flowDiv: a new pipeline for analyzing flow cytometric diversity

Flow cytometry (FCM) is a powerful analytical tool that is widely used worldwide, as it allows the depiction of the innate complexity of a vast range of biological systems in few seconds. It is a technique based on the spectroscopic properties of suspended particles that allows data to be graphically summarized by biplots, known as cytograms. Such versatility got raises to different analytical protocols which are commonly not interchangeable among expertise fields. In this sense, environmental sciences, in particular, faces major concerns when dealing with the adoption of non-specific protocols - a particularity essentially driven by the highly heterogeneous nature of environmental samples. Such intrinsic variety makes it difficult to adjust formal analytical protocols that both keep standardized mathematical rationales and retain a clear ecological meaning, namely when the focus of the analysis rely on the cytometric diversity - the quantitative evaluation of the differences among cytograms. Despite of the availability of promising tools conceived or adapted to approach cytometric diversity, most of them face common technical challenges, as perspective adjustment, dilution correction, resolution setup and enlightenment on the role of cytograms subregions to global diversity. To address such questions and harmonize formal mathematical rationales with coherent biological interpretation, we have developed flowDiv - a pipeline designed for environmental flow cytometry data analysis that handles data through consolidated macroecological methods to offer biologically apprehensive outputs. flowDiv was implemented using R language and has been published on CRAN (https://cran.r-project.org/web/packages/flowDiv/) with source code also available on GitHub (https://github.com/bmsw/flowDiv). Applied to a dataset from 31 freshwater bodies in Argentinian Patagonia, flowDiv uncovered significant aspects regrading environmental cytometric diversity, as its relation with taxonomic diversity and the role of environmental variables on cytometric diversity.

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flowDiv: a new pipeline for analyzing flow cytometric diversity

10.7287/peerj.preprints.26934v1 ◽

2018 ◽

Author(s):

Bruno M Wanderley ◽

Daniel SA Araújo ◽

María V Quiroga ◽

André M Amado ◽

Adrião DD Neto ◽

...

Keyword(s):

Flow Cytometry ◽

Taxonomic Diversity ◽

Spectroscopic Properties ◽

Flow Cytometry Data ◽

R Language ◽

Powerful Analytical Tool ◽

Freshwater Bodies ◽

Biological Interpretation ◽

Analytical Protocols

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A reappraisal of mesenchymal-to-epithelial transition within the endometrium

10.32469/10355/88111 ◽

2021 ◽

Author(s):

◽

Madelyn Spooner

Keyword(s):

Flow Cytometry ◽

Epithelial Cells ◽

Estrogen Receptor Alpha ◽

Endometrial Adenocarcinoma ◽

Developmental Time ◽

Flow Cytometry Data ◽

Mesenchymal To Epithelial Transition ◽

Damage Repair ◽

Vaginal Cytology

A controversial topic, mesenchymal-epithelial transition (MET) is thought to be a mechanism involved in regeneration of the uterine epithelial layer following pregnancy and menstruation. Little is known about this process though, requiring further exploration. Previously, MET was thought to only occur as a damage-repair mechanism following parturition and menses-like events in mouse models. However, in the current study we hypothesized that MET would also occur in other endometrial epithelialization events as a mechanism of homeostatic epithelial turnover. To identify mesenchymal-derived cells within the adult uterine epithelium, an Amhr2-Cre; Rosa26-EYFP reporter mouse line was used. Mice were staged by vaginal cytology prior to the isolation of uterine epithelial and stromal cells, which were then stained with EpCAM to identify epithelial cells and analyzed by flow cytometry. EpCAM+YFP+ cells were identified in all stages of the estrous cycle except diestrus, indicating a proportion of epithelial cells were derived from the stroma (i.e., mesenchyme). Up to 80 percent of the uterine epithelia was EpCAM+YFP+ during estrogen-dominant stages (proestrus and estrus) of the cycle, with negligible amounts found during progesterone-dominant stages (metestrus and diestrus), suggesting this population may be responsive to estrogen. Uteri were also evaluated direct fluorescence and immunofluorescence in tissue sections. Immunofluorescence for EpCAM, forkhead box protein A2 (FOXA2), Ki67, estrogen receptor alpha (ESR1), and progesterone receptor (PGR) was performed to assess epithelial characteristics and potential functionality. To further investigate the role of MET in epithelialization and assess the temporal origin of mesenchymal-derived epithelial cells, we evaluated key postnatal (P) developmental time points using Amhr2-Cre; Rosa26-tTA; H2B-GFP reporter mice. Flow cytometry data indicated that MET may initially occur immediately after birth at P 0.5, with results varying from negligible amounts (0.21 percent) to approximately 82 percent. Similar results were found at P 3, but with decreasing variation; the highest EpCAM+GFP+ population representing approximately 50 percent of the epithelium. Between P 3 and early adenogenesis at P 8, this population decreased to average less than 2 percent. By the completion of adenogenesis initiation at P 14, approximately 10 percent of epithelial cells were EpCAM+GFP+, suggesting MET may occur during adenogenesis initiation and is maintained through P 21. Together, these results suggest that MET may be a more ubiquitous mechanism of epithelialization than originally thought and is likely hormone regulated. This research will help elucidate the role of MET in uterine epithelialization with potential for insights into dysregulation of MET in diseases such as endometrial adenocarcinoma and endometriosis.

