Changes In Cell Abundance Research Articles

Blood-based molecular and cellular biomarkers of early response to neoadjuvant PD-1 blockade in patients with non-small cell lung cancer

BackgroundDespite the improved survival observed in PD-1/PD-L1 blockade therapy, a substantial proportion of cancer patients, including those with non-small cell lung cancer (NSCLC), still lack a response.MethodsTranscriptomic profiling was conducted on a discovery cohort comprising 100 whole blood samples, as collected multiple times from 48 healthy controls (including 43 published data) and 31 NSCLC patients that under treatment with a combination of anti-PD-1 Tislelizumab and chemotherapy. Differentially expressed genes (DEGs), simulated immune cell subsets, and germline DNA mutational markers were identified from patients achieved a pathological complete response during the early treatment cycles. The predictive values of mutational markers were further validated in an independent immunotherapy cohort of 1661 subjects, and then confirmed in genetically matched lung cancer cell lines by a co-culturing model.ResultsThe gene expression of hundreds of DEGs (FDR p < 0.05, fold change < -2 or > 2) distinguished responders from healthy controls, indicating the potential to stratify patients utilizing early on-treatment features from blood. PD-1-mediated cell abundance changes in memory CD4 + and regulatory T cell subset were more significant or exclusively observed in responders. A panel of top-ranked genetic alterations showed significant associations with improved survival (p < 0.05) and heightened responsiveness to anti-PD-1 treatment in patient cohort and co-cultured cell lines.ConclusionThis study discovered and validated peripheral blood-based biomarkers with evident predictive efficacy for early therapy response and patient stratification before treatment for neoadjuvant PD-1 blockade in NSCLC patients.

Seasonal changes in cell size of the dominant diatom species in Lake Biwa

We examined the seasonal changes in cell size and abundance of the dominant diatoms Aulacoseira ambigua f. japonica, Aulacoseira granulata, and Fragilaria crotonensis, together with environmental variables, in the northern basin of Lake Biwa. The cell size of the diatoms decreased gradually with the increase in cell abundance, indicating that the population underwent vegetative reproduction followed by sexual reproduction. The observation revealed two diatom blooms in a year. The autumn bloom from early October to late November was primarily dominated by A. granulata, A. ambigua f. japonica, and F. crotonensis. The minimum cell size of the diatoms recorded was 30, 20, and 75 μm, respectively. F. crotonensis caused another bloom from late March to early May; the minimum cell size recorded was 68 μm. These results suggested that F. crotonensis undergoes sexual reproduction twice a year, while the other two diatom species undergo sexual reproduction once a year. Correlation matrices and redundancy analysis were performed to explore the relationship of various environmental factors to cell size reduction and restoration of the seasonally dominant diatoms, and it is suggested that water temperature and/or concentration of dissolved inorganic nitrogen are critical environmental variables for seasonal changes in cell abundance and/or size of the dominant diatom species. Our results demonstrated that each diatom species had distinct favorable ecological conditions for staging its life cycle.

A cell abundance analysis based on efficient PAM clustering for a better understanding of the dynamics of endometrial remodelling

BackgroundSingle-cell RNA sequencing (scRNA-seq) is a powerful tool for investigating cell abundance changes during tissue regeneration and remodeling processes. Differential cell abundance supports the initial clustering of all cells; then, the number of cells per cluster and sample are evaluated, and the dependence of these counts concerning the phenotypic covariates of the samples is studied. Analysis heavily depends on the clustering method. Partitioning Around Medoids (PAM or k-medoids) represents a well-established clustering procedure that leverages the downstream interpretation of clusters by pinpointing real individuals in the dataset as cluster centers (medoids) without reducing dimensions. Of note, PAM suffers from high computational costs and memory requirements.ResultsThis paper proposes a method for differential abundance analysis using PAM as a clustering method and negative binomial regression as a statistical model to relate covariates to cluster/cell counts. We used this approach to study the differential cell abundance of human endometrial cell types throughout the natural secretory phase of the menstrual cycle. We developed a new R package -scellpam-, that incorporates an efficient parallel C++ implementation of PAM, and applied this package in this study. We compared the PAM-BS clustering method with other methods and evaluated both the computational aspects of its implementation and the quality of the classifications obtained using distinct published datasets with known subpopulations that demonstrate promising results.ConclusionsThe implementation of PAM-BS, included in the scellpam package, exhibits robust performance in terms of speed and memory usage compared to other related methods. PAM allowed quick and robust clustering of sets of cells with a size ranging from 70,000 to 300,000 cells. https://cran.r-project.org/web/packages/scellpam/index.html. Finally, our approach provides important new insights into the transient subpopulations associated with the fertile time frame when applied to the study of changes in the human endometrium during the secretory phase of the menstrual cycle.

Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.

IDENTIFICATION AND INVESTIGATION OF SENESCENT CELLS IN SKELETAL MUSCLE AGING

Abstract Skeletal muscle aging is marked by the loss and atrophy of resident fibers, and the accumulation of functionally diverse cell types including fibroblasts, adipocytes, and immune cells. Senescent cells amass in multiple tissues with advancing age where they contribute to aging, chronic disease, and physical decline. The role of senescence in mediating muscle aging has become a popular and sometimes contentious topic. However, to date, this concept has not been methodically tested. In this study, we characterized the changes in cell abundance and, importantly, cell-specific transcriptional profiles with skeletal muscle aging using scRNAseq. Interestingly, we identified a small population of p16 positive fibro-adipogenic progenitors (FAPs) which, upon further investigation using immunohistochemical methods, were found to express other senescence markers. This subpopulation of FAPs did not exhibit elevation in p21 levels with age. Instead, terminally differentiated myofibers were the source of the p21 increase. Myofibers with high p21 expression exhibit a strong inflammatory phenotype, which includes activated p53 signaling pathways together with strong cytokine-cytokine receptor interactions. We further identified large amounts of cross-talk between different cell types, suggesting that senescent FAPs and myofibers could contribute to skeletal muscle aging in a paracrine manner. Importantly, these observations in mice were confirmed in human samples, suggesting the strong translational power of these findings.

IDENTIFICATION AND INVESTIGATION OF SENESCENT CELLS IN SKELETAL MUSCLE AGING

Abstract Skeletal muscle aging is marked by the loss and atrophy of resident fibers, and the accumulation of functionally diverse cell types including fibroblasts, adipocytes, and immune cells. Senescent cells amass in multiple tissues with advancing age where they contribute to aging, chronic disease, and physical decline. The role of senescence in mediating muscle aging has become a popular and sometimes contentious topic. However, to date, this concept has not been methodically tested. In this study, we characterized the changes in cell abundance and, importantly, cell-specific transcriptional profiles with skeletal muscle aging using scRNAseq. Interestingly, we identified a small population of p16 positive fibro-adipogenic progenitors (FAPs) which, upon further investigation using immunohistochemical methods, were found to express other senescence markers. This subpopulation of FAPs did not exhibit elevation in p21 levels with age. Instead, terminally differentiated myofibers were the source of the p21 increase. Myofibers with high p21 expression exhibit a strong inflammatory phenotype, which includes activated p53 signaling pathways together with strong cytokine-cytokine receptor interactions. We further identified large amounts of cross-talk between different cell types, suggesting that senescent FAPs and myofibers could contribute to skeletal muscle aging in a paracrine manner. Importantly, these observations in mice were confirmed in human samples, suggesting the strong translational power of these findings.

IDENTIFICATION AND INVESTIGATION OF SENESCENT CELLS IN SKELETAL MUSCLE AGING

Abstract Skeletal muscle aging is marked by the loss and atrophy of resident fibers, and the accumulation of functionally diverse cell types including fibroblasts, adipocytes, and immune cells. Senescent cells amass in multiple tissues with advancing age where they contribute to aging, chronic disease, and physical decline. The role of senescence in mediating muscle aging has become a popular and sometimes contentious topic. However, to date, this concept has not been methodically tested. In this study, we characterized the changes in cell abundance and, importantly, cell-specific transcriptional profiles with skeletal muscle aging using scRNAseq. Interestingly, we identified a small population of p16 positive fibro-adipogenic progenitors (FAPs) which, upon further investigation using immunohistochemical methods, were found to express other senescence markers. This subpopulation of FAPs did not exhibit elevation in p21 levels with age. Instead, terminally differentiated myofibers were the source of the p21 increase. Myofibers with high p21 expression exhibit a strong inflammatory phenotype, which includes activated p53 signaling pathways together with strong cytokine-cytokine receptor interactions. We further identified large amounts of cross-talk between different cell types, suggesting that senescent FAPs and myofibers could contribute to skeletal muscle aging in a paracrine manner. Importantly, these observations in mice were confirmed in human samples, suggesting the strong translational power of these findings.

