Fraction Of Subpopulation Research Articles

Tetrahedral DNA-Based Ternary Recognition Ratiometric Fluorescent Probes for Real-Time In Situ Resolving Lysosome Subpopulations in Living Cells via Cl-, Ca2+, and pH.

Lysosomes are multifunctional organelles vital for cellular homeostasis with distinct subpopulations characterized by varying levels of Cl-, Ca2+, and H+. In situ visualization of these parameters is crucial for lysosomal research, yet developing probes that can simultaneously detect multiple ions remains challenging. Herein, we developed a lysosome-targeting ternary recognition ratiometric fluorescent probe based on tetrahedral DNA nanostructures (TDNs) to analyze lysosome subpopulations by Cl-, Ca2+, and pH. The TDN probe is assembled from four single-stranded DNAs, each end-modified with responsive fluorophores (Pr-Cl for Cl-, Pr-Ca for Ca2+, and Pr-pH for pH) or a reference fluorophore (Cy5). The fluorophores are integrated at the vertices of the rigid TDN to minimize mutual interference, and their fixed stoichiometry establishes a robust ternary recognition ratiometric fluorescence sensor for in situ resolution of lysosome subpopulations in living cells. Accordingly, a rise in lysosome subpopulations 2/6 characterized by low [Cl-], medium/high [Ca2+], and high pH was observed in the Niemann-Pick disease model cells but seldom observed in the control group. Conversely, there was a marked decline in the fraction of subpopulations 1/4/5 characterized by high [Cl-], medium to low [Ca2+], and pH. These changes were substantially reversed upon treatment. The probe holds great promise for studying lysosome subpopulations and the diagnosis and treatment of related diseases.

Percoll discontinuous density gradient centrifugation method for the fractionation of the subpopulations of Mycobacterium smegmatis and Mycobacterium tuberculosis from in vitro cultures

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis.•The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density.•The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis.•Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.

Abstract 4712: Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized

Abstract A close association between active fibroblastic foci (desmoplasia) and distant metastasis in the lung adenocarcinoma (LUAD) has been established decades ago and yet, the pro- and anti-tumor properties of cancer-associated fibroblasts (CAFs) are still being resolved in the hope of preventing LUAD progression. To better understand the role of stromal cells in progression, there is a growing interest in 3D patient-derived models for their translational and throughput advantages. In parallel, the emergence of multiplexed spatial biology platforms is advancing translational oncology forward by providing a more global understanding of the tumor microenvironment (TME). Despite the enthusiasm for these new technologies, there are no study showing the applicability and significance of using multiplexed spatial biology to characterized 3D patient-derived models. Here, we modeled spatial heterogeneity by assembling EGFR mutated patient-derived organoids (PDOs) with fibroblasts harvested from the tumor edge (tumor-adjacent fibroblasts; TAFs) or tumor core (tumor core fibroblasts; TCFs) referred here as tumor-stroma assembloids. Using these assembloids, we generated an extensive dataset with CODEX imaging technology and used a quantitative approach to dissect tumor-stroma crosstalk. Our results show that TAFs expressing the myofibroblast marker aSMA were strictly found at the periphery of the hydrogel dome containing the assembloid, while aSMA- fibroblasts were found strictly in the center. In contrast, we did not observe a similar spatial organization in TCF assembloids. Next, we used CELESTA, an unsupervised machine learning algorithm that uses prior knowledge of the cell expression and spatial information to dissect cell heterogeneity, then performed co-localization analyses. We also leveraged the EGFR mutation status of the PDOs and observed the protective effect of the fibroblasts from erlotinib. Both assembloid conditions showed drastically different tumor-stroma spatial changes including gains and losses of colocalizations even though erlotinib did not significantly change the subpopulation fractions, cell heterogeneity, nor cell density. The top tumor-stroma colocalization pairs that persisted after erlotinib involved either myofibroblasts and PanCK-Vim+MUC1+ cancer cells or were found in the center of the assembloids, shielded by a stromal barrier of myofibroblasts. Taken together, our results show that the assembloids undergo drastic spatial reorganization following erlotinib treatment even though subpopulation fractions and cell heterogeneity remain unchanged. This study provided new insights on tumor-stroma crosstalk in the LUAD and motivates the use 3D patient-derived models coupled with multiplex imaging to answer complex spatial biology questions. Citation Format: Gina Bouchard, Irene Li, Weiruo Zhang, Sylvia Plevritis. Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4712.

