Individual Eukaryotic Cells Research Articles

High-Throughput Host-Microbe Single-Cell RNA Sequencing Reveals Ferroptosis-Associated Heterogeneity during Acinetobacter baumannii Infection.

Interactions between host and bacterial cells are integral to human physiology. The complexity of host-microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high-resolution approaches to capture their full complexity. The latest breakthroughs in single-cell RNA sequencing (scRNA-seq) have opened up a new era of studies in host-pathogen interactions. Here, we first report a high-throughput cross-species dual scRNA-seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom-seq). Using reference cells, scRandom-seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom-seq witnessed polarization of THP-1 derived-macrophages and the intracellular A.b-induced ferroptosis-stress in host cells. The inhibition of ferroptosis by Ferrostatin-1 (Fer-1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom-seq provides a high-throughput cross-species dual single-cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host-microbe interactions in infection systems and tumor micro-environments.

High‐Throughput Host–Microbe Single‐Cell RNA Sequencing Reveals Ferroptosis‐Associated Heterogeneity during Acinetobacter baumannii Infection

AbstractInteractions between host and bacterial cells are integral to human physiology. The complexity of host–microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high‐resolution approaches to capture their full complexity. The latest breakthroughs in single‐cell RNA sequencing (scRNA‐seq) have opened up a new era of studies in host–pathogen interactions. Here, we first report a high‐throughput cross‐species dual scRNA‐seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom‐seq). Using reference cells, scRandom‐seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom‐seq witnessed polarization of THP‐1 derived‐macrophages and the intracellular A.b‐induced ferroptosis‐stress in host cells. The inhibition of ferroptosis by Ferrostatin‐1 (Fer‐1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom‐seq provides a high‐throughput cross‐species dual single‐cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host–microbe interactions in infection systems and tumor micro‐environments.

PERSPECTIVE: The comet assay as a method for assessing DNA damage in cryopreserved samples

The preservation of the nuclear genome's integrity is paramount for the viability and overall health of cells, tissues, and organisms. DNA, being susceptible to damage under physiological conditions and vulnerable to both endogenous and environmental factors, faces constant threats. To assess DNA damage and repair within individual eukaryotic cells, the comet assay presents itself as a versatile, gel electrophoresis-based, relatively simple, and highly sensitive method. Originally designed to monitor DNA damage and repair within populations of mammalian cells, the comet assay has now found applications across diverse domains, including yeast, protozoa, plants, and invertebrates. This technique has proven invaluable in cryopreservation studies, serving as a valuable adjunct for determining suitable cryopreservation protocols. These protocols encompass choices related to cryoprotectants, sample preparation, as well as storage conditions in terms of time and temperature. In the realm of animal cryopreservation research, the comet assay stands as a gold-standard method for assessing DNA integrity. Nevertheless, when applied in plant-oriented investigations, additional efforts are essential due to the distinct nature of plant cells and associated technical challenges. This review elucidates the fundamental principles underlying the comet assay, discusses its current iterations, and delineates its applications in the cryopreservation of both animal and plant specimens. Moreover, we delve into the primary challenges confronting the comet assay's utility as a monitoring tool in the context of plant sample cryopreservation.

Development of a Novel, Automated High-Throughput Device for Performing the Comet Assay.

A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Human Genome & Origin of Mitochondria

It had been believed that when individual eukaryotic cells engulfed aerobic and photosynthetic bacteria, the bacteria became endosymbionts with the phagocyte and evolved into mitochondria and chloroplasts respectively. The key objective of this study is to prove that the Endosymbiotic Theqory about the origins of mitochondria and chloroplasts is a misleading lie, being ridiculously laughable. Each of the micrographs or videos in the Figures, and each of the concrete evidences listed were designed to have targetful aims. The micrographs, videos, and concrete evidences were devised to serve as addressive findings (results). The human mitochondrial DNA is a transcript obtained when two separate spliced or mature RNAs (of DNA type) transcribed from the nuclear Genome of human oocyte have annealed into double-stranded transcript and exported into the cytoplasmic organelle called mitochondrion (plural, mitochondria) that mainly performs the task of producing energy (ATP) to power the genomic reactions of the cell. Lynn Margulis’s Endosymbiotic Theory which stated that mitochondrion & chloroplast found in eukaryotic cells had evolved from bacteria when they were engulfed by eukaryotic cells, was a misleading lie that would had been ridiculously laughable!!!!

