Biosynthesis of complement components by cultured rat hepatocytes.

Hepatocytes which had been isolated from the livers of Charles River rats were cultured in vitro. The cells were shown to synthesize albumin and the complement components C4, C2, C3 and B. Pulse-label studies with [35S]methionine showed that C4 and C3 were synthesized as single polypeptide chains. Pro-C4 did not appear to be converted into the plasma form of C4 intracellularly, whereas cell lysates contained the alpha- and beta-chains of plasma C3 as well as pro-C3. It is concluded that culture of rat hepatocytes in vitro provides a useful technique for studies of the synthesis of complement components.     

Increased intracellular calcium is required for neurite outgrowth induced by a synthetic peptide ligand of NCAM

We have recently identified a synthetic peptide, termed C3, capable of binding the first immunoglobulin-like module of neural cell adhesion molecule (NCAM) by means of combinatorial chemistry and shown that this NCAM ligand promotes neurite outgrowth. By means of single cell calcium imaging using the calcium-sensitive probe fura-2-acetomethyl ester, we here show that the C3-peptide induced an increase in intracellular calcium in primary hippocampal neurons and PC12-E2 cells, presumably requiring mobilization of calcium from both extracellular and intracellular stores. We further observed that C3-induced neurite outgrowth was inhibited by antagonists of voltage-dependent calcium channels as well as by an inhibitor of intracellular calcium mobilization, TMB-8. These findings demonstrate at the single cell level that a synthetic NCAM ligand directly can induce an increase in intracellular calcium and suggest that NCAM-dependent neurite outgrowth requires calcium mobilization from both extracellular and intracellular calcium stores. Thus, the C3-peptide may be regarded as a useful tool for the study of NCAM-dependent signal transduction. Furthermore, the peptide may be of considerable therapeutical interest for the treatment of neurodegenerative disorders.     

A novel method of encapsulating and cultivating adherent mammalian cells within collagen microcarriers

A novel method of preparing collagen microcarriers was developed and used to entrap adherent cells for cell culturing. This new technique involved seeding of cells in micro gel beads comprised of collagen fibrils dispersed in alginate. The gel beads were washed with phosphate buffered saline (PBS) to remove alginate and the resulting microspheres, about 300-500 microm in diameter, contained evenly distributed collagen fibrils which provided a 3D biomimetic environment for cell growth. The applicability of this microencapsulating system was demonstrated by its ability to support the growth of C2C12 myoblast cells. When seeded and cultured within the 3D collagen microcarriers, the population of C2C12 cells entrapped within the microcarriers increased by 1.5 folds in 7 days after inoculation. This encapsulation technique is potentially useful for culturing cells and especially useful for adherent cells that require a 3D fibrillar collagen environment.     

A Robust Method to Analyze Copy Number Alterations of Less than 100 kb in Single Cells Using Oligonucleotide Array CGH

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.     

Label-free observation of three-dimensional morphology change of a single PC12 cell by digital holographic microscopy

Observation of three-dimensional (3D) morphology changes of a single mammalian cell is very useful to understand cell response for various stimuli. Conventional techniques to evaluate morphology changes with sufficient precision and high temporal resolution are limited. For example, the confocal fluorescence microscope is available to take 3D morphology changes, whereas fluorescence microscopic observation requires labeling the cells with fluorescence dye. Recently, a novel imaging method based on digital holography was developed for nonlabeling microscopic observation of 3D morphology. Digital holographic microscopy has high potentiality in digital focusing properties, video-frequency capability, noninvasive operation, and so forth. It obtains a quantitative phase image of a living cell from a single recorded hologram, with interferometric accuracy, and surveys the rapid morphology change of a single cell. In this study, digital holographic microscopy was applied to monitor the 3D morphology change of an individual PC12 cell, a nerve model cell, subjected to high K(+) stimulation. Phase images of the rapidly swelling cell were acquired, and time lapse reconstruction of 3D cell morphology was performed from phase images. Our results demonstrate that digital holographic imaging is a powerful new tool for evaluation of cell response against various stimulants without any labeling reagent.     

Calculation of translational friction and intrinsic viscosity. I. General formulation for arbitrarily shaped particles.

A general method for calculating translational friction and intrinsic viscosity is developed through exploiting relations between hydrodynamics and electrostatics. An approximate relation xi = 6 pi eta 0C between the translational friction coefficient xi of a particle (eta 0: solvent viscosity) and its capacitance C was derived previously. This involved orientationally preaveraging the Oseen tensor, but the result was found to be very accurate. Based on preaveraging, we find that the intrinsic viscosity [eta] of a particle can be estimated from its polarizability alpha through [eta] = 3/4 alpha + 1/4 Vp, where Vp is the volume of the particle. Both the capacitance and the polarizability can be obtained in a single calculation using the boundary-element technique. An efficient approach is thus found for estimating [eta], a quantity that is very useful in practice because of its sensitivity to particle shape but is notoriously difficult to calculate. Illustrative calculations on ellipsoids, cylinders, and dumbbells demonstrate both the accuracy of the approximate relations and the efficiency of the present method.     