Automated On-Line Isolation and Fractionation Method for Subpopulations of Extracellular Vesicles.

Immunoaffinity chromatography (IAC) with selective antibodies immobilized on polymeric monolithic disk columns enables selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) can be used for further fractionation of relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Here we describe how the isolation and fractionation of subpopulations of extracellular vesicles can be achieved without the presence of lipoproteins using on-line coupled IAC-AsFlFFF. With the developed methodology, it is possible to have fast, reliable, and reproducible automated isolation and fractionation of challenging biomacromolecules from human plasma with a high purity and high yields of subpopulations.

An autonomous microchip for real-time, label-free immune cell analysis

Characterization of cell populations and identification of distinct subtypes based on surface markers are needed in a variety of applications from basic research and clinical assays to cell manufacturing. Conventional immunophenotyping techniques such as flow cytometry or fluorescence microscopy require immunolabeling of cells, expensive and complex instrumentation, skilled operators, and are therefore incompatible with field deployment and automated cell manufacturing systems. In this work, we introduce an autonomous microchip that can electronically quantify the immunophenotypical composition of a cell suspension. Our microchip identifies different cell subtypes by capturing each in different microfluidic chambers functionalized against the markers of the target populations. All on-chip activity is electronically monitored by an integrated sensor network, which informs an algorithm determining subpopulation fractions from chip-wide immunocapture statistics in real time. Moreover, optimal operational conditions within the chip are enforced through a closed-loop feedback control on the sensor data and the cell flow speed, and hence, the antibody-antigen interaction time is maintained within its optimal range for selective immunocapture. We apply our microchip to analyze a mixture of unlabeled CD4+ and CD8+ T cell sub-populations and then validated the results against flow cytometry measurements. The demonstrated capability to quantitatively analyze immune cells with no labels has the potential to enable not only autonomous biochip-based immunoassays for remote testing but also cell manufacturing bioreactors with built-in, adaptive quality control.

A Panoply of Rheumatological Manifestations in Patients with GATA2 Deficiency

Purpose: To characterize rheumatological manifestations of GATA2 deficiency. Methods: Single-center, retrospective review of 157 patients with GATA2 deficiency. Disease course, laboratory results, and imaging findings were extracted. In-person rheumatological assessments were performed on selected, available patients. A literature search of four databases was conducted to identify additional cases. Results: Rheumatological findings were identified in 28 patients, out of 157 cases reviewed (17.8%). Twenty-two of those patients (78.6%) reported symptom onset prior to or in conjunction with the molecular diagnosis of GATA2 deficiency. Notable rheumatological manifestations included: piezogenic pedal papules (PPP), joint hyperextensibility, early onset osteoarthritis, ankylosing spondylitis, and seronegative erosive rheumatoid arthritis. In peripheral blood of patients with rheumatological manifestations and GATA2 deficiency, CD4+ CD3+ helper T cells and naïve CD3+ CD4+ CD62L+ CD45RA+ helper T cell subpopulation fractions were significantly lower, while CD8+ cytotoxic T cell fractions were significantly higher, compared to those without rheumatological manifestations and with GATA2 deficiency. No changes in CD19, CD3, or NK populations were observed. Conclusion: GATA2 deficiency is associated with a broad spectrum of rheumatological disease manifestations. Low total helper T lymphocyte proportions and low naïve helper T cell proportions are associated with those most at risk of overt rheumatological manifestations. Further, PPP and joint hyperextensibility may explain some of the nonimmunologically-mediated joint problems encountered in patients with GATA2 deficiency. This catalogue suggests that rheumatological manifestations and immune dysregulation are relatively common in GATA2 deficiency.

Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images

Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro- and eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell counting by flow cytometry is often limited, for example when working with cells rigidly adhered either to each other or to external surfaces like bacterial biofilms or adherent cell lines and tissue samples. An alternative approach is provided by using fluorescent microscopy and confocal laser scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a given sample. For the quantitative assessment of cell fractions in microphotographs, we suggest a simple two-step algorithm that combines single cells selection and the statistical analysis. To facilitate the first step, we suggest a simple procedure that supports finding the balance between the detection threshold and the typical size of single cells based on objective cell size distribution analysis. Based on a series of experimental measurements performed on bacterial and eukaryotic cells under various conditions, we show explicitly that the suggested approach effectively accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) in all studied cases that are in good agreement with manual cell counting on microphotographs and flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive and user-friendly graphical interface for the initial adjustment of algorithm parameters to the microphotographs analysis as well as for the sequential analysis of homogeneous series of similar microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is freely available online at https://bitbucket.org/rogex/biofilmanalyzer/downloads/.