P033 Single-cell analysis of gut mucosal- and peripheral blood cells in ulcerative colitis patients undergoing vedolizumab treatment

Abstract Background Vedolizumab (VDZ), a monoclonal antibody that targets α4β7 integrin, was approved to treat moderate-to-severe ulcerative colitis (UC) based on the presumption that it blocks T cell recruitment to the inflamed intestinal mucosa. The clinical evidence suggests that up to 50% of UC patients do not achieve disease remission under VDZ treatment. This study aims to identify changes in cell abundances and molecular pathways associated with VDZ response in UC. To this end, we included anti-tumor necrosis factor (anti-TNF)-naïve and anti-TNF-exposed patients with active UC, and utilized single-cell RNA sequencing (scRNAseq) and high-dimensional flow cytometry (Cytek) to assess the peripheral blood and the gut mucosal compartments. Methods Gut mucosal biopsies from inflamed and non-inflamed regions, and peripheral blood mononuclear cells (PBMCs) were obtained from UC patients 2 wks before (t0) and 14 wks after (t4) the start of VDZ administration. Response to treatment was prospectively evaluated based on endoscopic assessment (defined as a decrease in total Mayo score between t0 and t4) and physician global assessment (PGA) that incorporates disease activity score and biochemical measurements. Results A total of 25 UC patients (pts) were included: 44% anti-TNF-naïve. Endoscopic response to VDZ was observed in 32% of UC pts, while 56% of pts showed response based on PGA. The VDZ response rate (by PGA) was higher in anti-TNF-naïve pts vs anti-TNF-exposed pts (82% vs 36% responders, respectively). A preliminary analysis was performed on samples from 8 (out of 25) UC pts, profiling &amp;gt;70,000 gut mucosal cells and &amp;gt;25,000 PBMCs. Within the mucosal compartment, at t0 we identified immune cells (50% of all captured cells), stromal cells (10%), and epithelial cells (40%). Upon inflammation, the proportion of immune cells increased to 70%, stromal cells to 20%, while epithelial cells depleted to 10%. Notably, all main identified immune cell lineages – T cells, B cells and myeloid cells – contributed to the expansion of the immune cell compartment in inflamed mucosa. In line with scRNAseq data, we identified all major immune cell populations and detected expression of both the classic gut-directed and the redundant trafficking integrins by Cytek. Conclusion The preliminary results substantiate our current understanding of VDZ biology in UC. We confirm that anti-TNF-naïve pts have a higher response rate to VDZ vs anti-TNF-exposed pts. With this unique cohort, our study has the power to further explore molecular mechanisms and pathways that underlie VDZ response at the single-cell level.

Phytoplankton growth at low temperatures: results from low temperature incubations

Abstract The response of phytoplankton to different temperatures has been studied for decades, and although the general relationship of growth to temperature is known to be exponential, the responses at very low temperatures (from −2 to +2°C) are poorly constrained. To address this uncertainty, we conducted a series of growth experiments with eight species of polar phytoplankton; each was grown in batch culture at temperatures ranging from −1.5 to 4°C, and growth rates determined from changes in cell abundance and chlorophyll. We found that different species responded differently to temperature, although the general response was an increase in growth rate at higher temperatures. The mean response from all taxa was less (ca. 35% less) than predicted from the classic [Eppley, R. W. (1972) Temperature and phytoplankton growth in the sea. Fish. Bull., 7, 1063–1085.] formulation. Maximum growth rates also were most reliable when determined from changes in cell abundance. If the variability in maximum growth rates among taxa is reflective of in situ responses to polar temperature increases, it will be challenging to predict the assemblage response to elevated in situ temperatures in future years.

The dynamics of cable bacteria colonization in surface sediments: a 2D view

Cable bacteria that are capable of transporting electrons on centimeter scales have been found in a variety of sediment types, where their activity can strongly influence diagenetic reactions and elemental cycling. In this study, the patterns of spatial and temporal colonization of surficial sediment by cable bacteria were revealed in two-dimensions by planar pH and H2S optical sensors for the first time. The characteristic sediment surface pH maximum zones begin to develop from isolated micro-regions and spread horizontally within 5 days, with lateral spreading rates from 0.3 to ~ 1.2 cm day−1. Electrogenic anodic zones in the anoxic sediments are characterized by low pH, and the coupled pH minima also expand with time. H2S heterogeneities in accordance with electrogenic colonization are also observed. Cable bacteria cell abundance in oxic surface sediment (0–0.25 cm) kept almost constant during the colonization period; however, subsurface cell abundance apparently increased as electrogenic activity expanded across the entire surface. Changes in cell abundance are consistent with filament coiling and growth in the anodic zone (i.e., cathodic snorkels). The spreading mechanism for the sediment pH–H2S fingerprints and the cable bacteria abundance dynamics suggest that once favorable microenvironments are established, filamentous cable bacteria aggregate or locally activate electrogenic metabolism. Different development dynamics in otherwise similar sediment suggests that the accessibility of reductant (e.g., dissolved phase sulfide) is critical in controlling the growth of cable bacteria.

