Transcriptional Profiling of Adult Neural Stem-Like Cells from the Human Brain

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.     

Massively Parallel Single Nucleus Transcriptional Profiling Defines Spinal Cord Neurons and Their Activity during Behavior

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Simultaneous Interferometric Measurement of Corrosive or Demineralizing Bacteria and Their Mineral Interfaces

Here, we report simultaneous surface profile measurements of several bacterial species involved in microbially influenced corrosion and their solid-surface interfaces by using vertical scanning interferometry. The capacity to nondestructively quantify microscale topographic changes beneath a single bacterium without its removal offers a unique opportunity to examine in vivo microbe-surface interactions.As microbiology advances, the relevance of bacterial surface interaction has become abundantly apparent, and the study of microbial communities attached to surfaces (also known as biofilms) has become a major focus. As biofilms form and develop, the surfaces to which they selectively attach may be altered through the activities of the resident microbes, as in microbially influenced corrosion, involving the release of chemicals or the deposition of electrochemically active minerals that accelerate surface demineralization and corrosion, with repercussions spanning from cavity formation in teeth to pit development on a ship''s hull (1-4, 6-8, 10, 11, 15, 17-20). Current methods for the characterization of altered surfaces require the removal of the causative bacteria, terminating the process. Noninvasive, visual methods that provide a quantitative understanding of a corroding solid surface and associated bacterial interactions are highly desirable.Here, we describe the use of vertical scanning interferometry (VSI) to directly measure the surface profiles of both microbes and the surface beneath them. VSI, a rapid, noncontact, minimally invasive technology, combines reflected-light microscopy with Mirau interference optics, using the wave properties of light to precisely define a vertical dimension, with resolutions of <1 μm laterally and <1 nm vertically (12-14). Light reflected from the sample (returned as interferometric fringes) is converted into graphical data called a correlogram and analyzed for peaks to extrapolate the height (z dimension) for every pixel of an image, resulting in a topographical map of a surface (see Fig. S1 and S2 in the supplemental material).Previously, we described bacterial imaging artifacts associated with light reflected from the surface beneath the bacteria (21). Expanding on techniques used to measure thin-film depth (16), we hypothesized that making minor changes in data acquisition and interpretation would allow us to use the light reflected from both the bacteria and their interface to (i) correct VSI imaging artifacts commonly seen when attempting to visualize bacteria on a surface and (ii) access a second set of fringes from the surface beneath a bacterium.To evaluate imaging defects on a larger scale, the evaporating edges of several different water drops on mirror steel were scanned by VSI (Fig. ​(Fig.1).1). Correlogram analyses of a series of points along each drop, moving from deep to shallow, revealed five factors key to imaging interfaces though transparent particles: (i) two correlogram peaks were acquired for every pixel of the video where water covered the polished steel, versus one correlogram peak per pixel for dry polished steel; (ii) as the water depth decreased, so did the distance between the two peaks; (iii) if the amplitude of peak 1 was greater than that of peak 2, then the expected three-dimensional (3-D) image height was obtained, and if the amplitude of peak 1 was less than that of peak 2, then the 3-D image height was lower than expected; (iv) based on a known location of the steel, by using the first peak, the actual height of the water''s surface was calculated; and (v) the second peak corresponded to the known location of the steel after correction for the known refractive index (see the supplemental material).Open in a separate windowFIG. 1.Measurement of the edge of a drop of water and its steel interface. Above the correlograms is a 3-D height map of the edge of a drop of water on polished steel, made using current software. Current software, clearly, has miscalculated the locations of points 3 to 5. Superimposed is a recalculation of the height of the water surface at all six points (displayed as red dots and a red line), based on the interpretation of the correlograms below. The black dots with the black line are the calculation of the location of the underlying steel surface after correction for the refractive index. Below are correlograms for the six points throughout the drop of water, moving from deep to shallow. These correlograms were used to calculate the depth of the water at each of the six points and the location of the steel. The black line in each correlogram represents the height of the steel after correction for the index of refraction. The measurements in red indicate the distance of the first peak from the steel surface, corresponding to the depth of the water at each of the six points. a.u., arbitrary units.In this approach, when a correlogram for a bacterium is acquired, the peak amplitude defines the surface reflectivity and the distance between peaks defines the thickness (the distance from the microbe surface to the interface) after correction for refractive index changes (see Fig. S3 in the supplemental material). Using facultative anaerobe Shewanella oneidensis MR-1, a metal oxide-reducing bacterium capable of protecting steel from corrosion (5), we measured bacterial height on polished mirror steel. Height measurements were highly variable; individual bacteria with no morphological abnormalities (as determined by environmental scanning electron microscopy) were commonly interpreted as structures that sat both above and below the steel surface (Fig. ​(Fig.2A),2A), and two-peak correlograms from each area supported the amplitude and height findings from the water experiments described above (Fig. ​(Fig.2B).2B). Measurements made via scanning near-field optical microscopy, a method that measures light passage through a medium, confirmed translucence variability within a single cell, consistent with VSI height measurement variability (data not shown).Open in a separate windowFIG. 