Single Cell Sorting Research Articles

A universal MHCII technology platform to characterize antigen-specific CD4+ T cells

CD4+ Tcells are critical to the immune system and perform multiple functions; therefore, their identification and characterization are crucial to better understanding the immune system in both health and disease states. However, current methods rarely preserve their exvivo phenotype, thus limiting our understanding of their invivo functions. Here we introduce a flexible, rapid, and robust platform for exvivo CD4+ Tcell identification. By combining MHCII allele purification, allele-independent peptide loading, and multiplexed flow cytometry technologies, we can enable high-throughput personalized CD4+ Tcell identification, immunophenotyping, and sorting. Using this platform in combination with single-cell sorting and multimodal analyses, we identified and characterized antigen-specific CD4+ Tcells relevant to COVID-19 and cancer neoantigen immunotherapy. Overall, our platform can be used to detect and characterize CD4+ Tcells across multiple diseases, with potential to guide CD4+ Tcell epitope design for any disease-specific immunization strategy.

Full-electric microfluidic platform to capture, analyze and selectively release single cells.

Current single-cell technologies require large and expensive equipment, limiting their use to specialized labs. In this paper, we present for the first time a microfluidic device which demonstrates a combined method for full-electric cell capturing, analyzing, and selectively releasing with single-cell resolution. All functionalities are experimentally demonstrated on Saccharomyces cerevisiae. Our microfluidic platform consists of traps centered around a pair of individually accessible coplanar electrodes, positioned under a microfluidic channel. Using this device, we validate our novel Two-Voltage method for trapping single cells by positive dielectrophoresis (pDEP). Cells are attracted to the trap when a high voltage (VH) is applied. A low voltage (VL) holds the already trapped cell in place without attracting additional cells, allowing full control over the number of trapped cells. After trapping, the cells are analyzed by broadband electrochemical impedance spectroscopy. These measurements allow the detection of single cells and the extraction of cell parameters. Additionally, these measurements show a strong correlation between average phase change and cell size, enabling the use of our system for size measurements in biological applications. Finally, our device allows selectively releasing trapped cells by turning off the pDEP signal in their trap. The experimental results show the techniques potential as a full-electric single-cell analysis tool with potential for miniaturization and automation which opens new avenues towards small-scale, high throughput single-cell analysis and sorting lab-on-CMOS devices.

Engineering Ag43 Signal Peptides with Bacterial Display and Selection

Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

BackgroundOrdered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development.ResultsHere, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data.ConclusionWe expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

Single-cell transcriptome analyses reveal microglia types associated with proliferative retinopathy.

Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and to characterize the transcriptional profiles and metabolic pathways of proangiogenic microglia in a mouse model of oxygen-induced PR (OIR). Using transcriptional single-cell sorting, we comprehensively mapped all microglia populations in retinas of room air (RA) and OIR mice. We have unveiled several unique types of PR-associated microglia (PRAM) and identified markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hypermetabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes, and proangiogenic Igf1. IHC staining shows that these PRAM were spatially located within or around neovascular tufts. These unique types of microglia have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis-related diseases.

Artificial intelligence-assisted automatic and index-based microbial single-cell sorting system for One-Cell-One-Tube.

Identification, sorting, and sequencing of individual cells directly from in situ samples have great potential for in-depth analysis of the structure and function of microbiomes. In this work, based on an artificial intelligence (AI)-assisted object detection model for cell phenotype screening and a cross-interface contact method for single-cell exporting, we developed an automatic and index-based system called EasySort AUTO, where individual microbial cells are sorted and then packaged in a microdroplet and automatically exported in a precisely indexed, "One-Cell-One-Tube" manner. The target cell is automatically identified based on an AI-assisted object detection model and then mobilized via an optical tweezer for sorting. Then, a cross-interface contact microfluidic printing method that we developed enables the automated transfer of cells from the chip to the tube, which leads to coupling with subsequent single-cell culture or sequencing. The efficiency of the system for single-cell printing is >93%. The throughput of the system for single-cell printing is ~120 cells/h. Moreover, >80% of single cells of both yeast and Escherichia coli are culturable, suggesting the superior preservation of cell viability during sorting. Finally, AI-assisted object detection supports automated sorting of target cells with high accuracy from mixed yeast samples, which was validated by downstream single-cell proliferation assays. The automation, index maintenance, and vitality preservation of EasySort AUTO suggest its excellent application potential for single-cell sorting.