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Immunomonitoring of Human Breast Milk Cells During HCMV-Reactivation

Frontiers in Immunology ◽

10.3389/fimmu.2021.723010 ◽

2021 ◽

Vol 12 ◽

Author(s):

Katrin Lazar ◽

Thorsten Kussmann ◽

Graham Pawelec ◽

Simone Pöschel ◽

Rangmar Goelz ◽

...

Keyword(s):

Flow Cytometry ◽

Breast Milk ◽

Human Breast Milk ◽

Mature Milk ◽

Phenotypic Properties ◽

Flow Cytometry Data ◽

Polychromatic Flow Cytometry ◽

Acetate Esterase ◽

Alpha Naphthyl Acetate Esterase

BackgroundBreast milk leukocytes may play a role in protecting the infant from pathogens. The dynamics and the role of lymphocytes in human cytomegalovirus (HCMV)-seropositive mothers shedding HCMV into breast milk during the first months postpartum (p.p.) are mostly unclear.MethodsBreast milk cells were analyzed by Pappenheim panoptic and alpha-naphthyl acetate esterase staining as well as by imaging and polychromatic flow cytometry to simultaneously establish their morphological and phenotypic properties. The latter were characterized in HCMV-seropositive and seronegative mothers´ breast milk cells at different time points p.p.ResultsPanoptic staining of breast milk cells revealed the presence of monocytes/macrophages, granulocytes and lymphocytes. Imaging flow cytometry data combining phenotypic and morphological analysis identified NKT-like cells, NK cells, epithelial cells, T cells and monocytes/macrophages. HCMV-seropositive but not -seronegative mothers had significantly higher T cell frequencies in mature milk.ConclusionsThe presence of lymphocyte subsets in breast milk may be more influenced by the HCMV-seropositivity of the mother than previously recognized.

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Exploring the Mechanism of Lingzhu San in Treating Febrile Seizures by Using Network Pharmacology

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207323666200902144348 ◽

2020 ◽

Vol 23 ◽

Author(s):

Xiao Zhou ◽

Xiao-Fei Zhang ◽

Dong-Yan Guo ◽

Yan-Jun Yang ◽

Lin Liu ◽

...

Keyword(s):

Febrile Seizures ◽

Network Pharmacology ◽

Biological Processes ◽

Active Compounds ◽

Potential Core ◽

R Language ◽

The Core ◽

Multiple Factors ◽

Therapeutic Mechanism

Objective: Lingzhu San (LZS) is a traditional Chinese medicine (TCM) prescription which can be effective in treating febrile seizures (FS) and has few researches on the mechanisms. In order to better guide the clinical use of LZS, we used the research ideas and methods of network pharmacology to find the potential core compounds, targets and pathways of LZS in the complex TCM system for the treatment of FS, and predict the mechanism. Materials and Methods: Databases such as BATMAN, TCMSP, TCMID, and SWISS TARGET are used to mine the active compounds and targets of LZS, and the target information of FS was obtained through GENECARDS and OMIM. Using Venny2.1.0 and Cytoscape software to locked the potential core compounds and targets of FS. The R language and ClusterProfiler software package were adopt to enrich and analyze the KEGG and GO pathways of the core targets and the biological processes and potential mechanisms of the core targets were revealed. Results: 187 active compounds and 2113 target proteins of LZS were collected. And 38 potential core compounds, 35 core targets and 775 metabolic and functional pathways were screened which involved in mediating FS. Finally, the role of the core compounds, targets and pivotal pathways of LZS regulated FS in the pathogenesis and therapeutic mechanism of FS was discussed and clarified. Conclusions: In this paper, the multi-compounds, multi-targets and multi-pathways mechanism of LZS in the treatment of FS was preliminarily revealed through the analysis of network pharmacology data, which is consistent with the principle of multi-compounds compatibility of TCM prescriptions and unified treatment of diseases from multiple angles, and it provides a new way for TCM to treat complex diseases caused by multiple factors.