The experimental clinical substantiation of treatment of patients with odontogenic phlegmon of maxillofacial area using Dаlargin in complex therapy.

The patients with odontogenic phlegmon of maxillofacial area suffer from immune depression at the surgical stage of treatment that can unfavorably affect their rehabilitation. The study analyzes possibility of application of medication Dalargin in complex treatment of patients during post-operation period. The experimental study established components of immune system affected by medication. Thereupon, Dalargin was applied in clinical practice of treatment of patients with course of disease of average severity. The study proved immunomodulatory characteristics of Dalargin effecting various fractions of sub-populations of lymphocytes and manifesting by way of decreasing of initially high indices and increasing of initially low indices that is of great importance during treatment of patients with odontogenic hormones, especially in case of severe course of disease.

Robust model-based analysis of single-particle tracking experiments with Spot-On.

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce 'Spot-On', an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

Single Amino Acid Variant Profiles of Subpopulations in the MCF-7 Breast Cancer Cell Line.

Cancers are initiated and developed from a small population of stem-like cells termed cancer stem cells (CSCs). There is heterogeneity among this CSC population that leads to multiple subpopulations with their own distinct biological features and protein expression. The protein expression and function may be impacted by amino acid variants that can occur largely due to single nucleotide changes. We have thus performed proteomic analysis of breast CSC subpopulations by mass spectrometry to study the presence of single amino acid variants (SAAVs) and their relation to breast cancer. We have used CSC markers to isolate pure breast CSC subpopulation fractions (ALDH+ and CD44+/CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF-7 breast cancer cell line. By searching the Swiss-CanSAAVs database, 374 unique SAAVs were identified in total, where 27 are cancer-related SAAVs. 135 unique SAAVs were found in the CSC population compared with the mature luminal cells. The distribution of SAAVs detected in MCF-7 cells was compared with those predicted from the Swiss-CanSAAVs database, where we found distinct differences in the numbers of SAAVs detected relative to that expected from the Swiss-CanSAAVs database for several of the amino acids.

Chronic effects of temperature on mortality in the Southeastern USA using satellite-based exposure metrics.

Climate change may affect human health, particularly for elderly individuals who are vulnerable to temperature changes. While many studies have investigated the acute effects of heat, only a few have dealt with the chronic ones. We have examined the effects of seasonal temperatures on survival of the elderly in the Southeastern USA, where a large fraction of subpopulation resides. We found that both seasonal mean temperature and its standard deviation (SD) affected long-term survival among the 13 million Medicare beneficiaries (aged 65+) in this region during 2000–2013. A 1 °C increase in summer mean temperature corresponded to an increase of 2.5% in death rate. Whereas, 1 °C increase in winter mean temperature was associated with a decrease of 1.5%. Increases in seasonal temperature SD also influence mortality. We decomposed seasonal mean temperature and its temperature SD into long-term geographic contrasts between ZIP codes and annual anomalies within ZIP code. Effect modifications by different subgroups were also examined to find out whether certain individuals are more vulnerable. Our findings will be critical to future efforts assessing health risks related to the future climate change.

A simple separation method for downstream biochemical analysis of aquatic microbes

In order to study the chemical composition of aquatic microbes it is necessary to obtain completely separated fractions of subpopulations. Size separation by filtration is usually unsuccessful because the smaller group of organisms contaminates the larger fractions due to being trapped on filter surfaces of nominally much larger pore sizes. Here we demonstrate that a simple sucrose density separation method allowed us to separate microorganisms of even subtle size differences and to determine their bulk biochemical composition (proteins, polysaccharides+nucleic acids, and lipids). Both autotrophs and heterotrophs (through anaplerotic pathways) were labeled with 14C-bicarbonate for biochemical fractionation. We provided proof of concept that eukaryotic microbes could be cleanly separated from prokaryotes in cultures and in field samples, enabling detection of differences in their biochemical makeup. We explored methodological issues regarding separation mechanisms, fixation, and pre-concentration via tangential flow filtration of oligotrophic marine waters where abundances of microorganisms are comparably low. By selecting an appropriate centrifugal force, two processes (i.e., isopycnal and rate–zonal separation) can be exploited simultaneously resulting in finely-separated density fractions, which also resulted in size separation. Future applications of this method include exploration of the stoichiometric, biochemical and genetic differences among subpopulations of microbes in a wide variety of aquatic environments.

