.7. Treatment with AA pH 2.7 for 150–300 min increased the range of recorded lag time values compared to 60 min, from 8.6 h up to 13.3 h, as well as the mean lag times in TSBYE. Modelling of the outgrowth kinetics comparing the two types of stress (oxidative vs acid) and the two systems of growth (colonial vs planktonic) revealed that low starting concentrations hindered the detection of viable L. monocytogenes cells, either due to VBNC induction or cell heterogeneity. • Stress history and type of growth affected the induction of VBNC state. • Low starting concentrations hindered the detection of viable L. monocytogenes cells. • Fluorescence-activated cell sorting can effectively reveal dormancy phenomena. [ABSTRACT FROM AUTHOR]

Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat -eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway. [Display omitted] • An unbiased GPCR atlas shows 179 distinct GPCRs present on DANs • 38 GPCRs are responsible for the vast majority of all GPCR transcripts detected in DANs • 41 GPCRs are expressed on midbrain DANs but not on midbrain non-DANs • FFAR4 is found on a subset of DANs and is involved in regulation of food and water intake Apuschkin et al. map the population-wide GPCR expression profile of dopamine neurons (DANs) to provide a framework for deciphering metabotropic DAN regulation. The authors highlight expression of the free fatty acid receptor 4 (FFAR4) on a subset of DANs and showcase this receptor as a food and water intake modulator. [ABSTRACT FROM AUTHOR]

The transcription factor Math6, mouse atonal homolog 6, belongs to the family of highly conserved basic helix–loop–helix transcription factors. It plays an important role in embryonic development and shows a wide expression pattern in murine tissues. The placenta, as a life-sustaining transient organ for the fetus, also depends on the expression of Math6. The adverse effects of deleting Math6 in mice, leading to deficient placental development and pregnancy loss, have already been demonstrated by us. Until now, detailed investigations regarding the specific mechanisms underlying the improper placental development in these murine mutants have failed, as the Math6 expression could not be confined to a specific cell type due to the lack of a highly specific Math6 antibody. To circumvent this problem, we used transgenic mice, where Math6 is marked with a Flag sequence that functions as a specific epitope. Tissues from these transgenic mice were used to establish immunohistochemical staining and fluorescence-activated cell sorting (FACS). The establishment of these methods yielded initial findings pertaining to the identification of Math6-expressing cell types and their localization. Our results reveal that Math6 shows a wide expression pattern in both maternal and fetal components of the murine placenta. It shows expression in various cell types, but predominantly in trophoblast giant cells, endothelial cells and macrophages. The largest subpopulation that we detected in the group of Math6-positive cells were identified as DBA+ uterine natural killer cells. These findings reveal information and a chance for further investigation on the involvement of Math6 in placental development and the molecular pathomechanisms of spontaneous abortion.

The clinical significance of PD-1 expression in circulating CD8+ T cells in patients with gastric cancer (GC) receiving chemotherapy remains unelucidated. Therefore, we aimed to examine its prognostic significance in blood samples of 68 patients with advanced GC who received platinum-based chemotherapy. The correlation between peripheral blood mononuclear cells, measured using fluorescence-activated cell sorting, was evaluated. Patients were divided into two groups according to the changes in PD-1+CD8+ T-cell frequencies between day 0 and 7. They were categorized as increased or decreased PD-1+CD8+ T-cell groups. The increased PD-1+CD8+ T-cell group showed longer progression-free survival (PFS) and overall survival (OS) than the decreased PD-1+CD8+ T-cell group (PFS: 8.7 months vs. 6.1 months, p = 0.007; OS: 20.7 months vs. 10.8 months, p = 0.003). The mean duration of response was significantly different between the groups (5.7 months vs. 2.5 months, p = 0.041). Multivariate analysis revealed that an increase in PD-1+CD8+ T-cell frequency was an independent prognostic factor. We concluded that the early increase in PD-1+CD8+ T-cell frequency is a potential predictor of favorable prognoses and durable responses in patients with advanced GC receiving chemotherapy.

The engineering of antibodies and antibody fragments for affinity maturation, stability, and other biophysical characteristics is a common aspect of therapeutic development. Maturation of antibodies in B cells during the adaptive immune response is the result of a process called somatic hypermutation (SHM), in which the activation-induced cytidine deaminase (AID) acts to introduce mutations into immunoglobulin (Ig) genes. Iterative selection and clonal expansion of B cells containing affinity-enhancing mutations drive an increase in the overall affinity of antibodies. Here we describe the use of SHM coupled with mammalian cell surface display for the maturation of antibodies in vitro and the complementarity of these methods with the mining of immune lineages using next-generation sequencing (NGS).

