Variation In Number Of Cells Research Articles

Getting the Right Clones in an Automated Manner: An Alternative to Sophisticated Colony-Picking Robotics.

In recent years, the design-build-test-learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli, promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum, were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a 'low-tech' method to be implemented in biofoundry settings without requiring additional devices.

Real-time assessment of relative mitochondrial ATP synthesis response against inhibiting and stimulating substrates (MitoRAISE)

BackgroundMitochondria are known to synthesize adenosine triphosphate (ATP) through oxidative phosphorylation. Understanding and accurately measuring mitochondrial ATP synthesis rate can provide insights into the functional status of mitochondria and how it contributes to overall cellular energy homeostasis. Traditional methods only estimate mitochondrial function by measuring ATP levels at a single point in time or through oxygen consumption rates. This study introduced the relative mitochondrial ATP synthesis response against inhibiting and stimulating substrates (MitoRAISE), designed to detect real-time changes in ATP levels as the cells respond to substrates.MethodsThe sensitivity and specificity of the MitoRAISE assay were verified under various conditions, including the isolation of mitochondria, variations in cell numbers, cells exhibiting mitochondrial damage, and heterogeneous mixtures. Using peripheral blood mononuclear cells (PBMCs), we analyzed MitoRAISE data from 19 patients with breast cancer and 23 healthy women.ResultsThe parameters observed in the MitoRAISE data increased depending on the quantity of isolated mitochondria and cell count, whereas it remained unmeasured in mitochondrial-damaged cell lines. Basal ATP, rotenone response, malonate response, and mitochondrial DNA copy numbers were lower in PBMCs from patients with breast cancer than in those from healthy women.ConclusionsThe MitoRAISE assay has demonstrated its sensitivity and specificity by measuring relative ATP synthesis rates under various conditions. We propose MitoRAISE assay as a potential tool for monitoring changes in the mitochondrial metabolic status associated with various diseases.

Automation preserves product consistency and quality for the formulation, fill, and finish of T cell-based therapies

Scaling up the manufacture of cell therapies can be complex and challenging. Maintaining critical quality attributes of the cell product during its final formulation and fill-finish into multiple containers can be especially difficult and laborious. Here, we tested the automated Finia™ Fill and Finish System to efficiently scale up the formulation and fill-finish of a T cell product, and then assessed cell quality and product consistency across different sub-lots filled during this expanded process. We found that this automated system could be effectively scaled to 4 times its singular capacity in a 2-h time interval, with variation in cell number and product volume less than 12% across all containers. Analysis of the different sub-lots of the final product revealed high cell viability and consistent T cell phenotype, with a high proportion of effector memory and central memory T cells and low expression of T cell senescence and exhaustion markers. The functionality of the T cell product was compared by measuring cytokine response after restimulation, with secreted levels of effector cytokines like IFN-γ and TNF-α being similar across the different sub-lots. Collectively, these results show that automation can scale up the formulation and fill-finish of a cell manufacturing process while maintaining the phenotype and functionality of the cell product. Better understanding of how to maintain product uniformity and quality during final manufacturing is important to the further scale-up and development of successful cell therapies.

ScRNA-Seq: First Atlas and Cellular Landscape of Lacrimal Sac: Implications in Primary Acquired Nasolacrimal Duct Obstruction Pathogenesis.