Quantitative dielectrophoretic tracking for characterization and separation of persistent subpopulations of Cryptosporidium parvum

Microbial persistence to antibiotics is attributed to subpopulations with phenotypic variations that cause a spread of susceptibility levels, leading to the recurrence of infections and stability of biofilms. Herein, persistent oocyst subpopulations identified by animal infectivity and excystation assays during the disinfection of Cryptosporidium parvum, a water-borne pathogen capable of causing enteric infections at ultra-low doses, are separated and characterized by quantitative dielectrophoretic tracking over a wide frequency range (10 kHz-10 MHz). To enable the simultaneous and facile dielectrophoretic tracking of individual oocysts, insulator constrictions in a microfluidic channel are utilized to spatially modulate the localized field over the extent needed for defining oocyst trajectories and for obtaining high-resolution displacement versus time measurements under both, positive and negative dielectrophoresis. In this manner, by obviating the need for averaging dielectrophoretic data over a large collection region, the force response is more sensitive to differences in electrophysiology from sub-population fractions. Hence, the electrophysiology of sensitive and persistent oocysts after heat and silver nanoparticle treatments can be quantified by correlating the force response at low frequencies (<100 kHz) to the integrity of the oocyst wall and at high frequencies (0.4-1 MHz) to the sporozoites in the oocyst. This label-free method can characterize heterogeneous microbial samples with subpopulations of phenotypically different alterations, for quantifying the intensity of alteration and fraction with a particular alteration type.

Mapping the Environmental Fitness Landscape of a Synthetic Gene Circuit

Gene expression actualizes the organismal phenotypes encoded within the genome in an environment-dependent manner. Among all encoded phenotypes, cell population growth rate (fitness) is perhaps the most important, since it determines how well-adapted a genotype is in various environments. Traditional biological measurement techniques have revealed the connection between the environment and fitness based on the gene expression mean. Yet, recently it became clear that cells with identical genomes exposed to the same environment can differ dramatically from the population average in their gene expression and division rate (individual fitness). For cell populations with bimodal gene expression, this difference is particularly pronounced, and may involve stochastic transitions between two cellular states that form distinct sub-populations. Currently it remains unclear how a cell population's growth rate and its subpopulation fractions emerge from the molecular-level kinetics of gene networks and the division rates of single cells. To address this question we developed and quantitatively characterized an inducible, bistable synthetic gene circuit controlling the expression of a bifunctional antibiotic resistance gene in Saccharomyces cerevisiae. Following fitness and fluorescence measurements in two distinct environments (inducer alone and antibiotic alone), we applied a computational approach to predict cell population fitness and subpopulation fractions in the combination of these environments based on stochastic cellular movement in gene expression space and fitness space. We found that knowing the fitness and nongenetic (cellular) memory associated with specific gene expression states were necessary for predicting the overall fitness of cell populations in combined environments. We validated these predictions experimentally and identified environmental conditions that defined a “sweet spot” of drug resistance. These findings may provide a roadmap for connecting the molecular-level kinetics of gene networks to cell population fitness in well-defined environments, and may have important implications for phenotypic variability of drug resistance in natural settings.

Subpopulation-Specific Metabolic Pathway Usage in Mixed Cultures as Revealed by Reporter Protein-Based 13 C Analysis

Most large-scale biological processes, like global element cycling or decomposition of organic matter, are mediated by microbial consortia. Commonly, the different species in such consortia exhibit mutual metabolic dependencies that include the exchange of nutrients. Despite the global importance, surprisingly little is known about the metabolic interplay between species in particular subpopulations. To gain insight into the intracellular fluxes of subpopulations and their interplay within such mixed cultures, we developed here a (13)C flux analysis approach based on affinity purification of the recombinant fusion glutathione S-transferase (GST) and green fluorescent protein (GFP) as a reporter protein. Instead of detecting the (13)C labeling patterns in the typically used amino acids from the total cellular protein, our method detects these (13)C patterns in amino acids from the reporter protein that has been expressed in only one species of the consortium. As a proof of principle, we validated our approach by mixed-culture experiments of an Escherichia coli wild type with two metabolic mutants. The reporter method quantitatively resolved the expected mutant-specific metabolic phenotypes down to subpopulation fractions of about 1%.

