An atlas of GPCRs in dopamine neurons: Identification of the free fatty acid receptor 4 as a regulator of food and water intake.

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.

Vibrational imaging of metabolites for improved microbial cell strains.

Significance: Biomanufacturing utilizes modified microbial systems to sustainably produce commercially important biomolecules for use in agricultural, energy, food, material, and pharmaceutical industries. However, technological challenges related to non-destructive and high-throughput metabolite screening need to be addressed to fully unlock the potential of synthetic biology and sustainable biomanufacturing. Aim: This perspective outlines current analytical screening tools used in industrial cell strain development programs and introduces label-free vibrational spectro-microscopy as an alternative contrast mechanism. Approach: We provide an overview of the analytical instrumentation currently used in the "test" portion of the design, build, test, and learn cycle of synthetic biology. We then highlight recent progress in Raman scattering and infrared absorption imaging techniques, which have enabled improved molecular specificity and sensitivity. Results: Recent developments in high-resolution chemical imaging methods allow for greater throughput without compromising the image contrast. We provide a roadmap of future work needed to support integration with microfluidics for rapid screening at the single-cell level. Conclusions: Quantifying the net expression of metabolites allows for the identification of cells with metabolic pathways that result in increased biomolecule production, which is essential for improving the yield and reducing the cost of industrial biomanufacturing. Technological advancements in vibrational microscopy instrumentation will greatly benefit biofoundries as a complementary approach for non-destructive cell screening.

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa.

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency. Bacillus subtilis spores were pressurized for 50 s - 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions. The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43-52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions. The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

Purification of Planarian Stem Cells Using a Draq5-Based FACS Approach.

Planarians are flatworms that have the remarkable ability to regenerate entirely new animals. This regenerative ability requires abundant adult stem cells called neoblasts, which are relatively small in size, sensitive to irradiation and the only proliferative cells in the animal. Despite the lack of cell surface markers, fluorescence-activated cell sorting (FACS) protocols have been developed to discriminate and isolate neoblasts, based on DNA content. Here, we describe a protocol that combines staining of far-red DNA dye Draq5, Calcein-AM and DAPI, along with a shortened processing time. This profiling strategy can be used to functionally characterize the neoblast population in pharmacologically-treated or gene knockdown animals. Highly purified neoblasts can be analyzed with downstream assays, such as in situ hybridization and RNA sequencing.

Human B Cell Receptor Repertoire Sequencing.

Next-generation sequencing has the potential to uncover the complex nature of B cell immunity by revealing the full complexity of B cell receptor (BCR) repertoires in health and disease. However, there are drawbacks which can compromise the validity of the repertoire analysis caused by quantitative bias and accumulation of sequencing errors during the library preparation and sequencing. Here, we provide an optimized protocol designed to minimize bias for reproducible and accurate preparation of human BCR repertoire libraries for high-throughput sequencing.

Isolation of Rare Antigen-Specific Memory B Cells via Antigen Tetramers.

Immunological memory, which sets the foundation for the adaptive immune response, plays a key role in disease protection and prevention. Obtaining a deeper understanding of the mechanisms underlying this phenomenon can aide in research aimed to improve vaccines and therapies. Memory B cells (MBCs) are a fundamental component of immunological memory but can exist in rare populations that prove challenging to study. By combining fluorescent antigen tetramers with multiple enrichment processes, a highly streamlined method for identifying and sorting antigen-specific MBCs from human blood and lymphoid tissues can be achieved. With the output of this process being viable cells, there is a multitude of downstream operations that can be used in conjunction with the antigen-specific cell sorting outlined in this chapter. Single-cell RNA-sequencing paired with B cell repertoire sequencing, which can be linked to distinct antigens in a high-throughput fashion, is a downstream application widely used in disease and vaccination research. Incorporation of this protocol can lead to a variety of applications and a diversity of outcomes aiding in a deeper understanding of how immunological memory not only forms but is recalled and impacted by infection and vaccination.

Multilineage-differentiating stress-enduring cells: a powerful tool for tissue damage repair.

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.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Raman cell sorting for single-cell research.

Cells constitute the fundamental units of living organisms. Investigating individual differences at the single-cell level facilitates an understanding of cell differentiation, development, gene expression, and cellular characteristics, unveiling the underlying laws governing life activities in depth. In recent years, the integration of single-cell manipulation and recognition technologies into detection and sorting systems has emerged as a powerful tool for advancing single-cell research. Raman cell sorting technology has garnered attention owing to its non-labeling, non-destructive detection features and the capability to analyze samples containing water. In addition, this technology can provide live cells for subsequent genomics analysis and gene sequencing. This paper emphasizes the importance of single-cell research, describes the single-cell research methods that currently exist, including single-cell manipulation and single-cell identification techniques, and highlights the advantages of Raman spectroscopy in the field of single-cell analysis by comparing it with the fluorescence-activated cell sorting (FACS) technique. It describes various existing Raman cell sorting techniques and introduces their respective advantages and disadvantages. The above techniques were compared and analyzed, considering a variety of factors. The current bottlenecks include weak single-cell spontaneous Raman signals and the requirement for a prolonged total cell exposure time, significantly constraining Raman cell sorting technology's detection speed, efficiency, and throughput. This paper provides an overview of current methods for enhancing weak spontaneous Raman signals and their associated advantages and disadvantages. Finally, the paper outlines the detailed information related to the Raman cell sorting technology mentioned in this paper and discusses the development trends and direction of Raman cell sorting.

