Capturing actin assemblies in cells using in situ cryo-electron tomography

Actin contributes to an exceptionally wide range of cellular processes through the assembly and disassembly of highly dynamic and ordered structures. Visualizing these structures in cells can help us understand how the molecular players of the actin machinery work together to produce force-generating systems. In recent years, cryo-electron tomography (cryo-ET) has become the method of choice for structural analysis of the cell interior at the molecular scale. Here we review advances in cryo-ET workflows that have enabled this transformation, especially the automation of sample preparation procedures, data collection, and processing. We discuss new structural analyses of dynamic actin assemblies in cryo-preserved cells, which have provided mechanistic insights into actin assembly and function at the nanoscale. Finally, we highlight the latest visual proteomics studies of actin filaments and their interactors reaching sub-nanometer resolutions in cells.     

Calcium channels and cancer stem cells

Cancer stem cells (CSC’s) have emerged as a key area of investigation due to associations with cancer development and treatment resistance, related to their ability to remain quiescent, self-renew and terminally differentiate. Targeting CSC’s in addition to the tumour bulk could ensure complete removal of the cancer, lessening the risk of relapse and improving patient survival. Understanding the mechanisms supporting the functions of CSC’s is essential to highlight targets for the development of therapeutic strategies. Changes in intracellular calcium through calcium channel activity is fundamental for integral cellular processes such as proliferation, migration, differentiation and survival in a range of cell types, under both normal and pathological conditions. Here in we highlight how calcium channels represent a key mechanism involved in CSC function. It is clear that expression and or function of a number of channels involved in calcium entry and intracellular store release are altered in CSC’s. Correlating with aberrant proliferation, self-renewal and differentiation, which in turn promoted cancer progression and treatment resistance. Research outlined has demonstrated that targeting altered calcium channels in CSC populations can reduce their stem properties and induce terminal differentiation, sensitising them to existing cancer treatments. Overall this highlights calcium channels as emerging novel targets for CSC therapies.     

Capturing B type acute lymphoblastic leukemia cells using two types of antibodies

One way to monitor minimal residual disease (MRD) is to screen cells for multiple surface markers using flow cytometry. In order to develop an alternative microfluidic based method, isolation of B type acute lymphoblastic cells using two types of antibodies should be investigated. The immunomagnetic beads coated with various antibodies are used to capture the B type acute lymphoblastic cells. Single beads, two types of beads and surface immobilized antibody were used to measure the capture efficiency. Both micro and nanosize immunomagnetic beads can be used to capture B type acute lymphoblastic cells with a minimum efficiency of 94% and maximum efficiency of 98%. Development of a microfluidic based biochip incorporating immunomagnetic beads and surface immobilized antibodies for monitoring MRD can be an alternative to current cost and time inefficient laboratory methods. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2737, 2019     

Functional role of adenosine via KATP channels in cerebral capillary endothelial cells and pericytes

Purinergic Signalling -     

Archaea with square cells

Two groups of microbiologists have independently isolated 'Walsby's square bacterium' from salt crystallizer ponds; its growth depends on pyruvate. Genetic analysis shows that the squares, discovered 25 years ago on the Sinai Peninsula, are archaea rather than bacteria. These transparent tile-like cells might have been dismissed as surface artefacts of salt crystals but for their gas vesicles--structures peculiar to prokaryotic organisms. Paradoxically, the square archaea are the dominant microorganisms in some hypersaline environments and might be globally important.     

Capturing clapper rails using thermal imaging technology

Capturing sufficient numbers of marsh birds for telemetry studies is difficult due to the secretive nature and inaccessible habitat of the birds. We experimented with thermal imaging technology to locate clapper rails and subsequently capture them from an airboat in South Carolina tidal marshes. This method produced a capture rate of up to 19.2 rails/hr, which exceeded other capture rates in the literature. We believe this technique will improve capture efficiency for clapper rails, and potentially other secretive marsh birds, enhancing opportunities for studying these important species. © 2011 The Wildlife Society.     