Modeling poliovirus replication dynamics from live time-lapse single-cell imaging data

Viruses experience selective pressure on the timing and order of events during infection to maximize the number of viable offspring they produce. Additionally, they may experience variability in cellular environments encountered, as individual eukaryotic cells can display variation in gene expression among cells. This leads to a dynamic phenotypic landscape that viruses must face to replicate. To examine replication dynamics displayed by viruses faced with this variable landscape, we have developed a method for fitting a stochastic mechanistic model of viral infection to time-lapse imaging data from high-throughput single-cell poliovirus infection experiments. The model’s mechanistic parameters provide estimates of several aspects associated with the virus’s intracellular dynamics. We examine distributions of parameter estimates and assess their variability to gain insight into the root causes of variability in viral growth dynamics. We also fit our model to experiments performed under various drug treatments and examine which parameters differ under these conditions. We find that parameters associated with translation and early stage viral replication processes are essential for the model to capture experimentally observed dynamics. In aggregate, our results suggest that differences in viral growth data generated under different treatments can largely be captured by steps that occur early in the replication process.

Single-Probe Mass Spectrometry Analysis of Metabolites in Single Cells.

Mass spectrometry (MS) is an indispensable analytical technique for bioanalysis. Based on the measurement of mass/charge ratios (m/z) of ions, MS can be used for sensitive detection and accurate identification of species of interest. In traditional studies, MS is utilized to measure analytes in prepared solutions or gas-phase samples. Benefited from recent development of sampling and ionization approaches, MS has been extensively applied to the analysis of broad ranges of biological samples. We have developed a new device, the Single-probe, that can be used for in situ, real-time MS analysis of metabolites inside individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip size smaller than 10μm, we can insert the Single-probe tip into single cells to extract intracellular compounds, which are analyzed using MS in real-time. We have successfully used the Single-probe MS technique to detect a variety of endogenous and exogenous cellular metabolites in individual eukaryotic cells. Single cell mass spectrometry (SCMS) is a new scientific technology that has the potential to reshape approaches in biological and pharmaceutical bioanalytical research.

Comet and micronucleus assays for analyzing DNA damage and genome integrity.

Detection of DNA damage in cells is fundamental for the study of DNA repair and genome-instability associated processes including carcinogenesis. Many studies often rely on cytotoxicity assays to estimate genotoxicity. However, measurements of cytotoxicity, a delayed outcome requiring high threshold genotoxicity to induce, does not provide information about the subtle, early genotoxic effects relevant for mechanistic understanding of DNA repair processes. Here describe how to combine two simple procedures for monitoring the presence of DNA damage in individual eukaryotic cells using: (1) the Comet assay for measuring initial DNA breaks and (2) the Micronucleus assay for detecting delayed outcome DNA breaks in dividing cells. We discuss the principles, experimental design considerations and troubleshooting tips for optimizing these methods. They require standard molecular biology instruments and a fluorescent microscope.

Abstract A16: Evaluation of the genotoxicity of ruthenium complex, trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6, by comet assay in vitro on breast cells and in vivo