Single-Cell Sequencing of 3' RNA Transcripts.

Single-cell RNA sequencing (scRNA-seq) enables the measurement of RNA expressed from individual cells within a tissue or population. RNA expression profiles may be used to draw conclusions about cellular states, cell subtypes within the population, responses to perturbations, and cellular behavior in the context of disease. Here we describe a method for scRNA-seq via single-cell encapsulation and capture of the polyadenosine tails at the 3' end of mRNA transcripts combined with cell and molecular barcoding, allowing for the sequencing of 3' untranslated regions in order to identify expressed genes from a cell.

Induction into viable but non culturable state and outgrowth heterogeneity of Listeria monocytogenes is affected by stress history and type of growth.

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.

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn's disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA sequencing.

A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for the development of hPSC-based therapeutics. In this study, single-cell RNA sequencing (scRNA-seq) was performed to decipher the heterogeneity during differentiation of three hPSC lines toward corneal limbal stem cells (LSCs). The scRNA-seq data revealed nine clusters encompassing the entire differentiation process, among which five followed the anticipated differentiation path of LSCs. The remaining four clusters were previously undescribed cell states that were annotated as either mesodermal-like or undifferentiated subpopulations, and their prevalence was hPSC line dependent. Distinct cluster-specific marker genes identified in this study were confirmed by immunofluorescence analysis and employed to purify hPSC-derived LSCs, which effectively minimized the variation in the line-dependent differentiation efficiency. In summary, scRNA-seq offered molecular insights into the heterogeneity of hPSC-LSC differentiation, allowing a data-driven strategy for consistent and robust generation of LSCs, essential for future advancement toward clinical translation.

Microbial consortium assembly and functional analysis via isotope labelling and single-cell manipulation of polycyclic aromatic hydrocarbon degraders.

Soil microbial flora constitutes a highly diverse and complex microbiome on Earth, often challenging to cultivation, with unclear metabolic mechanisms in situ. Here, we present a pioneering concept for the in situ construction of functional microbial consortia (FMCs) and introduce an innovative method for creating FMCs by utilizing phenanthrene as a model compound to elucidate their in situ biodegradation mechanisms. Our methodology involves single-cell identification, sorting, and culture of functional microorganisms, resulting in the formation of a precise in situ FMC. Through Raman-activated cell sorting-stable-isotope probing, we identified and isolated phenanthrene-degrading bacterial cells from Achromobacter sp. and Pseudomonas sp., achieving precise and controllable in situ consortia based on genome-guided cultivation. Our in situ FMC outperformed conventionally designed functional flora when tested in real soil, indicating its superior phenanthrene degradation capacity. We revealed that microorganisms with high degradation efficiency isolated through conventional methods may exhibit pollutant tolerance but lack actual degradation ability in natural environments. This finding highlights the potential to construct FMCs based on thorough elucidation of in situ functional degraders, thereby achieving sustained and efficient pollutant degradation. Single-cell sequencing linked degraders with their genes and metabolic pathways, providing insights regarding the construction of in situ FMCs. The consortium in situ comprising microorganisms with diverse phenanthrene metabolic pathways might offer distinct advantages for enhancing phenanthrene degradation efficiency, such as the division of labour and cooperation or communication among microbial species. Our approach underscores the importance of in situ, single-cell precision identification, isolation, and cultivation for comprehensive bacterial functional analysis and resource exploration, which can extend to investigate MFCs in archaea and fungi, clarifying FMC construction methods for element recycling and pollutant transformation in complex real-world ecosystems.

Programming Spatial Cell Sorting by Engineering Cadherin Intracellular Activity.

The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.

Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

Spectral analysis and sorting of microbial organisms using a spectral sorter.

This chapter discusses the problems related to the application of conventional flow cytometers to microbiology. To address some of those limitations, the concept of spectral flow cytometry is introduced and the advantages over conventional flow cytometry for bacterial sorting are presented. We demonstrate by using ThermoFisher's Bigfoot spectral sorter where the spectral signatures of different stains for staining bacteria are demonstrated with an example of performing unmixing on spectral datasets. In addition to the Bigfoot's spectral analysis, the special biosafety features of this instrument are discussed. Utilizing these biosafety features, the sorting and patterning at the single cell level is optimized using non-pathogenic bacteria. Finally, the chapter is concluded by presenting a novel, label free, non-destructive, and rapid phenotypic method called Elastic Light Scattering (ELS) technology for identification of the patterned bacterial cells based on their unique colony scatter patterns.

Accessible high-speed image-activated cell sorting.

Over the past six decades, fluorescence-activated cell sorting (FACS) has become an essential technology for basic and clinical research by enabling the isolation of cells of interest in high throughput. Recent technological advancements have started a new era of flow cytometry. By combining the spatial resolution of microscopy with high-speed cell sorting, new instruments allow cell sorting based on simple image-derived parameters or sophisticated image analysis algorithms, thereby greatly expanding the scope of applications. In this review, we discuss the systems that are commercially available or have been described in enough methodological and engineering detail to allow their replication. We summarize their strengths and limitations and highlight applications that have the potential to transform various fields in basic life science research and clinical settings.

A method for selective and efficient isolation of gray matter astrocytes from the spinal cord of adult mice.

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.

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.