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Background: Circulating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.Methods: We developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.Results: The microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines' metastatic ability.Conclusions: The microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes. [ABSTRACT FROM AUTHOR]

Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis. [ABSTRACT FROM AUTHOR]

Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks ( BacNet15 ) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (-omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria. [ABSTRACT FROM AUTHOR]

The article discusses the widespread use of Ibrutinib since its initial U.S. Food and Drug Administration approval for relapsed CLL 4 years ago. It notes that around 20% of treatment discontinuations in both the treatment-naive and relapsed or refractory cohorts were attributed to AEs over 5-year follow-up, which compares favorably to the 42% discontinuation rate at a median of 17 months in a real-world report of ibrutinib use.

New bacterial detection and imaging methods are desirable for diagnostics and healthcare applications, as well as in basic scientific research. We present a simple analytical platform for bacterial detection and imaging based upon attachment of amphiphilic carbon dots (CDs) to bacterial cells. We show that CDs functionalized with hydrocarbon chains readily bind to bacterial cells following short incubation and enable detection of bacteria through both fluorescence spectroscopy and microscopy. Importantly, we demonstrate that the intensity and spectral position of the carbon dots’ fluorescence depend upon bacterial species, providing a tool for distinguishing among bacteria even in cases of mixed bacterial populations. Moreover, bacterial labelling with the amphiphilic CDs enables visualization of physiological processes such as cell division. [ABSTRACT FROM AUTHOR]

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus. [ABSTRACT FROM AUTHOR]

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs. [ABSTRACT FROM AUTHOR]

Summary Over 30 years ago, ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein, we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one-carbon metabolism. Biochemical data confirm that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages. [ABSTRACT FROM AUTHOR]

Exopolymers are known to be useful in improving sand aggregation and the development of biological soil crusts (BSCs). A facultative bacterium KLBB0001 was isolated from BSCs in the Gurbantunggut Desert in northwestern China. With the strong effective production of extracellular polymeric substances (EPS), this strain exhibits a multifunctional role for sand stabilization and maintenance of water under laboratory conditions. Practical testing of the feasibility of its inoculation to speed up BSC recovery in the field was also conducted in this experiment. This strain stimulated the heterotrophic community assembly in the topsoil layer (0-2 cm) before the commencement of autotrophic cyanobacteria, while also significantly increasing the number of bacteria, actinomycetes, and content of total phosphorus, available nitrogen, and available phosphorus. However, the low nitrogenase activity (NA) (0.57 µmol/h) that was observed caused us to doubt the previous identification as Azotobacter Beijerinck that was based on physiological and biochemical properties. A phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain was a member of the genus Paenibacillus. It exhibited the closest phylogenetic affinity and highest sequence similarity to the strain Paenibacillus mucilaginosus VKPM B-7519 (sequence similarity 99.698%), which is well known as a typical silicate-weathering bacteria that releases lots of nutritional ions from minerals and the soil. Because P. mucilaginosus can excrete carbonic anhydrase (CA) to capture atmospheric CO2 through hydration of CO2, it is possible that KLBB0001 might use a similar strategy for heterotrophs in the BSCs to sequester CO2 from the air. Because of its potential role in the reestablishment of the BSC ecosystem due to its ability to improve water relations, sand stabilization, and chemical erosion, EPS-producing bacterial inoculation was concluded to be a suitable and effective treatment for BSC recovery, especially in environments with limited water and nutrients. [ABSTRACT FROM AUTHOR]

Bacillus subtilis has been used for over 50 years as a model organism for biochemistry, genetic, molecular biology and cell biology studies. More recently, its spore has been proposed as a platform to display heterologous proteins and as a vehicle for mucosal vaccination. We characterize here the spore surface of four human intestinal strains of B. subtilis, previously identified as able to grow anaerobically and form biofilm. These properties, lost in laboratory strains, are relevant for the colonization of human mucosal sites and likely to improve the efficiency of strains to be used for mucosal delivery. Our characterization is an essential preliminary step for the development of these intestinal strains as display systems and has indicated that spores of at least one of them are more efficient than the laboratory strain for the non-recombinant display of two model heterologous proteins. [ABSTRACT FROM AUTHOR]

Abstract: The impact of multi-well plate automation on bacterial flow cytometric analyses was investigated. Cell concentrations in up to 96 samples can be measured accurately, as long as a reproducible staining protocol and a total measurement time of below 80min is used. Fluorescence distribution in the samples may, however, display some variability. [Copyright &y& Elsevier]

Background: Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. Results: We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Conclusions: Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins. [ABSTRACT FROM AUTHOR]

The components of the bacterial division machinery assemble to form a dynamic ring at mid-cell that drives cytokinesis. The nature of most division proteins and their assembly pathway is known. Our knowledge about the biochemical activities and protein interactions of some key division elements, including those responsible for correct ring positioning, has progressed considerably during the past decade. These developments, together with new imaging and membrane reconstitution technologies, have triggered the ‘bottom-up’ synthetic approach aiming at reconstructing bacterial division in the test tube, which is required to support conclusions derived from cellular and molecular analysis. Here, we describe recent advances in reconstituting Escherichia coli minimal systems able to reproduce essential functions, such as the initial steps of division (proto-ring assembly) and one of the main positioning mechanisms (Min oscillating system), and discuss future perspectives and experimental challenges. [ABSTRACT FROM AUTHOR]

Summary: Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest. [ABSTRACT FROM AUTHOR]

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction. [ABSTRACT FROM AUTHOR]

In this issue of Developmental Cell , Isabella and Horne-Badovinac (2016) show that Rab10 directs site-specific secretion of basement membrane components, which assemble into fibrils that spool out to elongate the Drosophila egg chamber. These findings establish the basement membrane’s active role in tissue sculpting. [ABSTRACT FROM AUTHOR]

With the increase in scientific activity globally, the geographical focus of basic research is shifting away from the West. At the same time, multidisciplinary approaches are uncovering new layers in our understanding of how cells work. How will these trends affect cell biology in the near future? [ABSTRACT FROM AUTHOR]

Abstract: We designed and tested equipment to wash plugs following cell lysis in pulsed-field gel electrophoresis (PFGE). Our system can wash 30 plugs simultaneously in 1h using 15L of Tris–EDTA buffer, which makes plug washing for PFGE less labor-intensive. [Copyright &y& Elsevier]

Although proinflammatory cytokines such as TNF are critical for containment of tuberculosis, they can also exacerbate disease when produced at high levels. In this issue of Cell, Roca and Ramakrishnan demonstrate that high TNF production induces reactive oxygen species in infected macrophages, ultimately leading to macrophage necrosis and bacterial dissemination. [ABSTRACT FROM AUTHOR]

Bacteria lack many of the features that eukaryotic cells use to compartmentalize cytoplasm and membranes. In this issue, Schlimpert et al. describe a new mechanism of spatial confinment in the bacterium Caulobacter crescentus that prevents the exchange of soluble and membrane proteins between the stalk and cell body. [ABSTRACT FROM AUTHOR]

The article reports that a test has been conducted to explore the ability of bacterial cytological profiling (BCP) to further find out how antimicrobials work.