Flow Cytometric Sorting Research Articles

Induction of the apoptosis, degranulation and IL-13 production of human basophils by butyrate and propionate via suppression of histone deacetylation.

Allergic diseases are caused by dysregulated Th2 immune responses involving multiple effector cells including basophils. Short chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, exert immunomodulatory functions via activation of its receptors GPR41 and GPR43, and inhibition of the histone deacetylases (HDACs) activity. In allergic diseases, SCFAs suppress the activity of mast cells, eosinophils and type 2 innate lymphoid cells (ILC2) but enhance the function of Th2 cells. Here, we aimed to elucidate the function of SCFAs on human basophils. Human basophils were purified from healthy donors by flow cytometric sorting. The surface proteins, apoptosis and degranulation of basophils were analyzed by flow cytometric analysis. The mRNA expression was assayed using real-time PCR. Interleukin 4 (IL-4) and IL-13 were measured by ELISA. Histone acetylation was examined by western blot. GPR41 was expressed by basophils and was enhanced by IL-3. Acetate induced intracellular calcium influx in basophils which was suppressed by blocking GPR41. Propionate and butyrate, but not acetate, induced the expression of CD69 and IL-13. In addition, propionate and butyrate enhanced IgE-mediated basophil degranulation but inhibited basophil survival and IL-4secretion. Propionate and butyrate induced histone acetylation of basophils and suppression of HDACs activity mimicked the effects of propionate and butyrate on human basophils. Our findings demonstrate that propionate and butyrate may play a complex role in regulating basophil apoptosis, activation and degranulation via inhibiting HDACs activity. The in vivo effects of SCFAs on the regulation of basophil-associated allergic diseases need to be further explored.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Abstract P23: Detection of humoral and cellular responses to SARS-CoV-2 in COVID-19 convalescents

Abstract Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2. Citation Format: Anna Baranska, Marc Schuster, Marek Wieczorek, Sebastian Flade, Lennart Wessels, Burhan Karaca, Verena Traska, Mario Assenmacher, Gregor Winkels. Detection of humoral and cellular responses to SARS-CoV-2 in COVID-19 convalescents [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P23.

Chromosome analysis and sorting.

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow‐sorted fractions, and their suitability for downstream applications.

HCMV-controlling NKG2C+ NK cells originate from novel circulating inflammatory precursors

There is limited knowledge on the origin and development from CD34+ precursors of the ample spectrum of human natural killer (NK) cells, particularly of specialized NK subsets. This study sought to characterize the NK-cell progeny of CD34+DNAM-1brightCXCR4+ and of other precursors circulating in the peripheral blood of patients with chronic viral infections (eg, HIV, hepatitis C virus, cytomegalovirus reactivation). Highly purified precursors were obtained by flow cytometric sorting and cultured in standard NK-cell differentiation media (ie, SCF, FLT3, IL-7, IL-15). Phenotypic and functional analyses on progenies were performed by multiparametric cytofluorimetric assays. Transcriptional signatures of NK-cell progenies were studied by microarray analysis. Inhibition of cytomegalovirus replication was studied by PCR. Unlike conventional CD34+ precursors, Lin-CD34+DNAM-1brightCXCR4+ precursors from patients with chronic infection, rapidly differentiate into cytotoxic, IFN-γ-secreting CD94/NKG2C+KIR+CD57+ NK-cell progenies. An additional novel subset of common lymphocyte precursors was identified among Lin-CD34-CD56-CD16+ cells and characterized by expression of CXCR4 and lack of perforin and CD94. Lin-CD34-CD56-CD16+Perf-CD94-CXCR4+ precursors are also endowed with generation potential toward memory-like NKG2C+NK cells. Maturing NK-cell progenies mediated strong human cytomegalovirus-inhibiting activity. Microarray analysis confirmed a transcriptional signature compatible with NK-cell progenies and with maturing adaptive NK cells. During viral infections, precursors of adaptive NK cells are released and circulate in the peripheral blood.

Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

Cytogenetically Aberrant Cells in the Stem Cell Compartment (CD34+lin) in Acute Myeloid Leukemia

Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs.

Purification of membrane vesicles from Gram-positive bacteria using flow cytometry, after iodixanol density-gradient ultracentrifugation

Membrane vesicles (MVs) play biologically important roles in Gram-positive bacteria, and purification is essential for their study. Although high-performance flow cytometry has the capability to quantify and isolate specific small particles, it has not been examined for MV isolation. In this study, we used high-performance flow cytometry to analyze MV from Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, prepared by iodixanol density-gradient ultracentrifugation. Analysis of the quality of MV samples before and after sorting showed that the flow cytometric sorting provided higher purity and uniformity compared to gradient isolation alone. The MV purification method using flow cytometry should prove useful for applications requiring a very high purity of MV samples such as proteomic, metagenomic or lipidomic studies.

