A new validated mathematical model of the Wnt signalling pathway predicts effective combinational therapy by sFRP and Dkk

The Wnt signalling pathway controls cell proliferation and differentiation, and its deregulation is implicated in different diseases including cancer. Learning how to manipulate this pathway could substantially contribute to the development of therapies. We developed a mathematical model describing the initial sequence of events in the Wnt pathway, from ligand binding to β-catenin accumulation, and the effects of inhibitors, such as sFRPs (secreted Frizzled-related proteins) and Dkk (Dickkopf). Model parameters were retrieved from experimental data reported previously. The model was retrospectively validated by accurately predicting the effects of Wnt3a and sFRP1 on β-catenin levels in two independent published experiments (R(2) between 0.63 and 0.91). Prospective validation was obtained by testing the model's accuracy in predicting the effect of Dkk1 on Wnt-induced β-catenin accumulation (R(2)≈0.94). Model simulations under different combinations of sFRP1 and Dkk1 predicted a clear synergistic effect of these two inhibitors on β-catenin accumulation, which may point towards a new treatment avenue. Our model allows precise calculation of the effect of inhibitors applied alone or in combination, and provides a flexible framework for identifying potential targets for intervention in the Wnt signalling pathway.     

Highly conserved d-loop sequences in woolly mouse opossums Marmosa (Micoureus)

This study reports the occurrence of highly conserved d-loop sequences in the mitochondrial genome of the woolly mouse opossum genus Marmosa subgenus Micoureus (Mammalia, Didelphimorphia, Didelphidae). Sixty-six sequences of Marmosa (Micoureus) demerarae, Marmosa (Micoureus) constantiae, and Marmosa (Micoureus) paraguayanus were amplified using universal d-loop primers and virtually no genetic differences were detected within and among species. These sequences matched the control region of the mitochondrial marsupial genome. Analyses of qualitative aspects of these sequences revealed that their structural composition is very similar to the d-loop region of other didelphid species. However, the total lack of variability has not been reported from other closely related species. The data analyzed here support the occurrence of highly conserved d-loop sequences, and we found no support for the hypothesis that these sequences are d-loop-like nuclear pseudogenes. Furthermore, the control and flanking regions obtained with different primers corroborate the lack of variability of the d-loop sequences in the mitochondrial genome of Marmosa (Micoureus).     

Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis

HIV-1 encephalitis (HIVE) and its associated dementia can occur in up to 20% of infected individuals, usually when productive viral replication in brain mononuclear phagocytes (macrophages and microglia) and depletion of CD4(+) T lymphocytes are most significant. T cells control viral replication through much of HIV-1 disease, but how this occurs remains incompletely understood. With this in mind, we studied HIV-1-specific CTL responses in a nonobese diabetic (NOD)-SCID mouse model of HIVE. HIV-1-infected monocyte-derived macrophages (MDM) were injected into the basal ganglia after syngeneic immune reconstitution by HLA-A*0201-positive human PBL to generate a human PBL-NOD-SCID HIVE mouse. Engrafted T lymphocytes produced HIV-1gag- and HIV-1pol-specific CTL against virus-infected brain MDM within 7 days. This was demonstrated by tetramer staining of human PBL in mouse spleens and by IFN-gamma ELISPOT. CD8, granzyme B, HLA-DR, and CD45R0 Ag-reactive T cells and CD79alpha-positive B cells migrated to and were in contact with human MDM in brain areas where infected macrophages were abundant. The numbers of productively infected MDM were markedly reduced (>85%) during 2 wk of observation. The human PBL-NOD-SCID HIVE mouse provides a new tool for studies of cellular immune responses against HIV-1-infected brain mononuclear phagocytes during natural disease and after vaccination.     

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.     

Identification and relative quantitation of an orphan G-protein coupled receptor SREB2 (GPR85) protein in tissue using a linear ion trap mass spectrometer

SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2-overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate.     

Slowing of the HIV Epidemic in Ukraine: Evidence from Case Reporting and Key Population Surveys, 2005–2012

Background

Ukraine developed Europe''s most severe HIV epidemic due to widespread transmission among persons who inject drugs (PWID). Since 2004, prevention has focused on key populations; antiretroviral therapy (ART) coverage has increased. Recent data show increases in reported HIV cases through 2011, especially attributed to sexual transmission, but also signs of potential epidemic slowing. We conducted a data triangulation exercise to better analyze available data and inform program implementation.

Methods and Findings

We reviewed data for 2005 to 2012 from multiple sources, primarily national HIV case reporting and integrated biobehavioral surveillance (IBBS) studies among key populations. Annually reported HIV cases increased at a progressively slower rate through 2011 with recent increases only among older, more immunosuppressed individuals; cases decreased 2.7% in 2012. Among women <25 years of age, cases attributed to heterosexual transmission and HIV prevalence in antenatal screening declined after 2008. Reported cases among young PWID declined by three-fourths. In 2011, integrated biobehavioral surveillance demonstrated decreased HIV prevalence among young members of key populations compared with 2009. HIV infection among female sex workers (FSW) remains strongly associated with a personal history of injecting drug use (IDU).

Conclusions

This analysis suggests that Ukraine''s HIV epidemic has slowed, with decreasing reported cases and older cases predominating among those diagnosed. Recent decreases in cases and in prevalence support decreased incidence among young PWID and women. Trends among heterosexual men and men who have sex with men (MSM) are less clear; further study and enhanced MSM prevention are needed. FSW appear to have stable prevalence with risk strongly associated with IDU. Current trends suggest the Ukrainian epidemic can be contained with enhanced prevention among key populations and increased treatment access.     

Noggin Expression in the Adult Retina Suggests a Conserved Role during Vertebrate Evolution

Vertebrates share common mechanisms in the control of development and in the maintenance of neural and retinal function. The secreted factor Noggin, a BMP inhibitor, plays a crucial role in neural induction during embryonic development. Moreover, we have shown its involvement in retinal differentiation of pluripotent cells. Here we show Noggin expression in the adult retina in three vertebrate species. Four Noggin genes are present in zebrafish (Danio rerio; ZbNog1, 2, 3, 5), three in frog (Xenopus laevis; XenNog1, 2 and 4), and one in mouse (Mus musculus; mNog). Quantitative RT-PCR experiments show the presence of ZbNog3 and ZbNog5 mRNAs, but not ZbNog1 and ZbNog2, in the adult zebrafish retina. All three genes are expressed in the frog retina, and mNog in the mouse. Immunohistochemistry data show that Noggin proteins are predominantly localized in the Golgi apparatus of photoreceptors and in the fibers of the outer plexiform layer. Lower expression levels are also found in inner plexiform layer fibers, in ganglion cells, in the ciliary marginal zone, and in retinal pigmented epithelium. Our results show that Noggin has a specific cellular and sub-cellular expression in the adult vertebrate retina, which is conserved during evolution. In addition to its established role during embryonic development, we postulate that Noggin also exerts a functional role in the adult retina.