ACQUIRING SCANNING ELECTRON MICROSCOPICAL,LIGHT MICROSCOPICAL AND MULTIPLE GENE SEQUENCE DATA FROM A SINGLE DINOFLAGELLATE CELL1

We have developed a useful method to obtain light and scanning electron micrographs of a single dinoflagellate cell, prior to applying the cell to the single cell PCR technique. This method allows us to record detailed morphological information on any cell used for sequencing, which can be extremely important for the future identification of the organism, because cells used for single cell PCR usually cannot be retained. Furthermore, by applying multiple sets of PCR primers at the same time, we have successfully amplified and sequenced multiple genes (and DNA regions) simultaneously, even from a single cell. In this note, we demonstrate the methods of this technique by using two different types of dinoflagellates, i.e. an armored freshwater species, Peridinium willei Huitfeld‐Kaas, and an unarmored marine species, Akashiwo sanguinea (Hirasaka) Hansen and Moestrup. By rotating the cell, photographs of all aspects of a single cell can be taken even using the SEM. The genes and DNA regions sequenced in these examples include a region of the ribosomal DNA (SSU, ITS1, 5.8S, ITS2, and part of the LSU) as well as part of the mitochondrial DNA‐encoded gene, cox1. This technique can be applied to both photosynthetic and heterotrophic dinoflagellates and will accelerate biodiversity studies.     

Novel selection marker for mammalian cell transfection

The availability of selectable markers suitable for use in mammalian cells has permitted the analysis of the influence of the stable overexpression of single or multiple genes on specific cell properties. This powerful technique has led directly to many fundamental advances in molecular biology and increased our overall understanding of cell growth and regulatory events. Although a variety of selectable markers are currently available, some cell lines continue to be naturally resistant to certain markers, making direct selection difficult or not feasible. Thus, the characterization of additional cell selectable markers continues to be of interest. We have developed a novel selectable marker based on mitomycin C resistance that is suitable for stable transfection of mammalian cells. This system is based on the ability of the mcrA gene, isolatedfrom Streptomyces lavendulae, to confer mitomycin C resistance to both bacterial and mammalian cells by expression of the MCRA protein. Here we demonstrate that mcrA can be used as a selectable gene marker in Chinese hamster ovary cells when cells transfected with the mcrA gene are either pulsed or cultured continuously with mitomycin C This unique selection system may be of use for transfection of cells that are resistant to currently available selectable markers.     

Cell sorting analysis of geographically separated hypersaline environments

Biogeography of microbial populations remains to be poorly understood, and a novel technique of single cell sorting promises a new level of resolution for microbial diversity studies. Using single cell sorting, we compared saturated NaCl brine environments (32–35 %) of the South Bay Salt Works in Chula Vista in California (USA) and Santa Pola saltern near Alicante (Spain). Although some overlap in community composition was detected, both samples were significantly different and included previously undiscovered 16S rRNA sequences. The community from Chula Vista saltern had a large bacterial fraction, which consisted of diverse Bacteroidetes and Proteobacteria. In contrast, Archaea dominated Santa Pola’s community and its bacterial fraction consisted of the previously known Salinibacter lineages. The recently reported group of halophilic Archaea, Nanohaloarchaea, was detected at both sites. We demonstrate that cell sorting is a useful technique for analysis of halophilic microbial communities, and is capable of identifying yet unknown or divergent lineages. Furthermore, we argue that observed differences in community composition reflect restricted dispersal between sites, a likely mechanism for diversification of halophilic microorganisms.     

Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria.     

Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

The application of rule-based methods to class prediction problems in genomics.

We propose a method for constructing classifiers using logical combinations of elementary rules. The method is a form of rule-based classification, which has been widely discussed in the literature. In this work we focus specifically on issues that arise in the context of classifying cell samples based on RNA or protein expression measurements. The basic idea is to specify elementary rules that exhibit a locally strong pattern in favor of a single class. Strict admissibility criteria are imposed to produce a manageable universe of elementary rules. Then the elementary rules are combined using a set covering algorithm to form a composite rule that achieves a perfect fit to the training data. The user has explicit control over a parameter that determines the composite rule's level of redundancy and parsimony. This built-in control, along with the simplicity of interpreting the rules, makes the method particularly useful for classification problems in genomics. We demonstrate the new method using several microarray datasets and examine its generalization performance. We also draw comparisons to other machine-learning strategies such as CART, ID3, and C4.5.     

Characterization of a new type of variant of rat basophilic leukemia 2H3 cells presenting a different pattern of calcium signal

We selected a new type of variant (designated 3C7) derived spontaneously from parental RBL-2H3 cells. 3C7 cells showed lower contact inhibition, anchorage dependency, and serotonin release activity than those of RBL-2H3 cells. We conclude that 3C7 cells are a transformant of RBL-2H3 cell with greater malignancy. The production of inositol bisphosphate and the release of Ca2+ from intracellular stores induced by IgE-antigen stimulation were enhanced in 3C7. Oscillation of [Ca2+]i in individual 3C7 cells was observed by a digital imaging microscopic technique. We propose that 3C7 cells are a useful model system for studies on the mechanisms of stimulus-secretion coupling and the relationships between malignant alterations and disorders of signal transduction.     