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Exosomes from human umbilical cord mesenchymal stem cells attenuate the inflammation of severe steroid-resistant asthma by reshaping macrophage polarization

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02244-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bing Dong ◽

Chao Wang ◽

Jing Zhang ◽

Jinrong Zhang ◽

Yinuo Gu ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Quantitative Proteomics ◽

Macrophage Polarization ◽

Human Umbilical Cord ◽

Raw 264.7 ◽

Steroid Resistant ◽

Central Protein

Abstract Background Severe, steroid-resistant asthma (SSRA) is a serious clinical problem in asthma management. Affected patients have severe clinical symptoms, worsened quality of life, and do not respond to steroid, a mainstay steroid treatment of asthma. Thus, effective therapies are urgently needed. Exosomes derived from mesenchymal stem cell (MSC-Exo) has become attractive candidates for the lung inflammatory diseases through its immunomodulatory effects. In this study, we explored the therapeutic effects of MSC-Exo in SSRA and identified the therapeutic mechanism of MSC-Exo. Method Exosomes from human umbilical cord mesenchymal stem cell (hUCMSC) were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis and flow cytometry analysis. Effects of MSC-Exo on airway hyper responsiveness (AHR), inflammation, histopathology, and macrophage polarization in SSRA in mice were evaluated. Systematic depletion of macrophages determined the role of macrophages in the therapeutic effect of SSRA in mice. LPS-stimulated RAW 264.7 cell model was constructed to determine the underlying mechanism of MSC-Exo on macrophage polarization. qRT-PCR, Western blotting, immunofluorescence, and flow cytometry were performed to evaluate the expression of M1 or M2 markers. Tandem mass tags (TMT)-labeled quantitative proteomics were applied to explore the central protein during the regulation effect of MSC-Exo on macrophage polarization. Knockdown and overexpression of TRAF1 were used to further clarify the role of the central protein on macrophage polarization. Result We successfully isolated and characterized exosomes from hUCMSCs. We verified that the intratracheal administration of MSC-Exo reversed AHR, histopathology changes, and inflammation in SSRA mice. Systematic depletion of macrophages weakened the therapeutic effect of MSC-Exo. We found that MSC-Exo treatment inhibited M1 polarization and promoted M2 polarization in LPS-stimulated RAW 264.7 cells. Subsequently, tumor necrosis factor receptor-associated factor 1 (TRAF1) was determined as the central protein which may be closely related to the regulation of macrophage polarization from TMT-labeled quantitative proteomics analysis. Knockdown and overexpression of TRAF1 demonstrated that the effect of MSC-Exo treatment on macrophage polarization, NF-κB and PI3K/AKT signaling was dependent on TRAF1. Conclusion MSC-Exo can ameliorate SSRA by moderating inflammation, which is achieved by reshaping macrophage polarization via inhibition of TRAF1.

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The role of Sm2O3 on the structural, optical and spectroscopic properties of multi-component ternary borate glasses for orange-red emission applications

Journal of Non-Crystalline Solids ◽

10.1016/j.jnoncrysol.2020.120602 ◽

2021 ◽

Vol 554 ◽

pp. 120602

Author(s):

S. Hemalatha ◽

M. Nagaraja ◽

A. Madhu ◽

K. Suresh ◽

Srinatha N

Keyword(s):

Spectroscopic Properties ◽

Borate Glasses ◽

Red Emission

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THE ROLE OF MULTIPARAMETRIC FLOW CYTOMETRY IN DETECTING AND CHARACTERIZING GAMMA‐DELTA T‐CELL ENTITIES IN PERIPHERAL BLOOD. SINGLE‐CENTER CASE SERIES AND LITERATURE REVIEW