CD90 Is Dispensable for White and Beige/Brown Adipocyte Differentiation.

Brown adipose tissue (BAT) is a thermogenic organ in rodents and humans. In mice, the transplantation of BAT has been successfully used to combat obesity and its comorbidities. While such beneficial properties of BAT are now evident, the developmental and cellular origins of brown, beige, and white adipocytes have remained only poorly understood, especially in humans. We recently discovered that CD90 is highly expressed in stromal cells isolated from human white adipose tissue (WAT) compared to BAT. Here, we studied whether CD90 interferes with brown or white adipogenesis or white adipocyte beiging. We applied flow cytometric sorting of human adipose tissue stromal cells (ASCs), a CRISPR/Cas9 knockout strategy in the human Simpson-Golabi-Behmel syndrome (SGBS) adipocyte model system, as well as a siRNA approach in human approaches supports the hypothesis that CD90 affects brown or white adipogenesis or white adipocyte beiging in humans. Taken together, our findings call the conclusions drawn from previous studies, which claimed a central role of CD90 in adipocyte differentiation, into question.

An ultrahigh‐throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries

Uncultivable microbial communities provide enormous reservoirs of enzymes, but their experimental identification by functional metagenomics is challenging, mainly due to the difficulty of screening enormous metagenomic libraries. Here, we propose a reliable and convenient ultrahigh-throughput screening platform based on flow cytometric droplet sorting (FCDS). The FCDS platform employs water-in-oil-in-water double emulsion droplets serving as single-cell enzymatic micro-reactors and a commercially available flow cytometer, and it can efficiently isolate novel biocatalysts from metagenomic libraries by processing single cells as many as 108 per day. We demonstrated the power of this platform by screening a metagenomic library constructed from domestic running water samples. The FCDS assay screened 30 million micro-reactors in only 1 h, yielding a collection of esterase genes. Among these positive hits, Est WY was identified as a novel esterase with high catalytic efficiency and distinct evolutionary origin from other lipolytic enzymes. Our study manifests that the FCDS platform is a robust tool for functional metagenomics, with the potential to significantly improve the efficiency of exploring novel enzymes from nature.

Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells

Mesenchymal stem cells (MSCs) are expected to be useful in bone regeneration treatment for various diseases and conditions, including cleft lip and palate, fracture, and bone absorption. However, to date, MSCs have failed to produce satisfactory results in clinical settings. This is primarily due to the low rate of induced osteogenic differentiation. To realize MSC potential, it is necessary to establish methods for the isolation of MSC-derived living osteoblasts. However, no osteoblast markers have been reported to date. In an attempt to develop a method for the assessment of osteoblast differentiation, we established reporter human immortalized MSC (hiMSC) lines for in vitro monitoring of bone gamma-carboxyglutamate protein (BGLAP, osteocalcin) expression. To this end, we successfully knocked-in an enhanced green fluorescent protein (EGFP) gene cassette immediately downstream of the first ATG of BGLAP via CRISPR-Cas9, and established hiMSC lines expressing EGFP to monitor osteogenic differentiation. On differentiation day 7, EGFP-positive cells were collected by flow cytometric cell sorting, and the expression of EGFP and endogenous BGLAP was analyzed. During osteogenic differentiation, EGFP upregulation was found to correlate with expression of endogenous BGLAP. Moreover, mineralization was confirmed using Alizarin red-S staining after two weeks of osteogenic differentiation of the modified hiMSC lines. The modified hiMSC lines, as well as the derived differentiated osteoblasts obtained herein, are valuable tools for the monitoring osteoblast gene and protein expression, and can be used to develop novel methods for isolating living osteoblasts.

Seasonal and Geographical Transitions in Eukaryotic Phytoplankton Community Structure in the Atlantic and Pacific Oceans