Leveraging single-cell Raman spectroscopy and single-cell sorting for the detection and identification of yeast infections

Invasive fungal infection serves as a great threat to human health. Discrimination between fungal and bacterial infections at the earliest stage is vital for effective clinic practice; however, traditional culture-dependent microscopic diagnosis of fungal infection usually requires several days, meanwhile, culture-independent immunological and molecular methods are limited by the detectable type of pathogens and the issues with high false-positive rates. In this study, we proposed a novel culture-independent phenotyping method based on single-cell Raman spectroscopy for the rapid discrimination between fungal and bacterial infections. Three Raman biomarkers, including cytochrome c, peptidoglycan, and nucleic acid, were identified through hierarchical clustering analysis of Raman spectra across 12 types of most common yeast and bacterial pathogens. Compared to those of bacterial pathogens, the single cells of yeast pathogens demonstrated significantly stronger Raman peaks for cytochrome c, but weaker signals for peptidoglycan and nucleic acid. A two-step protocol combining the three biomarkers was established and able to differentiate fungal infections from bacterial infections with an overall accuracy of 94.9%. Our approach was also used to detect ten raw urinary tract infection samples. Successful identification of fungi was achieved within half an hour after sample obtainment. We further demonstrated the accurate fungal species taxonomy achieved with Raman-assisted cell ejection. Our findings demonstrate that Raman-based fungal identification is a novel, facile, reliable, and with a breadth of coverage approach, that has a great potential to be adopted in routine clinical practice to reduce the turn-around time of invasive fungal disease (IFD) diagnostics.

Effectiveness and Safety of Ultrasound-Guided Local Paricalcitol Injection in Treating Secondary Hyperparathyroidism in ESRD: A Retrospective Study.

To compare the safety and efficacy of percutaneous paricalcitol injection with intravenously administered paricalcitol in treating parathyroid hyperplasia in patients with secondary hyperparathyroidism (SHPT). This study was approved by the Ethics Committee of our institution. We retrospectively collected data on patients who received percutaneous paricalcitol injection (24 patients) and intravenously administered paricalcitol (22 patients) based on their intact parathyroid hormone (iPTH) level. Serum iPTH, calcium, phosphorus, and the volume of the parathyroid gland were measured at several indicated time points after treatment, and adverse events associated with the two treatments were evaluated. After 6 months of follow-up, we found that patients from the percutaneous injection group had significantly decreased levels of iPTH (from 1887.81 ± 726.81 pg/mL to 631.06 ± 393.06 pg/mL), phosphate (from 1.94 ± 0.36 mmol/L to 1.71 ± 0.34 mmol/L), and volume of the parathyroid gland (from 0.87 ± 0.50 cm3 to 0.60 ± 0.36 cm3), with relief from ostealgia within 48-72 h. In the intravenously administered group, the levels of iPTH decreased from 686.87 ± 260.44 pg/mL to 388.47 ± 167.36 pg/mL; while there was no significant change in phosphate levels, the volume of the parathyroid gland and ostealgia relief were observed at the end of follow-up. The serum calcium level did not significantly change, and no severe complications were observed in both groups. In vitro fluorescence-activated single cell sorting (FACS) analysis indicated that paricalcitol induced parathyroid cell apoptosis in a dose-dependent manner. Percutaneous paricalcitol injection is a selective treatment for SHPT in ESRD.

Single-cell Raman-activated sorting and cultivation (scRACS-Culture) for assessing and mining in situ phosphate-solubilizing microbes from nature