Flow-Based Cytometric Analysis of Cell Cycle via Simulated Cell Populations

We present a new approach to the handling and interrogating of large flow cytometry data where cell status and function can be described, at the population level, by global descriptors such as distribution mean or co-efficient of variation experimental data. Here we link the “real” data to initialise a computer simulation of the cell cycle that mimics the evolution of individual cells within a larger population and simulates the associated changes in fluorescence intensity of functional reporters. The model is based on stochastic formulations of cell cycle progression and cell division and uses evolutionary algorithms, allied to further experimental data sets, to optimise the system variables. At the population level, the in-silico cells provide the same statistical distributions of fluorescence as their real counterparts; in addition the model maintains information at the single cell level. The cell model is demonstrated in the analysis of cell cycle perturbation in human osteosarcoma tumour cells, using the topoisomerase II inhibitor, ICRF-193. The simulation gives a continuous temporal description of the pharmacodynamics between discrete experimental analysis points with a 24 hour interval; providing quantitative assessment of inter-mitotic time variation, drug interaction time constants and sub-population fractions within normal and polyploid cell cycles. Repeated simulations indicate a model accuracy of ±5%. The development of a simulated cell model, initialized and calibrated by reference to experimental data, provides an analysis tool in which biological knowledge can be obtained directly via interrogation of the in-silico cell population. It is envisaged that this approach to the study of cell biology by simulating a virtual cell population pertinent to the data available can be applied to “generic” cell-based outputs including experimental data from imaging platforms.

Invasion Threshold in Heterogeneous Metapopulation Networks

We study the dynamics of epidemic and reaction-diffusion processes in metapopulation models with heterogeneous connectivity patterns. In susceptible-infected-removed-like processes, along with the standard local epidemic threshold, the system exhibits a global invasion threshold. We provide an explicit expression of the threshold that sets a critical value of the diffusion/mobility rate below, which the epidemic is not able to spread to a macroscopic fraction of subpopulations. The invasion threshold is found to be affected by the topological fluctuations of the metapopulation network. The results presented provide a general framework for the understanding of the effect of travel restrictions in epidemic containment.

Effectiveness of population recovery projects based on captive breeding

A recovery programme for a population of Apollo butterflies in the Pieniny National Park was monitored over 12 years in order to assess its effectiveness. The programme was based on population ecology and metapopulation theory, and its object was to replenish wild populations with captive-reared individuals. Thanks to the adopted measures, the local population initially numbering only 30–50 individuals increased to ca. 1000 imagines, forming a functional metapopulation. Nevertheless, analysis of population dynamics parameters indicated that only some of the subpopulations were stable; a significant percentage of the subpopulation fractions represented sink populations whose survival depended either on an external supply of captive-reared individuals or on individuals from other source subpopulations. In some cases, intensively supplying the subpopulation with captive-reared individuals resulted in a decrease of that deme’s abundance. The results suggest that supplying captive-reared individuals to an endangered population may entail some risks and should be done with caution.

Large area multilayered electrode arrays for dielectrophoretic fractionation

A technique is presented for the fabrication of large-area (20×20mm) multilayered electrode structures with 10μm feature size. This technique has been used in the fabrication of 1000-electrode arrays powered by a series of interconnected bus-bars with quadrature-phased AC power supplies. These electrodes generate a travelling electric field which induces an asynchronous electrostatic linear force - Travelling Wave Dielectrophoresis - which can propel particles of micron to submicron scale and can be used for fractionation of subpopulations of such particles according to their dielectric properties.

Separation of T and B lymphocytes from human peripheral blood mononuclear cells using density perturbation methods

The fractionation of sub-populations of peripheral blood mononuclear cells has become an essential routine procedure and some of the main fractionation methods used today are immunomagnetic separations. We describe a less expensive method for the separation of subpopulations of mononuclear cells using density perturbation, which uses the binding of antibody-coated dense polystyrene beads to increase the density of specific sub-populations of cells. By incubating a total mononuclear fraction from human peripheral blood together with antibody-coated beads, in a commercially-available lymphocyte separation medium (Nycoprep 1.077), a depletion of 94.9 ± 1.68% of the T cells could be obtained by this procedure; a depletion of 69.7 ± 1.78% of the B cells was also achieved. These results indicate the potential for the separation of different sub-populations of peripheral blood mononuclear cells on the basis of the immunological identity of the surface of cells using density perturbation methods involving antibody-coated dense polystyrene beads.