Novel Applications of Magnetic Cell Sorting to Analyze Cell-Type Specific Gene and Protein Expression in the Central Nervous System

The isolation and study of cell-specific populations in the central nervous system (CNS) has gained significant interest in the neuroscience community. The ability to examine cell-specific gene and protein expression patterns in healthy and pathological tissue is critical for our understanding of CNS function. Several techniques currently exist to isolate cell-specific populations, each having their own inherent advantages and shortcomings. Isolation of distinct cell populations using magnetic sorting is a technique which has been available for nearly 3 decades, although rarely used in adult whole CNS tissue homogenate. In the current study we demonstrate that distinct cell populations can be isolated in rodents from early postnatal development through adulthood. We found this technique to be amendable to customization using commercially available membrane-targeted antibodies, allowing for cell-specific isolation across development and animal species. This technique yields RNA which can be utilized for downstream applications—including quantitative PCR and RNA sequencing—at relatively low cost and without the need for specialized equipment or fluorescently labeled cells. Adding to its utility, we demonstrate that cells can be isolated largely intact, retaining their processes, enabling analysis of extrasomatic proteins. We propose that magnetic cell sorting will prove to be a highly useful technique for the examination of cell specific CNS populations.     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.     

Parallel detection of ancient pathogens via array-based DNA capture

DNA capture coupled with next generation sequencing is highly suitable for the study of ancient pathogens. Screening for pathogens can, however, be meticulous when assays are restricted to the enrichment of single organisms, which is common practice. Here, we report on an array-based DNA capture screening technique for the parallel detection of nearly 100 pathogens that could have potentially left behind molecular signatures in preserved ancient tissues. We demonstrate the sensitivity of our method through evaluation of its performance with a library known to harbour ancient Mycobacterium leprae DNA. This rapid and economical technique will be highly useful for the identification of historical diseases that are difficult to characterize based on archaeological information alone.     

Toward selection of internalizing antibodies from phage libraries

Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).     

Monoclonal antibodies directed against rat liver epithelial cell lines selectively recognize bile duct epithelium in livers of adult rats

Monoclonal antibodies directed against antigens on rat liver epithelial cell lines were prepared. Three antibodies, 4C3, 19C6, and 3C2, recognized surface antigens present (although in different quantities) on eight epithelial cell lines tested, irrespective of whether they were normal or transformed. For MAb 3C2, the primary antigen common to all but one cell line showed a Mr of 135 kD. In paraffin sections of liver tissue, two antibodies, 40 and 19C6, reacted exclusively with bile duct epithelium, whereas the MAb 3C2 additionally reacted with sinusoidal endothelium and the endothelium of the portal venules. In sections of livers from rats exposed to diethylnitrosamine, the MAb 19C6 selectively stained bile duct-like structures in cholangiomas, while other preneoplastic and neoplastic lesions were not stained. These results demonstrate that the monoclonal antibodies obtained may prove useful for investigating cell lineages related to propagable liver epithelial cell lines and suggest that these cells may be derived from terminal bile ductular cells.Abbreviations ABTS2 2,2azinobis(3-ethylbenzthiazolinesulfonic) acid - ARL adult rat liver - DEN diethylnitrosamine - FCS fetal calf serum - MAb monoclonal antibody - PAP peroxidase antiperoxidase     

Laser tweezers as a biophotonic tool to investigate the efficacy of living sickle red blood cells in response to optical deformation

A laser tweezer technique based on single and/or dual-laser beams is proposed as a biophotonic tool to trap single cells and investigate their biophysical and biomechanical characteristics. Optical deformability and changes in size and cellular morphology of living and nonliving cells can be measured using the proposed technique. Representative results of red blood cell (RBC) optical deformability of 20 homozygous patients with sickle cell disease, including follow-up patients after treating with hydroxyurea (HU) for at least 3 months and 20 healthy control groups, are presented and compared. Shape recovery of deformed RBCs and relaxation time are recorded for each RBC. Results showed that healthy blood and patients treated with HU demonstrate significantly higher optical deformability and degree of optical elongation with morphological change of RBCs than untreated patients. Moreover, the healthy control group and patients treated with HU exhibited faster relaxation time for RBCs than untreated patients. A trapping power that reaches 180 mW caused no observable photo-damage at a wavelength 1064 nm.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12551-021-00790-0.     

Double-labeling and high precision comparison of complex protein patterns on two-dimensional polyacrylamide gels

Combination of double-labeling and two-dimensional polyacrylamide gel electrophoresis allows high precision comparison of proteins from two different cultured cell lines. Extracts of cell lines labeled with ¹⁴C and ³H, respectively, are mixed and analyzed on a single two-dimensional gel. Fluorography detects both isotopes, but autoradiography through carbon paper detects only ¹⁴C. Proteins can be classified into classes common to both cell lines and those specific to each cell line. The method should be useful for a wide range of biochemical studies.