Nitrogen and phosphorus limitation of oceanic microbial growth during spring in the Gulf of Aqaba

Bioassay experiments were performed to identify how growth of key groups within the microbial community was simultaneously limited by nutrient (nitrogen and phosphorus) availability during spring in the Gulf of Aqaba's oceanic waters. Measurements of chlorophyll a (chl a) concentration and fast repetition rate (FRR) fluorescence generally demonstrated that growth of obligate phototrophic phytoplankton was co-limited by N and P and growth of facultative aerobic anoxygenic photoheterotropic (AAP) bacteria was limited by N. Phytoplankton exhibited an increase in chl a biomass over 24 to 48 h upon relief of nutrient limitation. This response coincided with an increase in photosystem II (PSII) photochemical efficiency (F v /F m), but was preceded (within 24 h) by a decrease in effective absorption crosssection (σPSII) and electron turnover time (τ). A similar response for τ and bacterio-chl a was observed for the AAPs. Consistent with the up-regulation of PSII activity with FRR fluorescence were observations of newly synthesized PSII reaction centers via low temperature (77K) fluorescence spectroscopy for addition of N (and N + P). Flow cytometry revealed that the chl a and thus FRR fluorescence responses were partly driven by the picophytoplankton (æ10 μm) community, and in particular Synechococcus. Productivity of obligate heterotrophic bacteria exhibited the greatest increase in response to a natural (deep water) treatment, but only a small increase in response to N and P addition, demonstrating the importance of additional substrates (most likely dissolved organic carbon) in moderating the heterotrophs. These data support previous observations that the microbial community response (autotrophy relative to heterotrophy) is critically dependent upon the nature of transient nutrient enrichment. © Inter-Research 2009

Omacetaxine may have a role in chronic myeloid leukaemia eradication through downregulation of Mcl-1 and induction of apoptosis in stem/progenitor cells

Chronic myeloid leukaemia (CML) is maintained by a rare population of tyrosine kinase inhibitor (TKI)-insensitive malignant stem cells. Our long-term aim is to find a BcrAbl-independent drug that can be combined with a TKI to improve overall disease response in chronic-phase CML. Omacetaxine mepesuccinate, a first in class cetaxine, has been evaluated by clinical trials in TKI-insensitive/resistant CML. Omacetaxine inhibits synthesis of anti-apoptotic proteins of the Bcl-2 family, including (myeloid cell leukaemia) Mcl-1, leading to cell death. Omacetaxine effectively induced apoptosis in primary CML stem cells (CD34<sup>+</sup>38<sup>lo</sup>) by downregulation of Mcl-1 protein. In contrast to our previous findings with TKIs, omacetaxine did not accumulate undivided cells <i>in vitro</i>. Furthermore, the functionality of surviving stem cells following omacetaxine exposure was significantly reduced in a dose-dependant manner, as determined by colony forming cell and the more stringent long-term culture initiating cell colony assays. This stem cell-directed activity was not limited to CML stem cells as both normal and non-CML CD34<sup>+</sup> cells were sensitive to inhibition. Thus, although omacetaxine is not leukaemia stem cell specific, its ability to induce apoptosis of leukaemic stem cells distinguishes it from TKIs and creates the potential for a curative strategy for persistent disease

Spectral imaging in preclinical research and clinical pathology.

Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, pre-clinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle

The dynamics of genome replication using deep sequencing

Peer reviewedPublisher PD

Single-cell western blotting.

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution

Long-lasting cross-protection against influenza A by neuraminidase and M2e-based immunization strategies

There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus

Linked optical and gene expression profiling of single cells at high-throughput.

Single-cell RNA sequencing has emerged as a powerful tool for characterizing cells, but not all phenotypes of interest can be observed through changes in gene expression. Linking sequencing with optical analysis has provided insight into the molecular basis of cellular function, but current approaches have limited throughput. Here, we present a high-throughput platform for linked optical and gene expression profiling of single cells. We demonstrate accurate fluorescence and gene expression measurements on thousands of cells in a single experiment. We use the platform to characterize DNA and RNA changes through the cell cycle and correlate antibody fluorescence with gene expression. The platform's ability to isolate rare cell subsets and perform multiple measurements, including fluorescence and sequencing-based analysis, holds potential for scalable multi-modal single-cell analysis