Sequencing of single bacterial and archaeal cells is an important methodology that provides access to the genetic makeup of uncultivated microorganisms. We here describe the high-throughput fluorescence-activated cell sorting-based isolation of single cells from the environment, their lysis and strand displacement-mediated whole genome amplification. We further outline 16S rRNA gene sequence-based screening of single-cell amplification products, their preparation for Illumina sequencing libraries, and finally propose computational methods for read and contig level quality control of the resulting sequence data.

BackgroundVascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. During the optimization of endothelial cell (EC) inductions from mouse ESCs using our staged and chemically-defined induction methods, we found that cell seeding density but not VEGF treatment between 10 ng/mL and 40 ng/mL was a significant variable directing ESCs into FLK1+ VPCs during stage 1 induction. Here, we examine potential contributions from cell-to-cell signaling or cellular metabolism in the production of VPCs from ESCs seeded at different cell densities.MethodsUsing 1D 1H-NMR spectroscopy, transcriptomic arrays, and flow cytometry, we observed that the density-dependent differentiation of ESCs into FLK1+ VPCs positively correlated with a shift in metabolism and cellular growth.ResultsSpecifically, cell differentiation correlated with an earlier plateauing of exhaustive glycolysis, decreased lactate production, lower metabolite consumption, decreased cellular proliferation and an increase in cell size. In contrast, cells seeded at a lower density of 1,000 cells/cm2 exhibited increased rates of glycolysis, lactate secretion, metabolite utilization, and proliferation over the same induction period. Gene expression analysis indicated that high cell seeding density correlated with up-regulation of several genes including cell adhesion molecules of the notch family (NOTCH1 and NOTCH4) and cadherin family (CDH5) related to vascular development.ConclusionsThese results confirm that a distinct metabolic phenotype correlates with cell differentiation of VPCs.

Although single-cell RNA sequencing (scRNA-seq) is currently the gold standard for the analysis of cell-specific expression profiles, the options for processing, staining, and preserving fresh cells remain very limited. Immediate and correct tissue processing is a critical determinant of scRNA-seq success. One major limitation is the restricted compatibility of fixation approaches, which must not destabilize or alter antibody labeling or RNA content or interfere with cell integrity. An additional limitation is the availability of expensive, high-demand cell-sorting equipment to exclude debris and dead or unwanted cells before proceeding with sample sequencing. The goal of this study was to develop a method that allows cells to be fixed and stored prior to FACS sorting for scRNA-seq without compromising the quality of the results. Finally, the challenge of preserving as many living cells as possible during tissue processing is another crucial issue addressed in this study. Our study focused on pancreatic ductal adenocarcinoma samples, where the number of live cells is rather limited, as in many other tumor tissues. Harsh tissue dissociation methods and sample preparation for analysis can negatively affect cell viability. Using the murine pancreatic cancer model Pan02, we evaluated the semi-automated mechanical/enzymatic digestion of solid tumors by gentleMACS Dissociator and compared it with mechanical dissociation of the same tissue. Moreover, we investigated a type of cell fixation that is successful in preserving cell RNA integrity yet compatible with FACS and subsequent scRNA-sequencing. Our protocol allows tissue to be dissociated and stained in one day and proceeds to cell sorting and scRNA-seq later, which is a great advantage for processing clinical patient material. [ABSTRACT FROM AUTHOR]

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g−1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l−1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products. • A general method for industrial strain development of actinomycetes. • Protocol for fluorescence-activated cell sorting of actinomycetes. • Metabolic engineering for increased production of cholesterol oxidase. • Activation of a silent mutaxanthene gene cluster and yield enhancement. [ABSTRACT FROM AUTHOR]

Microalgae have become a promising novel and sustainable feedstock for meeting the rising demand for food and feed. However, microalgae-based products are currently hindered by high production costs. One major reason for this is that commonly cultivated wildtype strains do not possess the robustness and productivity required for successful industrial production. Several strain improvement technologies have been developed towards creating more stress tolerant and productive strains. While classical methods of forward genetics have been extensively used to determine gene function of randomly generated mutants, reverse genetics has been explored to generate specific mutations and target phenotypes. Site-directed mutagenesis can be accomplished by employing different gene editing tools, which enable the generation of tailor-made genotypes. Nevertheless, strategies promoting the selection of randomly generated mutants avoid the introduction of foreign genetic material. In this paper, we review different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis. Current challenges hampering strain improvement, selection, and commercialization will be discussed. The combination of these approaches with high-throughput technologies, such as fluorescence-activated cell sorting, as tools to select the most promising mutants, will also be discussed. [ABSTRACT FROM AUTHOR]