Hematological Oncology ◽

10.1002/hon.5_2881 ◽

2021 ◽

Vol 39 (S2) ◽

Author(s):

F Martín Moro ◽

I Martín Rubio ◽

R Alonso ◽

C López ◽

J Marquet ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Literature Review ◽

Peripheral Blood ◽

Case Series ◽

Single Center ◽

Gamma Delta ◽

Multiparametric Flow Cytometry ◽

Gamma Delta T Cell

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P0523PROTECTIVE ROLE OF REGULATORY B CELLS ON THE RENAL TISSUE REPAIRMEN AFTER ISCHEMIA REPERCUSSION INJURY

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0523 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Yokota Yunosuke ◽

Goh Kodama ◽

Sakuya Itou ◽

Yosuke Nakayama ◽

Nobukazu Komatsu ◽

...

Keyword(s):

Flow Cytometry ◽

Renal Function ◽

B Cells ◽

Interleukin 10 ◽

Renal Tissue ◽

Protective Role ◽

Regulatory B Cells ◽

Tubulointerstitial Injury ◽

Iri Model

Abstract Background and Aims Acute kidney injury (AKI), even if followed by renal recovery, is a risk factor for the future development of chronic kidney disease (CKD) and end- stage renal disease. It has been postulated that interleukin-10 (IL-10)-producing Regulatory B cells (Breg) play an important role for the tissue repairment in several tissues and organs. Basically, protective role of Breg has been reported in inflammatory bowel disease. In the kidney, it has been shown that IL-10 suppresses renal function decline and improves renal prognosis in IRI model, a typical model of AKI. However, the identity of Breg in the kidney and their origin have not been clarified. Further, how the Breg works during the transition from AKI to CKD is not known. Therefore, first we investigated whether Breg existed in renal tissue on the progression from AKI to CKD in IRI model mice. Further, we performed splenectomy, and examined the renal injury, Breg, and plasma IL-10 levels in this model. Method To examine the existence of Breg in the kidney of IRI model, we used 8-10 weeks-old GFP / IL-10 mice based on C57BL / 6J mice. They are reporter mice for IL-10 producing cells, and can visualize IL-10 producing cells under a fluorescence microscope without fluorescent immunostaining. We prepared following three groups, sham, IRI (unilateral), and IRI + SN (splenectomy) groups. Mice were anesthetized with chloral hydrate (4 g/kg,, intraperitoneal). After making a midline incision, exposed a blood vessel of the left renal pedicles and clamped it for 30 min by clips. one day, 7 days, and 14 days after the surgery, mice were sacrificed, and renal function and plasma IL-10 levels as well as tissue damages by PAS and Masson’s Trichrome staining were assessed. Tissue IL-10-producing cells were detected by flow cytometry. Results There was no difference of plasma IL-10 levels and renal tubulointerstitial injury in IRI group and IRI+SN group on day 1 after IRI. However, on day 7 and day 14, plasma IL-10 levels became gradually higher in IRI group, and SN decreased the increase in IL-10 levels. Tubulointerstitial injury was induced by IRI and SN further worsened tubular damages. Serum Cr and BUN levels were not different in three groups due to normal right kidney. On day 1, number of IL-10-producing B cells increased in the spleen and renal medulla in IRI group confirmed by flow cytometry, which was completely diminished by SN, suggesting that origin of the infiltrated Breg might be spleen, thereby being involved in the protective role in IRI injury in the kidney. Conclusion We report for the first time that Breg might be recruited from spleen by AKI, which may be one of the mechanisms to prevent the progression to CKD.

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Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00527.2016 ◽

2017 ◽

Vol 313 (5) ◽

pp. L899-L915 ◽

Cited By ~ 8

Author(s):

Fumiaki Kato ◽

Seiichiro Sakao ◽

Takao Takeuchi ◽

Toshio Suzuki ◽

Rintaro Nishimura ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cell ◽

Vascular Remodeling ◽

Time Dependent ◽

Causative Role ◽

Dependent Manner ◽

Vascular Endothelial ◽

Pulmonary Vascular Remodeling ◽

Pulmonary Arterial

Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of cross talk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.

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