Much is known about how broad eukaryotic phytoplankton groups vary according to nutrient availability in marine ecosystems. However, genus- and species-level dynamics are generally unknown, although important given that adaptation and acclimation processes differentiate at these levels. We examined phytoplankton communities across seasonal cycles in the North Atlantic (BATS) and under different trophic conditions in the eastern North Pacific (ENP), using phylogenetic classification of plastid-encoded 16S rRNA amplicon sequence variants (ASVs) and other methodologies, including flow cytometric cell sorting. Prasinophytes dominated eukaryotic phytoplankton amplicons during the nutrient-rich deep-mixing winter period at BATS. During stratification (‘summer’) uncultured dictyochophytes formed ∼35 ± 10% of all surface plastid amplicons and dominated those from stramenopile algae, whereas diatoms showed only minor, ephemeral contributions over the entire year. Uncultured dictyochophytes also comprised a major fraction of plastid amplicons in the oligotrophic ENP. Phylogenetic reconstructions of near-full length 16S rRNA sequences established 11 uncultured Dictyochophyte Environmental Clades (DEC). DEC-I and DEC-VI dominated surface dictyochophytes under stratification at BATS and in the ENP, and DEC-IV was also important in the latter. Additionally, although less common at BATS, Florenciella-related clades (FC) were prominent at depth in the ENP. In both ecosystems, pelagophytes contributed notably at depth, with PEC-VIII (Pelagophyte Environmental Clade) and (cultured) Pelagomonas calceolata being most important. Q-PCR confirmed the near absence of P. calceolata at the surface of the same oligotrophic sites where it reached ∼1,500 18S rRNA gene copies ml–1 at the DCM. To further characterize phytoplankton present in our samples, we performed staining and at-sea single-cell sorting experiments. Sequencing results from these indicated several uncultured dictyochophyte clades are comprised of predatory mixotrophs. From an evolutionary perspective, these cells showed both conserved and unique features in the chloroplast genome. In ENP metatranscriptomes we observed high expression of multiple chloroplast genes as well as expression of a selfish element (group II intron) in the psaA gene. Comparative analyses across the Pacific and Atlantic sites support the conclusion that predatory dictyochophytes thrive under low nutrient conditions. The observations that several uncultured dictyochophyte lineages are seemingly capable of photosynthesis and predation, raises questions about potential shifts in phytoplankton trophic roles associated with seasonality and long-term ocean change.

A single-cell polony method reveals low levels of infected Prochlorococcus in oligotrophic waters despite high cyanophage abundances

Long-term stability of picocyanobacteria in the open oceans is maintained by a balance between synchronous division and death on daily timescales. Viruses are considered a major source of microbial mortality, however, current methods to measure infection have significant methodological limitations. Here we describe a method that pairs flow-cytometric sorting with a PCR-based polony technique to simultaneously screen thousands of taxonomically resolved individual cells for intracellular virus DNA, enabling sensitive, high-throughput, and direct quantification of infection by different virus lineages. Under controlled conditions with picocyanobacteria-cyanophage models, the method detected infection throughout the lytic cycle and discriminated between varying infection levels. In North Pacific subtropical surface waters, the method revealed that only a small percentage of Prochlorococcus (0.35–1.6%) were infected, predominantly by T4-like cyanophages, and that infection oscillated 2-fold in phase with the diel cycle. This corresponds to 0.35–4.8% of Prochlorococcus mortality daily. Cyanophages were 2–4-fold more abundant than Prochlorococcus, indicating that most encounters did not result in infection and suggesting infection is mitigated via host resistance, reduced phage infectivity and inefficient adsorption. This method will enable quantification of infection for key microbial taxa across oceanic regimes and will help determine the extent that viruses shape microbial communities and ecosystem level processes.

DNA replication and chromosome positioning throughout the interphase in three-dimensional space of plant nuclei.

Despite much recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains surprisingly limited. Notably, it is not clear how these processes could be affected by the size of a plant's nuclear genome. In this study, DNA replication timing and interphase chromosome positioning were analyzed in seven Poaceae species that differ in their genome size. To provide a comprehensive picture, a suite of advanced, complementary methods was used: labeling of newly replicated DNA by ethynyl-2'-deoxyuridine, isolation of nuclei at particular cell cycle phases by flow cytometric sorting, three-dimensional immunofluorescence in situ hybridization, and confocal microscopy. Our results revealed conserved dynamics of DNA replication in all species, and a similar replication timing order for telomeres and centromeres, as well as for euchromatin and heterochromatin regions, irrespective of genome size. Moreover, stable chromosome positioning was observed while transitioning through different stages of interphase. These findings expand upon earlier studies in suggesting that a more complex interplay exists between genome size, organization of repetitive DNA sequences along chromosomes, and higher order chromatin structure and its maintenance in interphase, albeit controlled by currently unknown factors.

2014 – FLUORESCENT CELL CYCLE TIMER ENABLED ANALYSIS OF NORMAL AND INEFFECTIVE ERYTHROPOIESIS

Aberrant cell cycle speed during hematopoiesis defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. We utilized a unique live-cell reporter of cell cycle speed using a Histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein, distinguishing faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence. In transgenic mice expressing this reporter, we characterized the stress erythropoietic response of spleen and bone marrow following phenylhydrazine-induced hemolytic anemia. Using flow cytometric sorting of erythroblast stages, we found that all stages divide more rapidly after the hemolytic insult, not only early progenitors as previously thought. We also observe that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. And the last nucleated cell stage, orthochromatophilic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate anemia. Mass spectrometry of sorted populations shows upregulation of cell cycle and metabolic pathway related genes. Using this system in in vitro human CD34+ primary cell culture, we assessed the erythropoietic response to known treatments of β-thalassemia and Diamond Blackfan Anemia, like Epo and dexamethasone. We found an increase in cell cycling rates with an increase in dexamethasone concentration but not that of Epo. These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. This system can help in understanding stress hematopoiesis, allowing identification of cellular factors that facilitate response to external stress.