Abstract Cancer remains a major public health problem, accounting for one in six deaths worldwide. One of the most common types of cancer in women is breast cancer, which is extremely heterogeneous and composed of different subtypes. The majority of drugs currently used in cancer treatment show no selectivity for tumor cells, provoking undesired side effects. Cisplatin, discovered more than 40 years ago, and successive generations of platinum-based antitumor drugs have demonstrated that metal complexes may play an important role in the cancer treatment. Metals have unique characteristics that can be exploited by the chemistry coordination for the design of new drugs with fewer side effects. In the last two decades, ruthenium complexes have been widely studied and gained prominence for cancer treatment due to their unique characteristics, such as high rate of ligand exchange, accessible oxidation states, and the ability to mimic the iron to bind biologic molecules. This study aimed to evaluate the genotoxicity effects of the ruthenium complex trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 in vitro and in vivo by the comet assay, which can be used to measure DNA damage in individual eukaryotic cells. Briefly, for in vitro assays, MDA-MB-231 malignant breast cells and MCF-10A nonmalignant breast cells were seeded (1x105/1000µL) into sterile 12-well plates. Cells were allowed to grow at 37°C in 5% CO2 for 24h and then treated or not (control) with different concentrations (2, 4 or 8µM) of the ruthenium complex or positive control cisplatin (8µM) for 1h. After this period, cells were harvested, centrifuged and resuspended in 500μL of culture medium. For in vivo assays, the animals, swiss mice, were kept in a climate-controlled environment (22±2°C) with a 12/12h light/dark cycle and food and water ad libitum. The Animal Bioethics Committee (CEUA/UNESP FAMEMA, Marília, São Paulo, Brazil) approved this study (protocol no. 828/2016). Mice were divided into five groups with five animals in each group. The ruthenium complex (12.5, 25 or 50mg/kg), positive control doxorubicin (20mg/kg), or negative control (vehicle - DMSO) were administered intraperitoneally for 3 days at 24h interval. On the third day, peripheral blood from the tail vein was collected after 4h after the administration of the last treatment. Then both, cell suspension or blood, were mixed with 120µL of 0.5% low-melting point agarose and dropped on slides precoated with 1.5% normal melting point agarose and taken to a lysis solution for 1h. After denaturation (20min) and alkaline electrophoresis (25V, 300mA, 20min) the slides were neutralized and fixed. The slide staining was performed with GelRed® (Uniscience) and comet analysis of at least 150 random cells per group was done visually according to comet tail size using a fluorescence microscope (Olympus BX61-TRF5). In vitro results of genotoxic evaluation of trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 demonstrate that this complex was able to induce significant DNA damage in MDA-MB-231 cells at the highest concentrations (4 and 8μM), as well as the positive control, cisplatin (8μM). However, the complex did not cause DNA damage in MCF-10A cells; only cisplatin induced damage to these cells. The results of genotoxic evaluation of in vivo assays indicated that only the group of animals treated with the highest dose of the complex (50mg/kg) and the animals treated with doxorubicin presented DNA damage. These results demonstrate that this complex causes DNA damages in vitro with more specificity for tumor cells and that only in the highest dose it causes DNA damages in vivo. Therefore, more studies should be performed to evaluate its potential for breast cancer treatment. Citation Format: Amanda Blanque Becceneri, Cecília Patrícia Popolin, Ana Maria Plutin, Edson Luis Maistro, Alzir Azevedo Batista, Márcia Regina Cominetti. Evaluation of the genotoxicity of ruthenium complex, trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6, by comet assay in vitro on breast cells and in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A16.

Plasma membrane repair

Tissue wound repair has been studied extensively. It involves the coordinated activation of several intracellular and intercellular pathways, as well as remodeling from the sequential recruitment of different cell types to the wound site. There is, however, an equally important process that happens at the single cell level, when the integrity of the plasma membrane is compromised. Individual eukaryotic cells can rapidly repair their plasma membrane after injury, through a process that restores internal homeostasis and prevents cell death. Despite its importance, investigations of this fascinating mechanism have been limited. Only recently have we begun to understand that plasma-membrane repair resembles tissue healing, in the sense that it also involves sequential, highly localized remodeling steps that ultimately eliminate all traces of the injury.

Genotoxic Effect of Insect Growth Regulators on Different Stages of Peach Fruit Fly,Bactrocera zonata(Saunders) (Diptera: Tephritidae)

The present study aimed to evaluate the genotoxic effect of insect growth regulators (IGRs) pyriproxyfen and novaluron on the fully grown larvae and 1-day-old pupae of Bactrocera zonata. The genotoxic investigations occurred on the whole body cells of adults that emerged from the fully grown larvae in sand treated with lethal concentrations (LC50 = 0.96, 11.4ppm of pyriproxyfen and novaluron, respectively) and those emerged from the 1-day-old pupae in sand treated with lethal concentrations (LC50 = 4.5, 6200 ppm of pyriproxyfen and novaluron, respectively). Comet assay is the technique used in this study. The comet assay (SCGE - single-cell gel electrophoresis) is often used in measurements of the level of DNA strand breaks in individual eukaryotic cells as a rapid, sensitive and inexpensive method. However, the IGR is considered to have little human toxicity because humans do not make chitin and do not make or use the hormones that insects use in moulting, but it was noticed that IGR-treated insects resulted a significant increase in DNA damage values expressed as tail length (TL), tail moment (TM) and DNA tail%in the body cells compared to the control insects. These results mean that workers should be careful when using IGRs in the field due to the conservation of genes and pathways affecting key biological processes between flies and humans.