Due to the challenges in detecting in situ activity and cultivating the not-yet-cultured, functional assessment and mining of living microbes from nature has typically followed a ‘culture-first’ paradigm. Here, employing phosphate-solubilizing microbes (PSM) as model, we introduce a ‘screen-first’ strategy that is underpinned by a precisely one-cell-resolution, complete workflow of single-cell Raman-activated Sorting and Cultivation (scRACS-Culture). Directly from domestic sewage, individual cells were screened for in-situ organic-phosphate-solubilizing activity via D2O intake rate, sorted by the function via Raman-activated Gravity-driven Encapsulation (RAGE), and then cultivated from precisely one cell. By scRACS-Culture, pure cultures of strong organic PSM including Comamonas spp., Acinetobacter spp., Enterobacter spp. and Citrobacter spp., were derived, whose phosphate-solubilizing activities in situ are 90–200% higher than in pure culture, underscoring the importance of ‘screen-first’ strategy. Moreover, employing scRACS-Seq for post-RACS cells that remain uncultured, we discovered a previously unknown, low-abundance, strong organic-PSM of Cutibacterium spp. that employs secretary metallophosphoesterase (MPP), cell-wall-anchored 5′-nucleotidase (encoded by ushA) and periplasmic-membrane located PstSCAB-PhoU transporter system for efficient solubilization and scavenging of extracellular phosphate in sewage. Therefore, scRACS-Culture and scRACS-Seq provide an in situ function-based, ‘screen-first’ approach for assessing and mining microbes directly from the environment.

Single-cell sorting using integrated pneumatic valve droplet microfluidic chip

Single-cell analysis and sorting have been increasingly prevalent in scientific study and industry in recent years. Droplet-based microfluidic systems are significant tools for single cell separation due to the advantages of high-throughput screening flux and relatively simple chip manufacturing. Herein, this work integrated single-cell droplet generation, incubation, and sorting functions into a single microfluidic chip using pneumatic microvalve for single-cell screening of hybridoma cells. The chip realized the generation, storage, incubation, and assay sorting of droplets in order by adjusting the switch of the microvalve. Numerical methods were used to simulate the effects of membrane size and pressure on valve deformation. Incubation results showed that fluorescent signals were generated in the droplets containing both hybridoma cells and microspheres under the control of pneumatic microvalves. In addition, the purity of target droplets was increased from 4.87% before sorting to 89.70% after sorting using fluorescence-activated droplet sorting platform, demonstrating the feasibility of the integrated chip with a pneumatic valve for single-cell screening. The integrated chip is proved to be operated efficiently with minimum cell loss and human labor through reducing the risk of droplet fusion and facilitating the development of automated devices. So, the present study provides an integrated design for microdroplet-based single-cell sorting with various applications, such as single-cell sequencing, antibody screening, and directed evolutionary studies.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

Efficient reprogramming of human fibroblasts using RNA reprogramming with DAPT and iDOT1L under normoxia conditions.

IntroductionHuman induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells expressing a defined set of transcription factors. The advent of autologous iPSCs has enabled the generation of patient-specific iPSC lines and is expected to contribute to the exploration of cures and causes of diseases, drug screening, and tailor-made regenerative medicines. Efficient control of hiPSC derivation is beneficial for industrial applications. However, the mechanisms underlying somatic cell reprogramming remain unknown, while reprogramming efficiency remains extremely low, especially in human cells.Methods and resultsWe previously reported that chemical inhibition of the NOTCH signaling pathway and DOT1L promoted the generation of hiPSCs from keratinocytes, but the mechanisms and effect of this double inhibition on other types of cells remain to be investigated. Here, we found that the NOTCH/DOT1L inhibition markedly increased iPSC colony generation from human fibroblast cells via mRNA reprogramming, and mesenchymal to epithelial transition (MET)-related genes are significantly expressed in the early phase of the reprogramming. We successfully derived hiPSC lines using a single-cell sorting system under efficient reprogramming conditions.ConclusionsThis user-friendly reprogramming approach paves the way for the development of hiPSC derivations in industrial applications of disease modeling and drug screening.

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

Using gel microdroplets to develop a simple high-throughput screening platform for oleaginous microorganisms

The oleaginous yeast Lipomyces starkeyi is expected to be a new lipid source since this microorganism is capable of accumulating more than 85% lipid per dry cell weight. For effective utilization of oleaginous yeast, mutants with improved lipid production compared to the wild-type have been screened by methods such as single-cell sorting and Percoll density gradient centrifugation. Because these methods need to reculture all mutated oleaginous yeasts together in a flask, it is difficult to evaluate the growth of each individual mutant. Thus, screening for the slow-growing mutants with high-throughput has never been performed by conventional methods. In this study, we developed a high-throughput method using gel microdroplets (GMD). With this method, the growth and lipid production of L. starkeyi can be evaluated simultaneously. L. starkeyi grew in GMD and the size of these microcolonies was evaluated by scattered light. Finally, a mutant with a 10-fold delay in growth compared to the wild-type was obtained. Analysis of genetic information in this mutant could reveal valuable information about critical genes involved in the growth of these microorganisms, which could then be utilized further.