The aim of this study was to describe the transcriptional changes of individual cellular components in the lacrimal sac in patients with primary acquired nasolacrimal duct obstruction (PANDO) and attempt to construct the first lacrimal sac cellular atlas to elucidate the potential mechanisms that may drive the disease pathogenesis. Lacrimal sac samples were obtained intra-operatively during the endoscopic dacryocystorhinostomy (EnDCR) procedure from five patients. Single-cell RNA sequencing was performed to analyze each individual cell population including epithelial and immune cells during the early inflammatory and late inflammatory phases of the disease. Eleven cell types were identified among 25,791 cells. T cells and B cells were the cell populations with the greatest variation in cell numbers between the two phases and were involved in immune response and epithelium migration-related pathways. The present study showed that epithelial cells highly expressed the genes of senescence-associated secretory phenotype (SASP) and were involved in influencing the inflammation, neutrophil chemotaxis, and migration during the late inflammatory stage. Enhanced activity of CXCLs-CXCRs between the epithelial cells and neutrophils was noted by the cell-cell communication analysis and is suspected to play a role in inflammation by recruiting more neutrophils. The study presents a comprehensive single-cell landscape of the lacrimal sac cells in different phases of PANDO. The contribution of T cells, B cells, and epithelial cells to the inflammatory response, and construction of the intercellular signaling networks between the cells within the lacrimal sac has further enhanced the present understanding of the PANDO pathogenesis.

Abstract 4943: Benchmarking unpaired single-cell RNA and single-cell ATAC integration

Abstract The integration of single-cell RNA-sequencing (scRNA-seq) and single-cell ATAC-sequencing (scATAC-seq) data offers a unique opportunity to gain a comprehensive view of cellular identity with defining features and to infer gene-regulatory relationships. Despite the emergence of technologies that simultaneously capture both the gene expression and chromatin accessibility of individual cells (paired data), the practical challenges of these approaches (e.g., the unavailability in previous samples and prohibitive cost) have led researchers to turn to the existing trove of single-modality data generated from independent biological samples (unpaired data). Various computational tools have been developed to integrate these unpaired single modality datasets. However, the comparative performance of these tools has not been comprehensively evaluated, and a standard benchmark pipeline is still lacking. To address these challenges, we used pseudo-unpaired scRNA-seq and scATAC-seq data derived from publicly available paired single-cell multi-omics datasets to benchmark 14 publicly available integration methods. The primary goal of unpaired single-cell multi-omics integration is to narrow the omics gap while preserving cell type diversity. We therefore focused on pair-wise cell distance and cluster performance in the joint latent space constructed by various integration tools in our benchmarking pipeline. To ensure the robustness of these computational approaches, we examined their stability across a variety of scenarios, including variations in cell number, cell types, and biological and technical batch effects. A number of the integration methods tested produced promising results. While the widely used Seurat package was recently reported to have the best performance (Lee et al., Genome Biology 2023), other computational tools such as scVI, Cobolt, scJoint, scglue and scBridge performed equally well or better in reducing omics differences and facilitating the identification of cell clusters. Notably, scglue and Cobolt demonstrated strong performance in aligning the same cell from different modalities, and discrete clusters emerged in the joint latent space using scJoint and scBridge. These findings suggest that it may not be strictly necessary to use paired multi-omics data to guide integration to achieve favorable results. Our freely available benchmarking pipeline will empower researchers to identify the optimal data integration methods for their specific data, facilitate the benchmarking of new methods, and contribute to future method development in the field. Citation Format: Jiani Chen, Wanzi Xiao, Eric Zhang, Xiang Chen. Benchmarking unpaired single-cell RNA and single-cell ATAC integration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4943.

A rapid workflow for neuron counting in combined light sheet microscopy and magnetic resonance histology.

Information on regional variation in cell numbers and densities in the CNS provides critical insight into structure, function, and the progression of CNS diseases. However, variability can be real or a consequence of methods that do not account for technical biases, including morphologic deformations, errors in the application of cell type labels and boundaries of regions, errors of counting rules and sampling sites. We address these issues in a mouse model by introducing a workflow that consists of the following steps: 1. Magnetic resonance histology (MRH) to establish the size, shape, and regional morphology of the mouse brain in situ. 2. Light-sheet microscopy (LSM) to selectively label neurons or other cells in the entire brain without sectioning artifacts. 3. Register LSM volumes to MRH volumes to correct for dissection errors and both global and regional deformations. 4. Implement stereological protocols for automated sampling and counting of cells in 3D LSM volumes. This workflow can analyze the cell densities of one brain region in less than 1 min and is highly replicable in cortical and subcortical gray matter regions and structures throughout the brain. This method demonstrates the advantage of not requiring an extensive amount of training data, achieving a F1 score of approximately 0.9 with just 20 training nuclei. We report deformation-corrected neuron (NeuN) counts and neuronal density in 13 representative regions in 5 C57BL/6J cases and 2 BXD strains. The data represent the variability among specimens for the same brain region and across regions within the specimen. Neuronal densities estimated with our workflow are within the range of values in previous classical stereological studies. We demonstrate the application of our workflow to a mouse model of aging. This workflow improves the accuracy of neuron counting and the assessment of neuronal density on a region-by-region basis, with broad applications for studies of how genetics, environment, and development across the lifespan impact cell numbers in the CNS.