Comparison of Rapid Methods Used to Determine the Concentration, Size Structure and Species Composition of Algae

Traditionally, the abundance, cell size distribution and species identification of algae are determined by microscopic counts. In recent years, various rapid methods have been developed for routine algal studies. However, each of these methods has its drawbacks. It is important for aquatic ecologists to understand the advantages, disadvantages, and limitations of these methods. We compared the sensitivity of three rapid methods (multichannel fluorimeter FluoroProbe, imaging flow cytometer FlowCam, and CASY particle counter) to changes in cell abundance of three algae species (Chlorella vulgaris Beyerinck, Arthrospira platensis Gomont, and Nostoc sp.). We also assessed the ability of rapid methods to estimate the cell abundance of different species in the mixed samples. All instruments showed high sensitivity to changes in the cell abundance of different algae species and a mixture of these species. Any one of these methods, once calibrated, can be reliably used to estimate the abundance of a single-species/laboratory culture of microalgae. At the same time, FlowCam, without preliminary calibration, recorded the cell abundance closest to microscopic counts. When analysing a mixture of three microalgae differing in their cell sizes and spectral characteristics, FluoroProbe showed the highest accuracy in assessing the proportions of species in the mixture and FlowCam – in assessing their abundance. To study mixtures of algae and/or natural phytoplankton communities, it is advisable to use jointly a flow cytometer and a multichannel fluorimeter. The images of algae saved by the flow cytometer, if necessary, can be used to identify them, with a certain accuracy, to the species. Information on cells size and spectral characteristics obtained by two methods will be detailed enough to perform such common tasks as studying trophic interactions between phyto- and zooplankton or creating warning systems to inform of unwanted blooms of phytoplankton and their individual groups (for example, cyanobacteria)

Common diseases alter the physiological age-related blood microRNA profile

Aging is a key risk factor for chronic diseases of the elderly. MicroRNAs regulate post-transcriptional gene silencing through base-pair binding on their target mRNAs. We identified nonlinear changes in age-related microRNAs by analyzing whole blood from 1334 healthy individuals. We observed a larger influence of the age as compared to the sex and provide evidence for a shift to the 5’ mature form of miRNAs in healthy aging. The addition of 3059 diseased patients uncovered pan-disease and disease-specific alterations in aging profiles. Disease biomarker sets for all diseases were different between young and old patients. Computational deconvolution of whole-blood miRNAs into blood cell types suggests that cell intrinsic gene expression changes may impart greater significance than cell abundance changes to the whole blood miRNA profile. Altogether, these data provide a foundation for understanding the relationship between healthy aging and disease, and for the development of age-specific disease biomarkers.

Seasonal impact of grazing, viral mortality, resource availability and light on the group-specific growth rates of coastal Mediterranean bacterioplankton

Estimation of prokaryotic growth rates is critical to understand the ecological role and contribution of different microbes to marine biogeochemical cycles. However, there is a general lack of knowledge on what factors control the growth rates of different prokaryotic groups and how these vary between sites and along seasons at a given site. We carried out several manipulation experiments during the four astronomical seasons in the coastal NW Mediterranean in order to evaluate the impact of grazing, viral mortality, resource competition and light on the growth and loss rates of prokaryotes. Gross and net growth rates of different bacterioplankton groups targeted by group-specific CARD-FISH probes and infrared microscopy (for aerobic anoxygenic phototrophs, AAP), were calculated from changes in cell abundances. Maximal group-specific growth rates were achieved when both predation pressure and nutrient limitation were experimentally minimized, while only a minimal effect of viral pressure on growth rates was observed; nevertheless, the response to predation removal was more remarkable in winter, when the bacterial community was not subjected to nutrient limitation. Although all groups showed increases in their growth rates when resource competition as well as grazers and viral pressure were reduced, Alteromonadaceae consistently presented the highest rates in all seasons. The response to light availability was generally weaker than that to the other factors, but it was variable between seasons. In summer and spring, the growth rates of AAP were stimulated by light whereas the growth of the SAR11 clade (likely containing proteorhodopsin) was enhanced by light in all seasons. Overall, our results set thresholds on bacterioplankton group-specific growth and mortality rates and contribute to estimate the seasonally changing contribution of various bacterioplankton groups to the function of microbial communities. Our results also indicate that the least abundant groups display the highest growth rates, contributing to the recycling of organic matter to a much greater extent than what their abundances alone would predict.