2.Comparison of VSI images of S. oneidensis MR-1 on steel that measure above and below a polished steel surface. (A) Comparison of images of a single MR-1 bacterium that measures both as a pit in the steel and as a bump on the steel by VSI (bottom panel) and measures normally under an environmental scanning electron microscope (ESEM) (top panel). (B) Comparison of correlograms for a bacterium that falsely measures as a pit and a bacterium that measures normally (as a bump). If the amplitude of peak 1 (red arrowheads) is smaller than that of peak 2 (black arrowheads), then the bacterium appears as a pit (left panel). If the amplitude of peak 1 is larger than that of peak 2, then the bacterium appears as a bump on the surface of the steel (right panel). In both the environmental scanning electron microscope and VSI images, two additional bacteria that appear normally are displayed to the left for comparison. a.u., arbitrary units.To expand the range of VSI data acquisition to conditions that were suboptimal in reflectivity but were known to be capable of corroding or demineralizing a particular substrate, we measured S. oneidensis MR-1 on calcite (Fig. ​(Fig.3A),3A), Streptococcus mutans UA159 on hydroxyapatite (Fig. ​(Fig.3B),3B), and a sulfate-reducing bacterium isolated from environmental sludge on steel (Fig. ​(Fig.3C);3C); each of these organisms is known to be involved in the corrosion or demineralization of the respective contacting substrate. VSI measurements of the bacteria (confirmed to be accurate by atomic force microscopy) consistently resulted in two correlogram peaks. Current VSI software can improperly calculate the pixel height of a transparent particle on a surface, resulting in height measurement errors, such as the false pit in the VSI image in Fig. ​Fig.3C.3C. Each calculated bacterial height, based on the first correlogram peak, yielded an expected measurement, regardless of 3-D height map defects generated with current VSI software (Fig. ​(Fig.3;3; data not shown).Open in a separate windowFIG. 3.Measurement of demineralizing or corrosive bacteria and their interface with a biologically relevant substrate. For all image sections, the left panels show overlapping 3-D measurements of bacteria made by VSI (top) and an atomic force microscope (AFM; bottom). The red arrowheads in both images mark the pixel where the correlogram (right) was acquired from a VSI scan. The red arrowheads to the right of the correlograms highlight the peak indicative of the bacterial surface. The black arrowheads highlight the peak indicative of the interface. (A) S. oneidensis MR-1 on calcite. The black arrowhead highlights the peak indicative of the calcite surface. (B) S. mutans UA159 on hydroxyapatite. The black arrowhead highlights the peak indicative of the hydroxyapatite surface. (C) Sulfate-reducing bacteria on steel. The black arrowhead highlights the peak indicative of the steel surface. a.u., arbitrary units.In this study, we have shown how to (i) correct imaging artifacts generated with current software and (ii) simultaneously measure a bacterial surface and the surface on which it sits, without removing the potentially active surface-modifying bacteria. This constitutes the first step toward our goal of directly measuring surface modifications (dissolution or deposition) as they occur. With the adaptation of VSI for higher resolution, minor software adjustments, and calibration improvements, we should be able not only to measure dissolution beneath a single cell in real time but also to pinpoint the location where microbially influenced corrosion actually begins (if any exists). For more detail on making accurate measurements, see the supplemental material.Clearly, the ability to noninvasively study the microbe or particle-solid interface in real time has broad relevance, ranging from microbial ecology to medicine to material sciences and semiconductors. By understanding the nature of microbe-solid interface interactions, it may be possible to have an impact on processes such as microbially influenced corrosion, caused by sulfate-reducing bacteria that electrochemically corrode steel, or medical processes, such as the demineralization of tooth enamel by S. mutans lactic acid secretions (6, 9, 20). By directly visualizing real-time height changes in the microbe-mineral interface and refractive index changes to a bacterial population using modifications proposed in this study, we should be able to gain insight into the processes by which bacterial biofilms modify their interactive surfaces.     

Forms and Fates of Rhizobial Bacteria Present in the Infected Host Cells of Nodules

There were two forms of rhizobial bacteria present in infected host cells of nodules. One was bacteroids which were enclosed in peribacteroid membrane originated from the infected host cells. The other was rhizobia as vegetative cells. The infected host cells were occupied by most of the bacteroids and a certain number of the vegetative cells respectively. With the nodule senescence, there were two kinds of fate of the bacteria: The bacteroids degenerated togather with the infected host cells at the same time and further disintegrated completely, so it is not possible that the disintegrated bacteroids could be returned into soil to revive: the vegetative cells did not disintegrate and die when the infected host cells senesced, eventually could be turned back into soil. The vegetative cells may play an important role, on the one hand, in cycle between legume and soil, on the other hand, maintain rhizobia in natural balance of population ecosystem.     

Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 _{(55AAAAAAA61)} and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.     

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2'',7''-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.     

Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.