ComProliM: A cell growth assay robust to initial cell number in co-culture system

Cell growth is conventionally quantified using CCK-8 or MTT assays, but these methods display considerable sensitivity to initial cell quantities. Inherent sampling errors during cell counting and seeding make it impossible to achieve an absolute equivalence of initial cell numbers, potentially confounding the results of CCK-8 or MTT assays. In the present study, we introduce a novel cell proliferation assay, ComProliM, predicated on cell competition theory. Both numeral simulations and empirical testing demonstrate that ComProliM index (CPMI) reliably represents cell growth rate and is not influenced by variations in initial cell number. Intriguingly, two adherent cells of differing fluorescence states are co-cultured, suggesting that ComProliM can be successfully employed in co-culture system cell proliferation assays, including, for instance, the exploration of subclone interactions. We anticipate ComProliM will provide a viable alternative for quantifying adherent cell growth rates, particularly in cases where conventional methodologies deliver inconsistent or ambiguous results.

Quantitative trait loci on chromosomes 9 and 19 modulate AII amacrine cell number in the mouse retina.

Sequence variants modulating gene function or expression affect various heritable traits, including the number of neurons within a population. The present study employed a forward-genetic approach to identify candidate causal genes and their sequence variants controlling the number of one type of retinal neuron, the AII amacrine cell. Data from twenty-six recombinant inbred (RI) strains of mice derived from the parental C57BL/6J (B6/J) and A/J laboratory strains were used to identify genomic loci regulating cell number. Large variation in cell number is present across the RI strains, from a low of ∼57,000 cells to a high of ∼87,000 cells. Quantitative trait locus (QTL) analysis revealed three prospective controlling genomic loci, on Chromosomes (Chrs) 9, 11, and 19, each contributing additive effects that together approach the range of variation observed. Composite interval mapping validated two of these loci, and chromosome substitution strains, in which the A/J genome for Chr 9 or 19 was introgressed on a B6/J genetic background, showed increased numbers of AII amacrine cells as predicted by those two QTL effects. Analysis of the respective genomic loci identified candidate controlling genes defined by their retinal expression, their established biological functions, and by the presence of sequence variants expected to modulate gene function or expression. Two candidate genes, Dtx4 on Chr 19, being a regulator of Notch signaling, and Dixdc1 on Chr 9, a modulator of the WNT-β-catenin signaling pathway, were explored in further detail. Postnatal overexpression of Dtx4 was found to reduce the frequency of amacrine cells, while Dixdc1 knockout retinas contained an excess of AII amacrine cells. Sequence variants in each gene were identified, being the likely sources of variation in gene expression, ultimately contributing to the final number of AII amacrine cells.

Fruit shape loci sun, ovate, fs8.1 and their interactions affect seed size and shape in tomato.