Dissecting heterogeneous cell populations across drug and disease conditions with PopAlign

Single-cell measurement techniques can now probe gene expression in heterogeneous cell populations from the human body across a range of environmental and physiological conditions. However, new mathematical and computational methods are required to represent and analyze gene-expression changes that occur in complex mixtures of single cells as they respond to signals, drugs, or disease states. Here, we introduce a mathematical modeling platform, PopAlign, that automatically identifies subpopulations of cells within a heterogeneous mixture and tracks gene-expression and cell-abundance changes across subpopulations by constructing and comparing probabilistic models. Probabilistic models provide a low-error, compressed representation of single-cell data that enables efficient large-scale computations. We apply PopAlign to analyze the impact of 40 different immunomodulatory compounds on a heterogeneous population of donor-derived human immune cells as well as patient-specific disease signatures in multiple myeloma. PopAlign scales to comparisons involving tens to hundreds of samples, enabling large-scale studies of natural and engineered cell populations as they respond to drugs, signals, or physiological change.

Single-Cell RNA Sequencing of Calvarial and Long-Bone Endocortical Cells.

Single-cell RNA sequencing (scRNA-Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-Seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-Seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-Seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-Seq on freshly recovered long bone endocortical cells from mice that received either vehicle or sclerostin-neutralizing antibody for 1 week. We were unable to detect significant changes in bone anabolism-associated transcripts in immature and mature osteoblasts recovered from mice treated with sclerostin-neutralizing antibody; this might be a consequence of being underpowered to detect modest changes in gene expression, because only 7% of the sequenced endocortical cells were osteoblasts and a limited portion of their transcriptomes were sampled. We conclude that scRNA-Seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single-cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required. © 2020 American Society for Bone and Mineral Research.

Populations of the red tide forming dinoflagellate Noctiluca scintillans (Macartney): A comparison between the Black Sea and the northern Adriatic Sea

Populations of Noctiluca scintillans (hereafter Noctiluca) were compared from two regions: the northeastern-central Black Sea and the northern Adriatic Sea. In both seas samples were collected in near-shore waters 2–3 times per month during 2004–2012. For analysis of feeding activities and seasonal dynamics additional cruise data on the open waters of the Black Sea were used. Comparison between the two populations shows similarity in size structure with two classes 401–500μm and 501–600μm being the most numerous. Seasonal changes in cell abundance in both seas demonstrated a regular annual maximum with the peak period of high abundances in May–June with additional sporadic peaks in other seasons. In spring the average number of food vacuoles in the cell (1.78) and the proportion of feeding cells in populations (79%) in the Adriatic Sea were similar to those in the Black Sea (1.58 and 76%). In September–October, these parameters were lower both in the Adriatic Sea (0.69 and 49%) and in the Black Sea (1.46 and 65%) demonstrating that Noctiluca was better provided with food in spring. Among biotic parameters (wet phytoplankton biomass, chlorophyll biomass and zooplankton species) only the concentration of the eggs of Calanus euxinus was significantly positively correlated with abundance of Noctiluca. The possible effect of a high concentration of copepod eggs on the growth of Noctiluca in the peak period is discussed. An obvious negative relationship was observed between Noctiluca cell numbers in the peak period and wind velocity in both seas. The most significant negative correlation was observed between the number of windy hours per month (velocity more than 5–6ms−1) and cell concentrations in the Black Sea (r=−0.92) and in the northern Adriatic Sea (r=−0.67). On this basis, a new hypothesis has been proposed and discussed: in connection with features of the food behavior of Noctiluca, its outbursts during the peak period are controlled by the wind. An evident positive relationship was observed between the number of Noctiluca in the peak period and its quantity in the preceding months in both seas. Thus, we suggest that abundance data during early spring and weather forecasts (winds) may be used for medium-term prediction of Noctiluca outbursts and red tides.