Ion channels in death and differentiation of prostate cancer cells

Plasma membrane ion channels contribute to virtually all basic cellular processes, including such crucial ones for maintaining tissue homeostasis as proliferation, differentiation, and apoptosis. Enhanced proliferation, aberrant differentiation, and impaired ability to die are the prime reasons for abnormal tissue growth, which can eventually turn into uncontrolled expansion and invasion, characteristic of cancer. Prostate cancer (PCa) cells express a variety of plasma membrane ion channels. By providing the influx of essential signaling ions, perturbing intracellular ion concentrations, regulating cell volume, and maintaining membrane potential, PCa cells are critically involved in proliferation, differentiation, and apoptosis. PCa cells of varying metastatic ability can be distinguished by their ion channel characteristics. Increased malignancy and invasiveness of androgen-independent PCa cells is generally associated with the shift to a 'more excitable' phenotype of their plasma membrane. This shift is manifested by the appearance of voltage-gated Na(+) and Ca(2+) channels which contribute to their enhanced apoptotic resistance together with downregulated store-operated Ca(2+) influx, altered expression of different K(+) channels and members of the Transient Receptor Potential (TRP) channel family, and strengthened capability for maintaining volume constancy. The present review examines channel types expressed by PCa cells and their involvement in metastatic behaviors.     

Capturing data from three-dimensional surfaces using fuzzy landmarks

Anatomical landmarks are defined as biologically meaningful loci that can be unambiguously defined and repeatedly located with a high degree of accuracy and precision. The neurocranial surface is characteristically void of such loci. We define a new class of landmarks, termed fuzzy landmarks, that will allow us to represent the form of the neurocranium. A fuzzy landmark represents the position of a biological structure that is precisely delineated, but occupies an area that is larger than a single point in the observer's reference system. In this study, we present a test case in which the cranial bosses are evaluated as fuzzy landmarks. Five fuzzy landmarks (the cranial bosses) and three traditional landmarks were placed repeatedly by a single observer on three-dimensional (3D) computed tomography (CT) surface reconstructions of pediatric dry skulls and skulls of pediatric patients, and directly on four of the same dry skulls using a 3Space digitizer. Thirty landmark digitizing trials from CT scans show an average error of 1.15 mm local to each fuzzy landmark, while the average error for the last ten trials was 0.75 mm, suggesting a learning curve. Data collected with the 3Space digitizer was comparable. Measurement error of fuzzy landmarks is larger than that of traditional landmarks, but is acceptable, especially since fuzzy landmarks allow inclusion of areas that would otherwise go unsampled. The information obtained is valuable in growth studies, clinical evaluation, and volume measurements. Our method of fuzzy landmarking is not limited to cranial bosses, and can be applied to any other anatomical features with fuzzy boundaries. Am J Phys Anthropol 107:113–124, 1998. © 1998 Wiley-Liss, Inc.     

Capturing and profiling adult hair follicle stem cells

The hair follicle bulge possesses putative epithelial stem cells. Characterization of these cells has been hampered by the inability to target bulge cells genetically. Here, we use a Keratin1-15 (Krt1-15, also known as K15) promoter to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding enhanced green fluorescent protein (EGFP), which allow for lineage analysis and for isolation of the cells. We show that bulge cells in adult mice generate all epithelial cell types within the intact follicle and hair during normal hair follicle cycling. After isolation, adult Krt1-15-EGFP-positive cells reconstituted all components of the cutaneous epithelium and had a higher proliferative potential than Krt1-15-EGFP-negative cells. Genetic profiling of hair follicle stem cells revealed several known and unknown receptors and signaling pathways important for maintaining the stem cell phenotype. Ultimately, these findings provide potential targets for the treatment of hair loss and other disorders of skin and hair.     

Determination of Tn antigen released from cultured cancer cells by capillary electrophoresis

An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. In the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells.     

Transformation of intact cells of Saccharomyces cerevisiae by square wave pulses using castellated microelectrodes

Genetic transformation of intact cells of Saccharomyces cerevisiae with an expression vector pYES 2, to efficiencies of 10⁵ to 10⁶ by high voltage electroporation is presented. Prototrophic transformants of yeast expressing resistance to ampicillin were obtained by subjecting the mixture of cells and DNA to a single square wave pulse at an amplitude of 2.5, 2.75 and 3.0 kV/cm in combination with a pulse width of 4, 5 and 3 msec for the three different strains Y915, Y742 and INVSC 1 respectively. The critical factors and electrical parameters which determine the transformation efficiency were examined. This communication describes the optimal conditions for reproducible and high efficiency transformation of yeast by the method of electroporation.     