This study investigated the release and proteomic profile of tissue factor microparticles (TFMPs) prospectively (up to 6 months) following a myocardial infarction (MI) in a chronic porcine model to establish their utility in tracking cellular level activities that predict physiologic outcomes. Our animal groups (n = 6 to 8 each) consisted of control, noninfarcted (negative control); infarcted only (positive control); and infarcted animals treated with cardiac resynchronization therapy (CRT) and a β-blocker (BB) (metoprolol succinate). The authors found different protein profiles in TFMPs between the control, infarcted only group, and the CRT + BB treated group with predictive impact on the outward phenotype of pathological remodeling after an MI within and between groups. This novel approach of monitoring cellular level activities by profiling the content of TFMPs has the potential of addressing a shortfall of the current crop of cardiac biomarkers, which is the inability to capture composite molecular changes associated with chronic maladaptive signaling in a spatial and temporal manner.

Background: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics., Aim: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones., Methods: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed., Results: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes., Conclusions: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.

The capability of yeast cell factories to secrete industrially important enzymes into the extracellular environment can significantly reduce overall production costs. This impacts a wide range of sectors, including pharmaceuticals, food and feed, textiles, detergents, materials, and energy. Although recent advances in molecular biology and laboratory automation have expanded our capability to construct enormous strain variant libraries, it calls for technologies to effectively screen thousands of strains with phenotypes that are spatially removed from the original cell. In this work, we demonstrate the application of integrated double emulsion (DE) droplets and fluorescence-activated cell sorting (FACS) technology for the screening and selection of yeast single cells based on extracellular enzyme activity. Single Saccharomyces cerevisiae cells secreting cellulase enzyme, β-glucosidase, are compartmentalized within highly monodisperse DE droplets with a fluorogenic substrate. After single-cell encapsulation, cells are cultivated within the DE droplets to allow enzyme production. Single cell-laden DE droplets are sorted by FACS to identity strains with high β-glucosidase secretion. Moreover, encapsulated S. cerevisiae cells are successfully recovered from DEs after sorting. We expect that this user-friendly method will be combined with a range of synthetic biology techniques to optimize value-added product yield and stability. • Evaluation of single yeast cell growth and enzyme secretion by double emulsions. • Integration of double emulsion droplets with FACS for single-cell assays. • Identification of single yeast cells exhibiting superior enzyme secretion ability. [ABSTRACT FROM AUTHOR]

[Display omitted] • AIE-nanoprobes targeting EPS discern high-growing and high-photosynthetic algal strains; • High-purity algal strains were obtained through three sorting/subculturing cycles; • Sorted cells showed increased cell division and photosynthesis gene expression; • Sorted populations were stable after seven additional subculturing cycles; • Sorted cells showed a 1.2 to 1.8-fold increase in biomass, chlorophyll, and lipids; Biofuel production from microalgae has been greatly restricted by low biomass productivity and long-term photosynthetic efficacy. Here, a novel strategy for selecting high-growing, stress-resistant algal strains with high photosynthetic capacity was proposed based on biocompatible extracellular polymeric substances (EPS) probes with aggregation-induced emission (AIE) properties. Specifically, AIE active EPS probes were synthesized for in-situ long-term monitoring of the EPS productivity at different algal growth stages. By coupling the AIE-based fluorescent techniques, algal cells were classified into four diverse populations based on their chlorophyll and EPS signals. Mechanistic studies on the sorted algal cells revealed their remarkable stress resistance and high expression of cell division, biopolymer production and photosynthesis-related genes. The sorted and subcultured algal cells consistently exhibited relatively higher growth rates and photosynthetic capacities, resulting in an increased (1.2 to 1.8-fold) algal biomass production, chlorophyll, and lipids. This study can potentially open new strategies to boost microalgal-based biofuel production. [ABSTRACT FROM AUTHOR]