Seed size and shape are not only critical for plant reproduction and dispersal, but also important agronomic traits. Tomato fruit shape loci sun, ovate and fs8.1 regulate the morphology of fruit, flower, leaf and stem, and recently their functions in seed morphogenesis have also been noticed. However, mechanism underlying seed morphology variation has not been systematically investigated yet. Thus, using the near isogenic lines (NILs) harboring one, two or three of the fruit shape loci, histological, physiological and transcriptional bases of seed morphology change have been studied. sun and ovate showed potential abilities in decreasing seed size, whereas, fs8.1 had a potential ability in increasing this parameter. Interactions between two loci and the interaction among three loci all led to significant decrease of seed size. All the loci significantly down-regulated seed shape index (SSI), except for sun/fs8.1 double NIL, which resulted in the reductions in both seed length and width and finally led to a decreased trend of SSI. Histologically, seed morphological changes were mainly attributed to the cell number variations. Transcriptional and physiological analyses discovered that phytohormone-, cytoskeleton- as well as sugar transportation- and degradation-related genes were involved in the regulation of seed morphology by the fruit shape loci.

Experimental and theoretical study of 2-ethylfuran spherical expanding flame: Cellularization, intrinsic instability and self-acceleration

The intrinsic instability and cellularity of 2-ethylfuran (2-EF) spherically expanding flame were investigated using experimental and theoretical analysis. The development and saturation time of initial and secondary cracks, crack length, cell number, and average cell area on the flame front surface were quantified using image processing techniques. The results showed that the initial cracks grow linearly, and are mainly influenced by the equivalence ratio. As the equivalence ratio increases, the number of initial cracks increases and spreads widely, which influences the variations in cell number and average cell area. In addition, the effect of initial and secondary cracks on flame instability or cellularity was analyzed. It was indicated that the influence of initial cracks on flame cellularity mainly increases with increasing equivalence ratio, but the appearance of secondary cracks increases with increased equivalence ratio and initial pressure. Finally, the self-acceleration of the 2-EF cellularized flame was also investigated, which suggested that the flame self-acceleration increases with increased equivalence ratio and initial pressure.

Abstract 6214: Detecting and characterizing extrachromosomal DNA in medulloblastoma at single-cell resolution

Abstract Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. To assess the clinical import of ecDNA in MB, we applied computational methods to detect ecDNA to the genomes of a cohort of 468 MB patients and 31 MB model systems. Among patients, ecDNA was detected in 18% of tumors and carried threefold greater risk of mortality. Between sequential patient biopsies at initial diagnosis and subsequent relapse, we observed frequent structural variation at ecDNA loci and generation of new ecDNA sequences. Among model systems, ecDNA was found in 19 of 31 genomes (61%). Although ecDNA was far more prevalent among MB models than patients, the ecDNA genomic sequence was conserved between most patient-derived xenograft (PDX) models and their origin human tumors. EcDNA promotes high oncogene copy number, accelerated evolution of drug resistance and poor prognosis. Mechanistically, these behaviors are hypothesized to be consequences of intratumoral heterogeneity of ecDNA copy number; however, there are currently no methods to quantify the abundance and distribution of ecDNA across single cells in a tumor. We have previously identified a primary TP53-mutant SHH medulloblastoma tumor, which harbors multiple distinct lineages of ecDNA originating from different chromosomes. We have now simultaneously analyzed the accessible chromatin and gene expression in single cells of the human tumor and of a orthotopic mouse xenograft (PDX) derived for this tumor, using multiome single-cell ATAC-seq and gene expression (10X Genomics). By measuring scATAC-seq read depth at the ecDNA loci relative to the rest of the genome, we estimate ecDNA copy number in single cells. We observe substantial variation in the ecDNA copy number of cells in both the human tumor and the PDX, and strong association with transcription of ecDNA-amplified genes. We anticipate that this approach will illuminate mechanisms of ecDNA-mediated drug resistance, by revealing the sensitivity and population dynamics of ecDNA-containing cells under treatment pressure. Citation Format: Owen Chapman, Sunita Sridhar, Jon D. Larson, Robert J. Wechsler-Reya, Jill Mesirov, Lukas Chavez. Detecting and characterizing extrachromosomal DNA in medulloblastoma at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6214.

VvSUN may act in the auxin pathway to regulate fruit shape in grape.