Interrogating the variation of element masses and distribution patterns in single cells using ICP-MS with a high efficiency cell introduction system.

Cellular heterogeneity is an inherent condition of cell populations, which results from stochastic expression of genes, proteins, and metabolites. The heterogeneity of individual cells can dramatically influence cellular decision-making and cell fate. So far, our knowledge about how the variation of endogenous metals and non-metals in individual eukaryotic cells is limited. In this study, ICP-MS equipped with a high efficiency cell introduction system (HECIS) was developed as a method of single-cell ICP-MS (SC-ICP-MS). The method was applied to the single-cell analysis of Mn, Fe, Co, Cu, Zn, P, and S in human cancer cell lines (HeLa and A549) and normal human bronchial epithelial cell line (16HBE). The analysis showed obvious variation of the masses of Cu, Fe, Zn, and P in individual HeLa cells, and variation of Fe, Zn, and P in individual A549 cells. On the basis of the single-cell data, a multimodal distribution of the elements in the cell population was fitted, which showed marked differences among the various cell lines. Importantly, subpopulations of the elements were found in the cell populations, especially in the HeLa cancer cells. This study demonstrates that SC-ICP-MS is able to unravel the extent of variation of endogenous elements in individual cells, which will help to improve our fundamental understanding of cellular biology and reveal novel insights into human biology and medicine. Graphical abstract The variations of masses and distribution patterns of elements Mn, Fe, Co, Cu, Zn, P, and S in single cells were successfully detected by ICP-MS coupled with a high efficiency cell introduction system (HECIS).

Cell classification with low-resolution Raman spectroscopy (LRRS)

The identification of individual eukaryotic and prokaryotic cells is the backbone of clinical pathology and provides crucial information about the genesis and progression of a disease. While most commonly fluorescent-label based methods are applied, label-free methods, such as Raman spectroscopy, are elegant alternatives. A major disadvantage of Raman spectroscopy is the low signal yield resulting in long acquisition times, making it impractical for high-throughput clinical analysis. As a rule, Raman-based cell identification relies on high-resolution Raman spectra. This comes at a cost of detected Raman photons. In this letter we show that while the proper biochemical characterization of cells requires high-resolution Raman spectra, the proper classification of cells does not. By varying the slit-width between 50µm and 500µm it is possible to show that detected Raman signal from eukaryotic cells increased up to seven-fold. Raman-based cell classification was performed on three cancer cell lines: Jurkat, MiaPaca2, and Capan1, at three different resolutions 8cm-1 , 24cm-1 , and 48cm-1 . Moreover, we have simulated the resolution decrease due to low-diffraction gratings by binning neighboring pixels together. In both cases the cells were well classifiable using support vectors machine (SVM). For anyone working in the field of Raman spectroscopy this picture of Sir C.V. Raman is recognizable, even with reduced spatial resolution. Raman spectra of eukaryotic cells can also be recognized even with six fold reduced spectral resolution.

The single-probe: a miniaturized multifunctional device for single cell mass spectrometry analysis.

We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 μm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.

Use of the comet assay technique for quick and reliable prediction of in vitro response to chemotherapeutics in breast and colon cancer.

BackgroundDetermination of response to chemotherapy is a major requirement of personalized medicine. Resistance, whether developed or native, critically affects a treatment’s success. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. We assessed the use of comet assays in determining response to chemotherapeutic drugs that are widely used in breast and colon cancer.ResultsWe treated human breast and colon cancer cell lines with melphalan, cisplatin, mechlorethamine or doxorubicin, as monotherapies. Drug activities varied even in the same cancer types, further demonstrating the heterogeneity of different cancer types.ConclusionThe comet assay technique can provide reliable and quick results with minimum requirements and is applicable to a wide variety of drugs.