Reliability of rapid, semi‐automated assessment of plankton abundance, biomass, and growth rate estimates: Coulter Counter versus light microscope measurements

Here we determined (1) how accurately the semi‐automated Coulter Counter (CC) measured the abundance, volume, and growth rates of mono‐specific plankton cultures compared to light microscopy and (2) whether CC‐derived particle size‐abundance spectra of whole water samples provided a near‐real time assessment of plankton community composition. A significant, positive relationship was found between CC particle and microscope cell abundance and volume estimates for 16 phylogenetically diverse plankton species. However, abundance estimates of the 2 methods differed significantly from a 1:1 relationship, with CC counts on average 15% greater than microscopy counts. Growth rate estimates showed excellent agreement for both methods for all species and were consistent over time. No significant differences in abundance, growth rate, or size estimates were observed due to light levels (Scrippsiella trochoidea) or strain variation (Heterosigma akashiwo). CC‐based, ship‐board characterization of whole plankton communities, from Georges Bank, captured changes in size‐abundance spectra and thus provided a coarse picture of in‐situ community composition. In the laboratory, the CC appears particularly well‐suited to measuring relative changes in cell abundance, such as for growth and grazing rate measurements of mono‐specific laboratory cultures, or when predator and prey species are well separated by size differences. Although microscopy remains essential for plankton analyses, these results suggest that the CC can increase sample size, improving accuracy, and precision of studies requiring frequent sampling or high degrees of replication. Deployment of these automated techniques in the field may aid in quantifying spatial and temporal variability in plankton community structure by forthcoming ocean‐observing systems.

Monitoring Functions in Managed Microbial Systems by Cytometric Bar Coding

Cytometric monitoring of microbial community dynamics can be used to estimate stability of technical microbial processes like biogas production by analysis of segregated cell abundance changes. In this study, structure variations of a biogas community were cytometrically recorded over 9 months and found to be of diagnostic value for process details. The reactor regime was intentionally disturbed with regard to substrate overload or H(2)S accumulation. A single-cell based approach called cytometric bar coding (CyBar) for fast identification of reactive subcommunities was used. Functionality of specific subcommunities was uncovered by processing CyBar data with abiotic reactor parameters using Spearman's correlation coefficient. Twenty subcommunities showed a discrete and divergent behavior. For example, a 4-fold substrate overload increased the cell number of two acidogenic index subcommunities to 176 and 193% within three days. Supplementary analyses were done using DNA fingerprinting, cloning, and sequencing. Bioreactor perturbations were shown to create cell abundance changes in subcommunities rather than variations in their phylogenetic composition. The used workflow and macros are ready-to-use tools and allow on-site monitoring and interpretation of variation in microbial community functions within a few hours.

Molecular Analysis of theIn SituGrowth Rates of Subsurface Geobacter Species

Molecular tools that can provide an estimate of the in situ growth rate of Geobacter species could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate of Geobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins, rpsC and rplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance of rpsC correlated best (r(2) = 0.90) with specific growth rates. Therefore, expression patterns of rpsC were used to estimate specific growth rates of Geobacter species during an in situ uranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression of Geobacter rpsC, and the increase in the number of Geobacter cells estimated with fluorescent in situ hybridization compared well with specific growth rates estimated from levels of in situ rpsC expression. However, in later phases, cell number increases were substantially lower than predicted from rpsC transcript abundance. This change coincided with a bloom of protozoa and increased attachment of Geobacter species to solid phases. These results suggest that monitoring rpsC expression may better reflect the actual rate that Geobacter species are metabolizing and growing during in situ uranium bioremediation than changes in cell abundance.

Effects of Lugol's fixation on the size structure of natural nano-microplankton samples, analyzed by means of an automatic counting method

Accurate abundance and biomass measurements are essential steps for determining the role of nano-microplankton in the microbial food web. Owing to practical constraints, traditional microscope analysis of nano-microplankton requires preservation; but preservatives alter plankton samples and bias the measurements. The majority of studies on the effects of preservation have been based on cell cultures. However, new automatic counting systems offer the possibility to investigate the effect of fixatives on large numbers of natural samples. In the present study, cell counts of live and 1% Lugol’s preserved samples were compared at 115 stations located in the Bay of Biscay. Additionally, the effect of different Lugol’s concentration (1 and 5%) was studied. Analyses were performed with the FlowCAM (see Sieracki et al. in An imaging-in-flow system for automated analysis of marine microplankton. Mar. Ecol. Progress Ser., 168, 285-296, 1998), using plankton samples directly collected from the field. The results show that the analysis of natural samples preserved with a single fixative biases the abundance and biomass estimates of different size ranges of the nano- and microplankton, not only in the large sizes. This is due to changes in cell abundance, especially in the nanoplankton size range, and to the formation of aggregates.