Fruit shape is an essential agronomic feature in many crops. We identified and functionally characterized an auxin pathway-related gene, VvSUN. VvSUN, which belongs to the SUN/IQ67-DOMAIN (IQD) family, localizes to the plasma membrane and chloroplast and may be involved in controlling fruit shape through auxin. It is highly expressed in the ovary, and the expression level 1week before the anthesis stage is positively correlated with the fruit shape index. Functional analyses illustrated that VvSUN gene overexpression in tomato and tobacco plants changed fruit/pod shape. The VvSUN promoter directly bound to VvARF6 in yeast and activated ß-glucuronidase (GUS) activity by indole-3-acetic acid (IAA) treatments in grapevine leaves, indicating that VvSUN functions are in coordination with auxin. Further analysis of 35S::VvSUN transgenic tomato ovaries showed that the fruit shape changes caused by VvSUN were predominantly caused by variations in cell number in longitudinal directions by regulating endogenous auxin levels via polar transport and/or auxin signal transduction process variations. Moreover, enrichment of the 35S::VvSUN transgenic tomato differentially expressed genes was found in a variety of biological processes, including primary metabolic process, transmembrane transport, calcium ion binding, cytoskeletal protein binding, tubulin binding, and microtubule-based movement. Using weighted gene co-expression network analysis (WGCNA), we confirmed that this plant hormone signal transduction may play a crucial role in controlling fruit shape. As a consequence, it is possible that VvSUN acts as a hub gene, altering cellular auxin levels and the plant hormone signal transduction pathway, which plays a role in cell division patterns, leading to anisotropic growth of the ovary and, ultimately, an elongated fruit shape.

Bayesian Generalized Linear Model for Simulating Bacterial Inactivation/Growth Considering Variability and Uncertainty.

Conventional regression analysis using the least-squares method has been applied to describe bacterial behavior logarithmically. However, only the normal distribution is used as the error distribution in the least-squares method, and the variability and uncertainty related to bacterial behavior are not considered. In this paper, we propose Bayesian statistical modeling based on a generalized linear model (GLM) that considers variability and uncertainty while fitting the model to colony count data. We investigated the inactivation kinetic data of Bacillus simplex with an initial cell count of 105 and the growth kinetic data of Listeria monocytogenes with an initial cell count of 104. The residual of the GLM was described using a Poisson distribution for the initial cell number and inactivation process and using a negative binomial distribution for the cell number variation during growth. The model parameters could be obtained considering the uncertainty by Bayesian inference. The Bayesian GLM successfully described the results of over 50 replications of bacterial inactivation with average of initial cell numbers of 101, 102, and 103 and growth with average of initial cell numbers of 10–1, 100, and 101. The accuracy of the developed model revealed that more than 90% of the observed cell numbers except for growth with initial cell numbers of 101 were within the 95% prediction interval. In addition, parameter uncertainty could be expressed as an arbitrary probability distribution. The analysis procedures can be consistently applied to the simulation process through fitting. The Bayesian inference method based on the GLM clearly explains the variability and uncertainty in bacterial population behavior, which can serve as useful information for risk assessment related to food borne pathogens.

Tyrosine hydroxylase immunostaining in the central complex of dicondylian insects.

Dopamine acts as a neurohormone and neurotransmitter in the insect nervous system and controls a variety of physiological processes. Dopaminergic neurons also innervate the central complex (CX), a multisensory center of the insect brain involved in sky compass navigation, goal-directed locomotion and sleep control. To infer a possible influence of evolutionary history and lifestyle on the neurochemical architecture of the CX, we have studied the distribution of neurons immunoreactive to tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. Analysis of representatives from 12 insect orders ranging from firebrats to flies revealed high conservation of immunolabeled neurons. One type of TH-immunoreactive neuron was found in all species studied. The neurons have somata in the pars intercerebralis, arborizations in the lateral accessory lobes, and axonal ramifications in the central body and noduli. In all pterygote species, a second type of tangential neuron of the upper division of the central body was TH-immunoreactive. The neurons have cell bodies near the calyces and arborizations in the superior protocerebrum. Both types of neuron showed species-specific variations in cell number and in the innervated areas outside and inside the CX. Additional neurons were found in only two taxa: one type of columnar neuron showed TH immunostaining in the water strider Gerris lacustris, but not in other Heteroptera, and a tritocerebral neuron innervating the protocerebral bridge was immunolabeled in Diptera. The data show largely taxon-specific variations of a common ground pattern of putatively dopaminergic neurons that may be commonly involved in state-dependent modulation of CX function.