In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry.

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.

Highly efficient synchronization of sheep skin fibroblasts at G2/M phase and isolation of sheep Y chromosomes by flow cytometric sorting

At present, based on whole genome sequencing, sequences and genes annotation of the sheep (Ovis aries) Y chromosome are still absent. The isolation of Y chromosomes followed by sequencing has been approved as an effective approach to analyze this complex chromosome in other species. In this study, we established a highly efficient synchronization method for G2/M phase of sheep fibroblasts, which was successfully applied to flow-sorting chromosomes of sheep, with a focus on isolation and sequencing of the ovine Y chromosome. The isolated (~80,000) Y chromosomes were verified by fluorescence quantitative real-time polymerase chain reaction, further confirmed by fluorescence in situ hybridization, and amplified by the MALBAC method before next-generation sequencing. The sequence results indicated that 68.90% of reads were Y chromosome-related sequences as they are homologous to the bovine Y chromosome. The remaining 31.1% of reads were aligned to the sheep reference genome, including 13.57% reads to chromosome X and 6.68% to chromosome 17. Importantly, the paired-end reads that are properly aligned to the bovine Y sequence assembly accounted for 46.49%, indicating the success in the ovine Y chromosome isolation and the high quality of the Y chromosome sequences. This study not only set up a foundation for future sequencing, assembly and annotation of the ovine Y chromosome, but also provide a validated approach to overcoming difficulties in sequencing Y chromosome in other mammalian species.

Temporal patterns in the interaction between photosynthetic picoeukaryotes and their attached fungi in Lake Chaohu.

The combination of flow cytometric sorting and high-throughput sequencing revealed the broad existence of photosynthetic picoeukaryote attached fungi (PPE-attached fungi) in Lake Chaohu. The relative sequence abundance of attached fungi was negatively correlated with that of the photosynthetic picoeukaryotes (PPEs). PPE-attached fungal communities were mainly composed of Basidiomycota, Chytridiomycota and Ascomycota. Temperature, Si and PPE community structure are the most important driving factors for the temporal succession of PPE-attached fungal communities. In particular, PPE-attached fungi can be divided into three groups from high to low temperatures. Phylogenetic molecular ecological network results indicated that the connectivity and the total number of links in the network of the high-temperature group (> 21.82°C) are higher than those in the other two temperature groups (between 9.67 and 21.82°C, and < 9.67°C, respectively). Moreover, the interaction between PPE-attached fungi and the PPEs changed from antagonistic to cooperative, with the decline in temperature. The most abundant operational taxonomic units of PPE-attached fungi were affiliated with the Cladosporium, the most common saprophytic fungus, whereas most fungal hub taxa were Chytridiomycota, the main parasite fungi of phytoplankton.

CD109 mediates tumorigenicity and cancer aggressiveness via regulation of EGFR and STAT3 signalling in cervical squamous cell carcinoma

BackgroundCD109 was involved in the tumorigenesis and progression of various cancers via TGF-β1 signalling and STAT3 activation. As CD109 is strongly expressed in cervical squamous cell carcinoma, this study was conducted to investigate its functional characteristics in cervical cancer.MethodsCD109 expression was examined by immunohistochemistry (IHC) with cervical tissue microarray. The effects of CD109 expression were examined on migration, cell proliferation, spheroid formation and soft-agar colony-formation assay. Meanwhile, cervical cancer cell lines with high CD109 expression were chosen for the functional study using siRNA knockdown and CRISPR/Cas9 knockout.ResultsIHC demonstrated an upregulation of CD109 in the cell membrane of cervical squamous cell carcinoma. CD109( + ) cells isolated by flow-cytometric sorting displayed enhanced migration, cell proliferation, sphere-forming and anchorage-independent cell growth ability. In contrast, silencing of CD109 expression could reverse the in vitro and in vivo tumorigenic and aggressive properties. Furthermore, CD109 induced EGFR-mediated STAT3 phosphorylation known to be responsible for cell migration, proliferation and maintenance of CSC phenotype.ConclusionAbundant CD109( + ) populations in cervical cancer cells potentially contributed to carcinogenesis and aggressiveness, whereas silencing of CD109 expression could reverse those properties. CD109 mediates cervical tumorigenicity and aggressiveness via CD109/EGFR/STAT3 signalling.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.