Achillea fragrantissima (Forssk.) Sch.Bip Flower Dichloromethane Extract Exerts Anti-Proliferative and Pro-Apoptotic Properties in Human Triple-Negative Breast Cancer (MDA-MB-231) Cells: In Vitro and In Silico Studies.

The aggressive triple-negative breast cancer (TNBC) is a challenging disease due to the absence of tailored therapy. The search for new therapies involves intensive research focusing on natural sources. Achillea fragrantissima (A. fragrantissima) is a traditional medicine from the Middle East region. Various solvent extracts from different A. fragrantissima plant parts, including flowers, leaves, and roots, were tested on TNBC MDA-MB-231 cells. Using liquid chromatography, the fingerprinting revealed rich and diverse compositions for A. fragrantissima plant parts using polar to non-polar solvent extracts indicating possible differences in bioactivities. Using the CellTiter-Glo™ viability assay, the half-maximal inhibitory concentration (IC50) values were determined for each extract and ranged from 32.4 to 161.7 µg/mL. The A. fragrantissima flower dichloromethane extract had the lowest mean IC50 value and was chosen for further investigation. Upon treatment with increasing A. fragrantissima flower dichloromethane extract concentrations, the MDA-MB-231 cells displayed, in a dose-dependent manner, enhanced morphological and biochemical hallmarks of apoptosis, including cell shrinkage, phosphatidylserine exposure, caspase activity, and mitochondrial outer membrane permeabilization, assessed using phase-contrast microscopy, fluorescence-activated single-cell sorting analysis, Image-iT™ live caspase, and mitochondrial transition pore opening activity, respectively. Anticancer target prediction and molecular docking studies revealed the inhibitory activity of a few A. fragrantissima flower dichloromethane extract-derived metabolites against carbonic anhydrase IX, an enzyme reported for its anti-apoptotic properties. In conclusion, these findings suggest promising therapeutic values of the A. fragrantissima flower dichloromethane extract against TNBC development.

Moringa concanensis-Mediated Synthesis and Characterizations of Ciprofloxacin Encapsulated into Ag/TiO2/Fe2O3/CS Nanocomposite: A Therapeutic Solution against Multidrug Resistant E. coli Strains of Livestock Infectious Diseases.

Background: Multidrug resistant MDR bacterial strains are causing fatal infections, such as mastitis. Thus, there is a need for the development of new target-oriented antimicrobials. Nanomaterials have many advantages over traditional antibiotics, including improved stability, controlled antibiotic release, targeted administration, enhanced bioavailability, and the use of antibiotic-loaded nanomaterials, such as the one herein reported for the first time, appear to be a promising strategy to combat antibiotic-resistant bacteria. The use of rationally designed metallic nanocomposites, rather than the use of single metallic nanoparticles (NPs), should further minimize the bacterial resistance. Aim: Green synthesis of a multimetallic/ternary nanocomposite formed of silver (Ag), titanium dioxide (TiO2), and iron(III) oxide (Fe2O3), conjugated to chitosan (CS), in which the large spectrum fluoroquinolone antibiotic ciprofloxacin (CIP) has been encapsulated. Methods: The metallic nanoparticles (NPs) Ag NPs, TiO2 NPs, and Fe2O3 NPs were synthesized by reduction of Moringa concanensis leaf aqueous extract. The ternary junction was obtained by wet chemical impregnation technique. CIP was encapsulated into the ternary nanocomposite Ag/TiO2/Fe2O3, followed by chitosan (CS) conjugation using the ionic gelation method. The resulting CS-based nanoparticulate drug delivery system (NDDS), i.e., CIP-Ag/TiO2/Fe2O3/CS, was characterized in vitro by gold standard physical techniques such as X-ray diffractometry (XRD), field emission scanning electron microscopy (FESEM), Fourier-transform infrared (FTIR) spectroscopy. Pharmacological analyses (i.e., LC, EE, ex-vivo drug release behavior) were also assessed. Further, biological studies were carried out both ex vivo (i.e., by disk diffusion method (DDM), fluorescence-activated single cell sorting (FACS), MTT assay) and in vivo (i.e., antibacterial activity in a rabbit model, colony-forming unit (CFU) on blood agar, histopathological analysis using H&E staining). Results: The encapsulation efficiency (EE) and the loading capacity (LC) of the NDDS were as high as 94% ± 1.26 and 57% ± 3.5, respectively. XRD analysis confirmed the crystalline nature of the prepared formulation. FESEM revealed nanorods with an average diameter of 50–70 ± 12 nm. FTIR confirmed the Fe-O-Ti-CS linkages as well as the successful encapsulation of CIP into the NDDS. The zeta potential (ZP) of the NDDS was determined as 85.26 ± 0.12 mV. The antimicrobial potential of the NDDS was elicited by prominent ZIs against MDR E. coli (33 ± 1.40 mm) at the low MIC of 0.112 μg/mL. Morphological alterations (e.g., deformed shape and structural damages) of MDR pathogens were clearly visible overtime by FESEM after treatment with the NDDS at MIC value, which led to the cytolysis ultimately. FACS analysis confirmed late apoptotic of the MDR E. coli (80.85%) after 6 h incubation of the NDDS at MIC (p < 0.05 compared to untreated MDR E. coli used as negative control). The highest drug release (89% ± 0.57) was observed after 8 h using PBS medium at pH 7.4. The viability of bovine mammary gland epithelial cells (BMGE) treated with the NDDS remained superior to 90%, indicating a negligible cytotoxicity (p < 0.05). In the rabbit model, in which infection was caused by injecting MDR E. coli intraperitoneally (IP), no colonies were detected after 72 h of treatment. Importantly, the histopathological analysis showed no changes in the vital rabbit organs in the treated group compared to the untreated group. Conclusions: Taken together, the newly prepared CIP-Ag/TiO2/Fe2O3/CS nanoformulation appears safe, biocompatible, and therapeutically active to fight MDR E. coli strains-causing mastitis.