Variability of an Early Developmental Cell Population Underlies Stochastic Laterality Defects

SummaryEmbryonic development seemingly proceeds with almost perfect precision. However, it is largely unknown how much underlying microscopic variability is compatible with normal development. Here, we quantify embryo-to-embryo variability in vertebrate development by studying cell number variation in the zebrafish endoderm. We notice that the size of a sub-population of the endoderm, the dorsal forerunner cells (DFCs, which later form the left-right organizer), exhibits significantly more embryo-to-embryo variation than the rest of the endoderm. We find that, with incubation of the embryos at elevated temperature, the frequency of left-right laterality defects is increased drastically in embryos with a low number of DFCs. Furthermore, we observe that these fluctuations have a large stochastic component among fish of the same genetic background. Hence, a stochastic variation in early development leads to a remarkably strong macroscopic phenotype. These fluctuations appear to be associated with maternal effects in the specification of the DFCs.

Spatial and temporal evolution of quantitative magnetic resonance imaging parameters of peach and apple fruit - relationship with biophysical and metabolic traits.

Fruits are complex organs that are spatially regulated during development. Limited phenotyping capacity at cell and tissue levels is one of the main obstacles to our understanding of the coordinated regulation of the processes involved in fruit growth and quality. In this study, the spatial evolution of biophysical and metabolic traits of peach and apple fruit was investigated during fruit development. In parallel, the multi-exponential relaxation times and apparent microporosity were assessed by quantitative magnetic resonance imaging (MRI). The aim was to identify the possible relationships between MRI parameters and variations in the structure and composition of fruit tissues during development so that transverse relaxation could be proposed as a biomarker for the assessment of the structural and functional evolution of fruit tissues during growth. The study provides species-specific data on developmental and spatial variations in density, cell number and size distribution, insoluble and soluble compound accumulation and osmotic and water potential in the fruit mesocarp. Magnetic resonance imaging was able to capture tissue evolution and the development of pericarp heterogeneity by accessing information on cell expansion, water status and distribution at cell level, and microporosity. Changes in vacuole-related transverse relaxation rates were mostly explained by cell/vacuole size. The impact of cell solute composition, microporosity and membrane permeability on relaxation times is also discussed. The results demonstrate the usefulness of MRI as a tool to phenotype fruits and to access important physiological data during development, including information on spatial variability.

A transient decrease in mitochondrial activity contributes to establish the ganglion cell fate in retina adapted for high acuity vision

Although the plan of the retina is well conserved in vertebrates, there are considerable variations in cell type diversity and number, as well as in the organization and properties of the tissue. The high ratios of retinal ganglion cells (RGCs) to cones in primate fovea and bird retinas favor neural circuits essential for high visual acuity and color vision. The role that cell metabolism could play in cell fate decision during embryonic development of the nervous system is still largely unknown. Here, we describe how subtle changes of mitochondrial activity along the pathway converting uncommitted progenitors into newborn RGCs increase the recruitment of RGC-fated progenitors. ATOH7, a proneural protein dedicated to the production of RGCs in vertebrates, activates transcription of the Hes5.3 gene in pre-committed progenitors. The HES5.3 protein, in turn, regulates a transient decrease in mitochondrial activity via the retinoic acid signaling pathway few hours before cell commitment. This metabolic shift lengthens the progression of the ultimate cell cycle and is a necessary step for upregulating Atoh7 and promoting RGC differentiation.