Amine metabolomics of hyperglycemic endothelial cells using capillary LC-MS with isobaric tagging

Intracellular amine metabolite changes were quantified from hyperglycemic human aortic endothelial cells (HAECs) as a model for macrovascular complications of diabetes. Amines were selectively tagged using the N-hydroxysuccinimide ester (NHS) based isobaric tag DiART (Deuterium isobaric Amine Reactive Tag), synthesized in house. DiART labeling improved chromatographic resolution of derivatized amines, resulted in 100-fold signal-to-noise enhancement in mass spectrometry (MS) analyses, and allowed multiplex quantification of four samples concurrently through tandem MS fragmentation. Targeted measurement of 31 DiART-tagged amines demonstrated the limits of detection below 10 nM/100 amol and averaged RSDs less than 5%. Examination of endothelial cells exposed to short-term hyperglycemia resulted in significant changes to alanine, proline, glycine, serine, and glutamine compared to osmotic controls. Discovery of proline elevation in hyperglycemic endothelial cells suggests a role of proline in hyperglycemia-mediated oxidative stress. Exposure of endothelial cells to high glucose for 7 days resulted in reduced cell number and significant changes to 21 amines relative to cell number. Prominent amine elevation from long-term hyperglycemia include aminoadipate as a sign of lysine breakdown through oxidative stress; cystathionine, hypotaurine, and proline indicating an antioxidant response; and glutamine/glutamate as substrate level activators of additional metabolic pathways. This report is the first investigation of amine changes to hyperglycemic endothelial cells and offers new insights into the pathophysiology of diabetic complications.     

Capturing crop adaptation to abiotic stress using image-based technologies

Farmers and breeders aim to improve crop responses to abiotic stresses and secure yield under adverse environmental conditions. To achieve this goal and select the most resilient genotypes, plant breeders and researchers rely on phenotyping to quantify crop responses to abiotic stress. Recent advances in imaging technologies allow researchers to collect physiological data non-destructively and throughout time, making it possible to dissect complex plant responses into quantifiable traits. The use of image-based technologies enables the quantification of crop responses to stress in both controlled environmental conditions and field trials. This paper summarizes phenotyping imaging technologies (RGB, multispectral and hyperspectral sensors, among others) that have been used to assess different abiotic stresses including salinity, drought and nitrogen deficiency, while discussing their advantages and drawbacks. We present a detailed review of traits involved in abiotic tolerance, which have been quantified by a range of imaging sensors under high-throughput phenotyping facilities or using unmanned aerial vehicles in the field. We also provide an up-to-date compilation of spectral tolerance indices and discuss the progress and challenges in machine learning, including supervised and unsupervised models as well as deep learning.     

Rapid deoxygenation of red cells and hemoglobin solution using hollow capillary fibers

A newly designed capillary deoxygenator has been constructed by using microporous polypropylene hollow fibers sealed into an airtight plexiglass housing. Oxygenated red cell suspensions and hemoglobin solutions flowing through the hollow fibers were subjected to deoxygenation with a gas mixture composed of 95 percent N2 and 5 percent CO2 passed through the housing. At a given flow rate of the oxygenated fluid, the outgoing fluid pO2 varied directly with hematocrit and inversely with the residence time. With a deoxygenator composed of 144 parallel 100-micrometers fibers with an active length of 10 cm, 2 ml of blood at 10 percent hematocrit can be converted from arterial to venous pO2 in approximately 1 min. The design of this deoxygenator provides a method for rapid deoxygenation of blood without red cell membrane damage or hemolysis.     

Enhanced gene transfer into brain capillary endothelial cells using Antp-modified DNA-loaded nanoparticles

Brain capillary endothelial cells (BCECs) have been considered as one of the primary targets for cerebral gene therapy. However, the cells, well-known for their poor function of endocytosis, are difficult to be transfected by general non-viral vectors. The aim of this study was to enhance the efficiency of transfection and expression in BCECs of DNA/polymer nanoparticles with the modification of membrane-penetrating peptide, Antennapedia peptide (Antp) polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare Antp-modified DNA-loaded nanoparticles with a complex coacervation technique. After a 20-min transfection, the efficiency, in terms of transfection and expression, of DNA/PEI NP or DNA/PAMAM NP was enhanced significantly with the modification of Antp. After a 3-h transfection of DNA/Antp/PEI NP, there was no difference in cellular uptake but an enhancement in gene expression, compared to DNA/PEI NP alone. However, both the transfection and expression efficiency of DNA/PAMAM NP were enhanced using Antp. These observations suggest that Antp can increase the membrane-penetrating ability of DNA-loaded nanoparticles, which can be employed as novel non-viral gene vectors.     

SP600125 inhibits Kv channels through a JNK-independent pathway in cancer cells

Kv channels represent new important targets for the control of cancer growth and a better understanding of their regulating pathways in cancer cells is necessary to develop therapeutic strategies. In this study, we have addressed the putative modulation of Kv by MAP kinases through a pharmacological approach. We have found that the commonly used JNK inhibitor SP600125 strongly inhibits Kv channels through a JNK-independent pathway, likely interacting directly with the channels at the external side of the membrane. Our results indicate that the use of this compound may produce misleading conclusions for the role of JNK in cell cycle.