To improve the proliferation and activity of adoptive T cell by screening serum-free media for in vitro expansion of flow sorting T cells. Methods: Peripheral blood mononuclear cells from six healthy volunteers were prepared by gradient centrifugation using peripheral blood lymphocyte separation tube. The CD3+T cells from the peripheral blood mononuclear cells were then sorted by flow cytometry into four commonly Lymphocyte culture medium, including X-VIVO 15, KBM 581, TexMACS GMP serum-free media and RPMI 1640 with ten percent of fetal bovine serum complete medium. The status of the cultured cells and the proliferation ability of the cells in vitro was observed and recorded. On the 3rd, 6th and 8th days, viable cell counts were tested by trypan blue staining. On the 8th day, apoptosis was detected by the Annexin V: FITC Apoptosis Detection Kit. The immune phenotype of the expanded cells invitro was detected by the flow cytometer. Results: A small amount of cell debris was observed when X-VIVO 15, TexMACS GMP and RPMI 1640 with ten percent of fetal bovine serum medium were used as the receiving medium for flow cytometry, while a large number of dead cells were observed in samples sorted in KBM 581 (P<0.05). The number of cells expanded in X-VIVO 15 medium is more than that of the other three media, and proliferation testing shows that live cells proliferate rapidly in X-VIVO 15 medium. Rate of apoptosis is significantly lower than that of KBM 581 and TexMACS GMP (P<0.05). The expanded cells mainly show effect memory type in the four different cultural environments. The rate of effect memory T cells in X-VIVO 15 medium was higher than that in TexMACS GMP(P<0.05). The rate of terminal effector T cells in TexMACS GMP medium was higher than that in 10 % FBS/1640. Conclusion: Flow-sorted T cells show higher proliferation capacity, viability and memory phenotype in X-VIVO 15 media, suggesting that X-VIVO 15 media is suitable for in vitro expansion of cells after flow-sorting. [ABSTRACT FROM AUTHOR]

Whole tissue RNASeq is the standard approach for studying gene expression divergence in evolutionary biology and provides a snapshot of the comprehensive transcriptome for a given tissue. However, whole tissues consist of diverse cell types differing in expression profiles, and the cellular composition of these tissues can evolve across species. Here, we investigate the effects of different cellular composition on whole tissue expression profiles. We compared gene expression from whole testes and enriched spermatogenesis populations in two species of house mice, Mus musculus musculus and M. m. domesticus, and their sterile and fertile F1 hybrids, which differ in both cellular composition and regulatory dynamics. We found that cellular composition differences skewed expression profiles and differential gene expression in whole testes samples. Importantly, both approaches were able to detect large‐scale patterns such as disrupted X chromosome expression, although whole testes sampling resulted in decreased power to detect differentially expressed genes. We encourage researchers to account for histology in RNASeq and consider methods that reduce sample complexity whenever feasible. Ultimately, we show that differences in cellular composition between tissues can modify expression profiles, potentially altering inferred gene ontological processes, insights into gene network evolution, and processes governing gene expression evolution. [ABSTRACT FROM AUTHOR]

[Display omitted] • Winter strain of Tisochrysis lutea obtained from >180-day cold adaptation and two rounds of FACS selection. • Fx content increased from 1.11 to 4.24 mg g−1 during strain improvement. • TFA polar lipid fraction increased from 21 % to 55 % during strain improvement. • Fx productivity in winter strain at 15 °C was identical to original one at 30 °C. Tisochrysis lutea is an important microalgal species for fucoxanthin and docosahexaenoic acid (DHA) production with an optimum cultivation temperature of approximately 30 °C. The aim of the present work was to develop a winter strain with high productivity at 15 °C. The response of the original strain to a decrease in temperature from 30 °C to 15 °C was investigated in continuous turbidostat experiments. This was followed by adaptation for >180 days at 15 °C and 2 rounds of sorting for cells with high chlorophyll fluorescence (top 5%) using fluorescence-activated cell sorting (FACS). For the original strain the productivity of biomass, fucoxanthin, and DHA decreased by 92 %, 98 % and 85 % respectively when decreasing the temperature from 30 °C to 15 °C. In the sorted cold-adapted 'winter strain', biomass, fucoxanthin, and DHA productivities were similar to those at 30 °C. In addition, the fucoxanthin concentration increased from 1.11 to 4.24 mg g−1 dry weight and the polar lipid fraction in total fatty acids increased from 21 % to 55 %. The winter strain showed a robust and stable phenotype after one year of cultivation, expanding the outdoor fucoxanthin and lipid production seasons for this species. [ABSTRACT FROM AUTHOR]