Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 µM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

ETS1 loss in mice impairs cardiac outflow tract septation via a cell migration defect autonomous to the neural crest.

Ets1 deletion in some mouse strains causes septal defects and has been implicated in human congenital heart defects in Jacobsen syndrome, in which one copy of the Ets1 gene is missing. Here, we demonstrate that loss of Ets1 in mice results in a decrease in neural crest (NC) cells migrating into the proximal outflow tract cushions during early heart development, with subsequent malalignment of the cushions relative to the muscular ventricular septum, resembling double outlet right ventricle (DORV) defects in humans. Consistent with this, we find that cultured cardiac NC cells from Ets1 mutant mice or derived from iPS cells from Jacobsen patients exhibit decreased migration speed and impaired cell-to-cell interactions. Together, our studies demonstrate a critical role for ETS1 for cell migration in cardiac NC cells that are required for proper formation of the proximal outflow tracts. These data provide further insights into the molecular and cellular basis for development of the outflow tracts, and how perturbation of NC cells can lead to DORV.

The Rise of Induced Pluripotent Stem Cell Approach to Hair Restoration.

SUMMARY: The advent of pluripotent stem cells following the discovery of Shinya Yamanaka (2012 Nobel prize in Medicine) brought about a regenerative medicine approach to virtually every human condition including hair loss. It is now possible to reprogram somatic cells (eg, blood or skin cells) from a person experiencing hair loss to generate autologous induced pluripotent stem cells (iPSCs), which could be amplified and cryopreserved. Subsequently, these iPSCs could be differentiated into various cell types such as dermal papilla cells, epithelial cells, melanocytes, and other cell types constituting functional hair follicle. Transplantation of human iPSC-derived folliculogenic cells into the nude mice has successfully generated xenografts with hair outgrowth. Because iPSCs provide a virtually unlimited source of folliculogenic cells for de novo formation of hair follicles, this approach has major advantages over current surgical hair restoration procedures, which merely redistribute existing hair follicles from one part of the sculp to another. Combined with robotics and automation of the transplantation process, this novel regenerative medicine approach is well poised to make hair restoration a routine procedure affordable for everybody who can benefit from it.

Improving drug discovery using image-based multiparametric analysis of the epigenetic landscape.

High-content phenotypic screening has become the approach of choice for drug discovery due to its ability to extract drug-specific multi-layered data. In the field of epigenetics, such screening methods have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks and employs machine learning to accurately distinguish between such patterns. We validated the MIEL platform across multiple cells lines and using dose-response curves, to insure the fidelity and robustness of this approach for high content high throughput drug discovery. Focusing on noncytotoxic glioblastoma treatments, we demonstrated that MIEL can identify and classify epigenetically active drugs. Furthermore, we show MIEL was able to accurately rank candidate drugs by their ability to produce desired epigenetic alterations consistent with increased sensitivity to chemotherapeutic agents or with induction of glioblastoma differentiation.

Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

hESC derived neuro-epithelial rosettes recapitulate early mammalian neurulation events; an in vitro model.

The in vitro neuralization of hESCs has been widely used to generate central and peripheral nervous system components from neural precursors (Bajpai et al., 2009; Curchoe et al., 2010), most often through an intermediate "rosette" stage. Here we confirm that hESC derived neuro-epithelial rosettes express many characteristics of the developing embryonic neural plate (Aaku-Saraste et al., 1996), characterized by expression of the tight junction proteins ZO-1 and N-Cadherin. Moreover, neuro-epithelial rosettes display a characteristic acetylated alpha tubulin cytoskeletal arrangement (similar to that observed in the developing embryonic neural plate) (Bhattacharyya et al., 1994). Demonstrated here for the first time MKLP was observed in a hESC model system. We found MKLP expression in small particles in between mitotic spindles, large particles aggregating in the lumen of neuroepithelial rosettes, and we did not observe MKLP in the nucleus of hESC derived neural precursors as previously described in the HeLa cell line. We observed MKLP+ particles in aggregations in the lumen of "early" rosette structures. Furthermore, we observed that MKLP+ particle aggregations can also be lost from the lumens of hESC derived neuro-epithelial rosettes, similar to a phenomenon observed in the developing neural tube in vivo (Marzesco et al., 2005). We determined that this loss of MKLP+ particles occurs from "late" as opposed to "early" stage neuro-epithelial rosettes (characterized by junction type). Disrupting the apical-basal polarization of "early" stage rosettes with a 1% Matrigel overlay (Krtolica et al., 2007) nearly ablates MKLP particle aggregation in the lumen of rosettes, demonstrating that the apical-basal polarity of early NE cells is necessary for lumenal MKLP particle aggregation. We conclude that early hESC derived neuro-epithelial rosettes can model early neurulation events, such as the transition from neural plate like cells to neural tube like cells (i.e., symmetric to asymmetric NE cell division) demonstrated by polarized MKLP particle inheritance and distribution using junction type as a measure of stage.

Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo.

Maternal embryonic leucine zipper kinase (MELK) is expressed in several developing tissues, in the adult germ line, and in adult neural progenitors. MELK expression is elevated in aggressive undifferentiated tumors, correlating with poor patient outcome in human breast cancer. To investigate the role of MELK in mammary tumorigenesis in vivo, we used a MELK-green fluorescent protein (GFP) reporter mouse, which allows prospective isolation of MELK-expressing cells based on GFP fluorescence. We found that in the normal mammary gland, cells expressing high levels of MELK were enriched in proliferating cells that express markers of mammary progenitors. The isolation of cells with high levels of MELK in mammary tumors from MMTV-Wnt1/MELK-GFP bitransgenic mice resulted in a significant enrichment of tumorsphere formation in culture and tumor initiation after transplantation into mammary fat pads of syngeneic mice. Furthermore, using lentiviral delivery of MELK-specific shRNA and limiting dilution cell transplantations, we showed that MELK function is required for mammary tumorigenesis in vivo. Our findings identify MELK as a potential target in breast tumor-initiating cells.

Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells.

Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up-regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high-grade brain tumors.

Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1.

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Mammalian stem cells.

Stem cells are quickly coming into focus of much biomedical research eventually aiming at the therapeutic applications for various disorders and trauma. It is important, however, to keep in mind the difference between the embryonic stem cells, somatic stem cells and somatic precursor cells when considering potential clinical applications. Here we provide the review of the current status of stem cell field and discuss the potential of therapeutic applications for blood and Immune system disorders, multiple sclerosis, hypoxic-ischemic brain injury and brain tumors. For the complimentary information about various stem cells and their properties we recommend consulting the National Institutes of Health stem cell resources (http://stemcells.nih.gov/info/basics).

Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice.

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

Long-term persistence of a nonintegrated lentiviral vector in mouse hematopoietic stem cells.

OBJECTIVE: Lentiviral transduction is an established method for efficiently modifying the gene expression program of primary cells, but the ability of the introduced construct to persist as an episome has not been well studied. MATERIAL AND METHODS: Here we investigated this issue in lethally irradiated female mice injected with 300 or 3000 doubly sorted male lin(neg), Sca-1(high), c-kit(high), Thy-1.1(low) mouse bone marrow cells that had been exposed in vitro to self-inactivating lentivirus vector encoding a green fluorescence protein (GFP) cDNA. Seven to sixteen months later, bone marrow cells from primary mice were injected into secondary female recipients and another 8 months later into tertiary female recipients. Integration study was performed on individual spleen colonies by Southern blot analysis. Inverse polymerase chain reaction (PCR) and sequence of amplified vector-derived DNA was used to verify Southern blot results. RESULTS: Spleen colony-forming cell study revealed that a small fraction of the spleen colonies contained integrated provirus as shown by Southern blot analysis. Unexpectedly, many spleen colonies were found to contain a nonintegrated episomal form of the provirus, which was confirmed by an inverse PCR analysis. In some of the spleen colonies containing only the episomal form, GFP-expressing cells were also detected. Lentiviral sequences were present in hematopoietic tissues of primary mice but not in other tissues. CONCLUSIONS: These results demonstrate that lentiviral vectors produce episomal circles in hematopoietic stem cells that can be transferred through many cell generations and expressed in their progeny.

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations.

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.

Gene expression analysis of purified hematopoietic stem cells and committed progenitors.

Lifelong self-renewal is a unique property of somatic stem cells. Recently, several primitive multipotent yet committed (non-self-renewing) hematopoietic progenitor populations were identified in mouse bone marrow. We have characterized the expression of 1200 selected mouse genes using the Atlas cDNA array in highly purified hematopoietic stem cells (HSCs) and 6 closely related progenitor populations: common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs), common lymphoid progenitors (CLPs), and pro-T and pro-B cells. Cluster analysis revealed that nearly half of all differentially expressed transcripts are associated with HSCs, supporting the notion of an active transcriptional status of HSCs. Genes found enriched in the HSC cluster encompass many developmentally regulated genes, some previously associated with HSC self-renewal. In contrast, genes that are enriched in committed progenitors are mostly associated with hematopoietic differentiation, immune regulation, and metabolism. Thus, the transition from HSCs toward committed progenitors correlates with the down-regulation of a large number of HSC-associated genes and progressive up-regulation of a limited number of lineage-specific genes. These genetic analyses revealed both quantitative and qualitative differences between the transcripts associated with HSCs versus downstream progenitors and produced a list of the candidate genes, potentially involved in HSC self-renewal.