The microbiome of a bacterivorous marine choanoflagellate contains a resource-demanding obligate bacterial associate

Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor. We analyse complete ‘Comchoano’ genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella’s that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

Single-cell metagenomics and metagenomics approaches reveal extracellular electron transfer of psychrophilic electroactive biofilms

Revealing the metabolic functions and the extracellular electron transfer (EET) process of psychrophilic electroactive biofilms (PEB) is important for understanding the functional adaptability of electroactive bacteria (EAB) to low temperatures. In this study, single cell sorting (SCS)-based metagenomics sequencing was used to characterize the composition and function of the PEB. Biofilm microbiome analysis showed that Geobacter, with a relative abundance of 62.64%, dominated the PEB enriched in the bioelectrochemical system (BES) at 4 °C. Both the metagenome (MAG) and single-cell metagenome (SCM) revealed that there were no obvious metabolic differences between the original biofilm and the sorted single cells. The presence of genes associated with type IV pilus, c-type cytochromes, and riboflavin indicated that the EET potential was maintained in the PEB at low temperatures. These results suggested that SCM provides an alternative approach to reconstruct the metabolic functions of uncultured and slow-growing EAB.

Single cell mutant selection for metabolic engineering of actinomycetes

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.

Comprehensive characterization and optimization of Caco-2 cells enabled the development of a miniaturized 96-well permeability assay

Assessment of compound permeability through a Caco-2 cell monolayer is a well-accepted model to evaluate its in-vivo permeability potential and transporter interaction. While this assay has commonly been conducted using a 24-well assay plate format, a miniaturised 96-well assay format is highly desirable to achieve greater capacity and higher efficiency. Previous attempts to convert this assay from 24-well to 96-well format at our lab, however, had met with varied efflux capacities and unacceptable efflux ratios for digoxin, a substrate of P-glycoprotein (Pgp), which indicated inadequate Pgp transporter expression in the 96-well format. These challenges in converting the assays were attributed to the heterogeneous and unstable nature of the Caco-2 cells. To overcome the challenges, single-cell sorting of Caco-2 cells was conducted by flow cytometry to obtain a more homogeneous and stable cell population. The sorted cells were then seeded to 96-well transwell plates and the Pgp expression under various cell culture conditions was monitored by a LC-MS/MS-based targeted proteomics method. Through cell sorting and direct Pgp expression measurement, Caco-2 cells with adequate and sustained Pgp expression in a 96-well format were obtained, which led to the successful development and implementation of a 96-well Caco-2 assay with significant efficiency gain and faster turnaround time than the historical 24-well assay.