Variations in mucous cell numbers in gills of Atlantic salmon (Salmo salar) presmolt in commercial freshwater farms in Norway

Fish gills are heavily exposed to the external milieu and may react against irritants with different cellular responses. We describe variations in mucous cell counts in gills from healthy Atlantic salmon (Salmo salar) presmolts in five recirculating aquaculture system (RAS) farms and one flow-through farm. Based on certain criteria, mucous cells were histologically quantified in a defined lamellar region of the gills and the counts were analysed. Immunohistochemistry (IHC) was used to investigate epithelial responses. The median number of total mucous cells in the defined region was 59 per fish. Between the farms, the medians varied from 31 to 101 with the lowest in the flow-through farm. A regression model was fitted with "total mucous cells" as the dependent variable and with "fish length" and "fish farm" as independent variables. The proportion of variation in mucous cell counts explained by the model was twice as high when "fish farm" was included compared to only "fish length." IHC revealed proliferative responses in coherence with high mucous cell numbers. Conclusively, the variation in mucous cell counts depends on combined farm-related factors. Establishing a baseline for mucous cell counts is fundamental in the development of high-throughput monitoring programmes of gill health in farmed fish.

0031 Photoperiodic Effects on Diurnal Rhythms in the Immune System, Rest-Activity Behavior, and Cortisol Revealed in a Diurnally Active Large Animal Model - The Domestic Pig

Abstract Introduction Diurnal variations in immune cell number and function are regarded important for immune competence and are thought to be mediated by rest-activity rhythms and hormones. Moreover, the photoperiod is also known to modulate the immune system and considered to affect seasonal disease susceptibility. Whereas few studies investigated seasonal effects, the present study is the first investigating the specific effect of the photoperiod on diurnal rhythms in cell numbers of peripheral leukocyte types in any species. Methods Domestic pigs were held either under long day (LD, 16L:8D, lights-on 07:00-23:00, n=9) or short day conditions (SD, 8L:16D, lights-on 07:00-15:00, n=11) and fed concentrate at 07:30 + 14:00 (ad libitum hay/water). Blood samples were taken every 2 h over periods of 50 h via indwelling vein catheters. Rest-activity behavior of the pigs was analyzed using continuous camera recordings. Results Cosinor analyses (p<.05) revealed photoperiodic differences in diurnal rhythms of cell numbers of various peripheral leukocyte subtypes, rest-activity behavior, and cortisol concentration. Cell numbers of total leukocytes, NK cells, T cells, and eosinophils in blood, rest-activity behavior, and cortisol concentration peaked earlier relative to lights-on under SD (p<.05). Relative amplitudes in rest-activity behavior and cell counts of total leukocytes, NK cells, T cells, and monocytes were higher under SD (p<.05). However, there was no photoperiodic effect on diurnal rhythms in neutrophil counts and mesor in any leukocyte type. Generalized linear mixed models revealed associations of leukocyte counts with rest-activity behavior and cortisol concentration in most cell types (p<.05). Moreover, the found photoperiodic effects on diurnal rhythms in rest-activity behavior and cortisol concentration are in agreement with research in humans and primates. Conclusion The present study revealed photoperiodic effects on diurnal rhythms in the immune system, rest-activity behavior, and cortisol concentration in pigs and strengthens the importance of the domestic pig as suitable model for chronoimmunology. Support German Research Foundation DFG (grant provided to SS, grant number SCHM3162/1-1), Federal Ministry of Education and Research, Germany (grant number 01PL16003), Faculty of Agricultural Sciences, University of Hohenheim (scholarship provided to LE).

