Novel aspects of systems biology and clinical cytomics.

The area of Cytomics and Systems Biology became of great impact during the last years. In some fields of the leading cytometric techniques it represents the cutting edge today. Many different applications/variations of multicolor staining were developed for flow- or slide-based cytometric analysis of suspensions and sections to whole animal analysis. Multispectral optical imaging can be used for studying immunological and tumorigenic processes. New methods resulted in the establishment of lipidomics as the systemic research of lipids and their behavior. All of these development push the systemic approach of the analysis of biological specimens to enhance the outcome in the clinic and in drug discovery programs.     

A nanophosphor-based method for selective DNA recovery in Synthosomes

A nanocompartment system composed of an ABA triblock copolymer, where A is poly(dimethylsiloxane) and B is poly(2-methyloxazoline), has been developed for selective recovery and detection of DNA. Translocation of TAMRA-labeled complementary primers into the nanocompartment system has been achieved through two deletion mutants (FhuA Delta1-129; FhuA Delta1-160) of the channel protein FhuA. Translocation was monitored by fluorescence resonance energy transfer through hybridization of the TAMRA-labeled primer to the complementary sequence of a nanophosphor-DNA-conjugate, which reduces its half-life (FhuA Delta1-129, 16.0% reduced; FhuA Delta1-160, 39.0% reduced).     

Thermal ecology of natterjack toads (Bufo calamita) in a semiarid landscape

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To monitor the seasonal variation of body temperature (t_b) in free-ranging adult Bufo calamita, 15 toads were radio-tracked at Mas de Melons (Catalonia, Spain) using temperature-sensitive transmitters implanted to the abdominal cavity.     

A metallopolymer, [Cu(abt)]∞ (abt, 2-aminobenzenethiol) with novel structural patterns resembling black phosphorus

A copper(I) complex of 2-aminobenzenethiol, [Cu(abt)]_∞ (1), has been synthesized and characterized. The crystal structure determination indicates a two-dimensional metallopolymeric network formed by edge and corner sharing [Cu(μ₃-S)N] coordination tetrahedra wherein the copper(I) centers are coordinated to three bridging thiolate donors and the amino group of 2-aminobenzenethiolate. The copper, the sulfur and the nitrogen atoms form sub-lattices that reveal independently striking similarities to the double-layers present in black phosphorus.     

Yttrium containing head-on complexes of silico- and germanotungstate: Synthesis, structure and solution properties

Two dimeric head-on complexes of yttrium containing silico- and germanotungstate were isolated from the one-pot reaction of Y(NO₃)₃·6H₂O with the lacunary Na₁₀[MW₉O₃₄]·16H₂O (M = Si and Ge) building blocks in an acetate buffer at pH 4.5. Both polyanions were structurally characterized using various solid-state analytics, such as single-crystal X-ray diffraction, single-crystal X-ray analysis shows that both polyanions crystallize in the monoclinic crystal system (S.G. P2₁/c). FT-IR spectroscopy, or thermogravimetric analysis. The stability of the polyanion in aqueous solution was studied by multinuclear NMR spectroscopy (¹⁸³W, ⁸⁹Y, ²⁹Si, ¹³C, and ¹H). As expected, the ¹⁸³W NMR spectra display six peaks in the intensity ratio of 4:4:2:4:4:4 which indicates that both polyanions exist as dimeric entities in aqueous solution.     

CD1 antigen presentation by human dendritic cells as a target for herpes simplex virus immune evasion

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying activation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down-regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus.     

The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A approximately 29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.     

Elucidation of the Mechanisms Underlying Hypo-osmotically Induced Turgor Pressure Regulation in the Marine Alga Valonia utricularis

Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 microM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 microM NPPB, regulation could only be inhibited by simultaneous external addition of 200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl(-) transport. At concentrations of about 100 microM, NPPB seems to selectively inhibit Cl(-) transporters in the tonoplast and K(+) transporters in the plasmalemma, whereas 300 microM NPPB inhibits K(+) and Cl(-) transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G(t) and G(p)) in low-Cl(-) and K(+)-free artificial seawater. Inhibition of turgor pressure regulation by 300 microM NPPB was accompanied by about 85% reduction of G(t) and G(p). Vacuolar addition of sulfate, an inhibitor of tonoplast Cl(-) transporters, together with external addition of DIDS and Ba(2+) (an inhibitor of K(+) transporters) also strongly reduced G(p) and G(t) but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.