Innovations in cytometry methods and technology

of existing methods and development ofnew techniques for upcoming biomedical problems are thecorner stones of scientific discovery. In many cases, these inno-vations are question driven. However, in other cases, the tech-nical advances are the driving force and lay the foundation fornew discoveries. Cytometry as a multi-disciplinary science isby nature the source for both innovative discoveries enabledby new methods and assays as well as by fascinating newinstrumentation (may it be hardware or software).One important approach for innovation is simplification.Kiesel and coworkers (this issue, page 317) developed a simplemicrofluidic cytometer that allows absolute counting of la-beled cells in suspension in the absence of elaborate optics.Their approach is termed ‘‘spatially modulated fluorescenceemission’’ and is based on mathematical correlation of eventsthat move behind a grid of bars of varying wideness that arepositioned between microfluidic channel and a light detectingcharge coupled device (CCD) array. The resulting data displaya high signal-to-noise ratio and yield data comparable to thoseobtained by traditional but more complex (and expensive)flow cytometers. Such cytometers of low complexity and pri-cing are dedicated for use in resource limited places, specia-lized on a limited diagnostic range and underline the effortstaken by cytometrists in this area (1,2).Alternatively, simplifying the preanalytics by the aban-donment of staining or even preparative procedures mayrender an additional insight into biological processes andreduce costs. One such label free approach is made possible byanalyzing the complex patterns of light scattering by singlecells in flow systems as demonstrated for microbes (3). Panand colleagues (this issue, page 284) went a step further andmeasured two-dimensional light scattering patterns of indivi-dual eukaryotic cells sprayed by an ink jet aerosol generator.By using autocorrelation analysis and modeling of cell scatter-ing, patterns can be obtained that may in the future lead tosimplified cell identification and discrimination between celltypes and between different states of cell activation and devel-opment.Both aforementioned studies used complex mathematicaltools to analyze their measurements and identify and charac-terize individual cells. Indeed, the complexity of data pro-duced by new instrumentation is generating the demand fornew ways to view and understand them. The work presentedby Malkassian and coworkers (this issue, page 263) is a verygood example. The authors used a submersible flow cytometer(4) that collects data from phytoplankton in a spatiotemporalmanner. These data provide a rich wealth of informationregarding shape, size, scattering, autofluorescence, amongothers. The mathematical approach proposed enables thefunctional analysis and classification to better understand thecomplexity and population dynamics of complex biocenosisin a changing environment. The application may be furtherextended to the temporal analysis of cytomes in health anddisease, for example, in clinical monitoring.Considering that cytometry is not exclusively restrictedto the analysis of single suspended events in flow but also toimage-based cytometry (alternatively, image cytometry, ima-ging cytometry, slide based cytometry... unfortunately, there isstill no unified idiom for cytometry on the microscope!) ofimmobilized specimens additional challenges arise. One ofthese challenges is the automated recognition of areas that arerelevant for analysis, such as areas covered by tissue fragmentsas in tissue micro arrays (5). This problem is particularly rele-vant for high-throughput image cytometry. Zeder and co-workers (this issue, page 306) addressed a new challenge: theanalysis of microbial communities in aquatic systems by sam-

The comet assay: a method to measure DNA damage in individual cells

We present a procedure for the comet assay, a gel electrophoresis-based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.

266 ORAL NCIC CTG MA17: Increasing benefit of letrozole with longer duration of treatment as measured by the hazard ratio of disease recurrence over time

A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH–LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow). River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed. Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH–LBA technique. In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix. The analysis could be performed on different levels of complexity. The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies. Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells. Consequently, application of the FISH–LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.

'Swell dialysis' demonstrates that adenylate cyclase in Trypanosoma brucei is regulated by calcium ions.

A new technique, 'swell dialysis', is described that allows the study of cellular enzymes in situ if they are located in the cytoplasm or on the cytoplasmic face of any membrane. The technique is potentially applicable to any population of individual eukaryotic cells that lack a cell wall. In this study swell dialysis has been applied to an examination of the regulation of adenylate cyclase in blood-stream forms of Trypanosoma brucei (427-12/ICI-060). Adenylate cyclase in this protozoon is shown to be controlled by Ca2+. There is a 16-fold stimulation of the cyclase at 100 microM Ca2+ over basal activity without added Ca2+. The ability of Ca2+ to stimulate adenylate cyclase is lost upon rupture of the cell, which is reminiscent of the loss of control of the adenylate cyclase in Escherichia coli by sugars of the phosphotransferase system when cell breakage occurs. The physiological function of the Ca2+ activation of adenylate cyclase in T. brucei has not been established but a possible role in the change of surface coat in bloodstream forms should be considered.