Simultaneously Monitoring the Metabolic Activity of Three Bacteria Utilizing a LAPS-Based Differential Sensor Set-up

Introduction On-line monitoring of the metabolic response of microorganisms can avoid costly and time-consuming disturbances at different stages in biofermentation processes. For instance, process downtimes in biogas plants caused by metabolically inactive microorganisms can induce irreversible hindrances and cost-intensive interventions, which can be prevented by utilizing an efficient monitoring system. A LAPS (light-addressable potentiometric sensor) can be a promising candidate for those monitoring systems, since it combines several advantages such as small size, possibility of mass fabrication, solid-state nature and short response time [1,2]. In general, LAPS with an appropriate recognition layer provides a spatially resolved detection of a certain analyte and can record 2D-chemical images of concentration changes of bio/chemical species on its flat surface [3]. In this work, a LAPS-based multi-chamber measuring system with 16 laser-diode modules was utilized as shown in Fig. 1 (left), which allows to simultaneously determine the extracellular acidification of three microorganisms: Escherichia coli (E. coli) K12, Corynebacterium glutamicum (C. glutamicum) ATCC13032, and Lactobacillus brevis (L. brevis) ATCC14869. With the knowledge about the acidification behavior of each microorganism in a multivariate-type analysis, a signal-response pattern can be obtained. In this context, the sensor signal and the status of the biofermentation process can be correlated. Materials and Methods Bacterial cultures were prepared and resuspensioned in phosphate-buffered saline (PBS) solution (0.2 mM/pH, pH 7.4). Different glucose concentrations were adjusted. A four-chamber LAPS measuring system was deployed for differential measurements, as assembled in Fig. 1. First, all four chambers were loaded with 100 μl of glucose solution, varying in each experiment between 0.042 mM and 5.0 mM. The salt bridge chamber was filled with 1 ml of 3 M KCl and was immersed into the four chambers, passing the bias voltage through a Ag/AgCl reference electrode to the sensor surface, containing the glucose solution. After a conditioning phase of 10 min with the particular glucose concentration, cells (200 µl) were added to the respective chambers. Chamber 1 was loaded with E. coli, chamber 2 with C. glutamicum and chamber 3 with L. brevis bacteria. Chamber 4 served as the reference chamber without cells containing only the diluted PBS solution. Four LDMs were considered for each chamber to read-out the sensor signals. The mean value of each chamber was used for further evaluations [4]. Results and Conclusions Fig. 1 (right) depicts exemplary the metabolic responses as correlation curves between the LAPS signal and various glucose concentrations for the three types of investigated bacteria at a constant cell number of 4.8 × 109 cells/ml. The black curve refers to the extracellular acidification of E. coli, the red curve to C. glutamicum, and the green one to L. brevis bacteria. It was observed that by increasing the glucose concentration, the overall cellular metabolism increases due to an increase of H+-ion activity on the pH-sensitive transducer layer of the LAPS chip during the acidification phase. The obtained results will be discussed in context of varying cell numbers as well as status of a selected biofermentation process.Fig. 1. Schematic illustration of four-chamber differential LAPS set-up (left). RE: Ag/AgCl reference electrode, SC: sealing cap, SB: salt bridge chamber, MC: multi-chamber structure fixed on the sensor surface; LAPS consisting of layer structure of Al/p-Si/SiO2/Ta2O5 and modulated light source (LS) based on 16 laser-diode modules; 1–3: active sensor chambers with cells; 4: reference sensor chamber without cells; Iphoto: photocurrent; Vbias: bias voltage; AC: alternating current; LDMs: laser-diode modules. Correlation curves between LAPS signal response and glucose concentration (right). Mean values and standard deviations of potential change values of E. coli (black), C. glutamicum (red), L. brevis (green) at different glucose concentrations (0.042, 0.085, 0.17, 0.20, 0.33, 0.40, 0.50, 0.68, 0.83, 1.20, 1.67, 2.50, 3.33, and 5 mM) at constant cell number (4.8 × 109 cells). Three independent measurements were performed to calculate mean potential change rate values. The blue arrow in the diagram indicates a particular glucose concentration (1.67 mM), which was exemplarily chosen to compare calibration curves of metabolic responses of the three microorganisms by cell number variations.