The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

The U7 snRNP and the hairpin binding protein: Key players in histone mRNA metabolism.

Animal replication-dependent histone mRNAs are subject to several post-transcriptional regulatory processes. Their non-polyadenylated 3' ends are formed preferentially during S phase by a unique nuclear cleavage event. This requires the base pairing between U7 snRNA and a histone spacer element 3' of the cleavage site. Cleavage occurs preferentially after adenosine, at a fixed distance from the hybrid region. A conserved RNA hairpin just upstream of the cleavage site is recognised by the hairpin binding protein (HBP) that acts as an auxiliary processing factor, stabilising the interaction of the histone pre-mRNA with the U7 snRNP. The interaction between HBP and the RNA hairpin is very stable and HBP is also found associated with histone mRNAs on polysomes. The hairpin and presumably, HBP are also required for nuclear export and translation of histone mRNA. Furthermore, histone mRNAs are selectively destabilised in the G2 phase or upon inhibition of DNA synthesis and this regulation is also associated with the hairpin. Recently, HBP-encoding cDNAs were isolated from various organisms. Human, mouse and Xenopus laevis HBPs are similar, while the Caenorhabditis elegans protein has significant homology to the others only in a central RNA binding domain.Copyright 1997 Academic Press Limited

Molecular and cellular basis of bone resorption.

Osteoclast research has an exciting history and a challenging future. More than 3 decades ago, it became evident that bone-resorbing osteoclasts are of hematopoietic origin and are ultimately linked to the "basic multicellular unit," where they team up with the other cell types, including bone-forming osteoblasts. Since 2 decades, we have learned about the signaling pathways controlling genes relevant for osteoclastogenesis and bone resorption. It took another decade until the hypothesized "osteoclast differentiation" factor was discovered and was translated into an approved pharmacologic strategy. Here, the focus is on another molecular target, cathepsin K, a cysteine protease being released by the osteoclast into the resorption compartment. Genetic deletion and pharmacological blocking of cathepsin K reduces bone resorption but with ongoing bone formation. This observation not only holds great promise to become a new pharmacologic strategy, but it also provides new insights into the coordinated work of cells in the "basic multicellular unit" and thus, bridges the history and future of osteoclast research. This article is a short primer on osteoclast biology for readers of the special issue on odanacatib, a cathepsin K inhibitor.

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

T- and B-cell responses and previous exposure to hepatitis B virus in 'anti-HBc alone' patients.

A serologic response to hepatitis B virus (HBV) defined as 'anti-HBc alone' is commonly observed, but its significance remains unclear. This study aimed to define the relationship between 'anti-HBc alone' serostatus and HBV infection, including HBV-specific T- and B-cell memory responses. We enrolled 31 'anti-HBc alone' patients. Total HBV DNA and cccDNA were tested by nested polymerase chain reaction (PCR) analysis in liver samples from 22 'anti-HBc alone' patients vs controls (chronic or resolved HBV infection), followed by HBsAg/HBcAg immunohistochemical (IHC) staining. IFN-γ secretion by HBV-specific T cells was compared in individuals who were 'anti-HBc alone' (n = 27), resolved HBV (n = 21), chronic HBV (n = 24) and 12 healthy controls using enzyme-linked immunospot (ELISpot) assays. An HBsAg-IgG B-cell ELISpot assay was performed in 'anti-HBc alone' patients before and after one dose of recombinant HBsAg vaccine. The majority (23/31, 74.2%) of the 'anti-HBc alone' individuals were co-infected with HCV. Infrequent intrahepatic total HBV DNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T-cell responses were similar between 'anti-HBc alone' individuals and HBV resolvers. Circulating HBV-memory B-cell responses were detected in all 'anti-HBc alone' individuals, consistent with an HBsAg-specific memory pool. After one HBV vaccine dose, increased anti-HBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P = 0.0226). 'Anti-HBc alone' individuals showed HBV-specific T-cell and memory B-cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

The Epicardium in the Embryonic and Adult Zebrafish

The epicardium is the mesothelial outer layer of the vertebrate heart. It plays an important role during cardiac development by, among other functions, nourishing the underlying myocardium, contributing to cardiac fibroblasts and giving rise to the coronary vasculature. The epicardium also exerts key functions during injury responses in the adult and contributes to cardiac repair. In this article, we review current knowledge on the cellular and molecular mechanisms underlying epicardium formation in the zebrafish, a teleost fish, which is rapidly gaining status as an animal model in cardiovascular research, and compare it with the mechanisms described in other vertebrate models. We moreover describe the expression patterns of a subset of available zebrafish Wilms' tumor 1 transgenic reporter lines and discuss their specificity, applicability and limitations in the study of epicardium formation.

[Lineage tracing of epicardial cells during development and regeneration].

Tracing the history of individual cells during embryonic morphogenesis in a structure as complex as the cardiovascular system is one of the major challenges of developmental biology. It involves determining the relationships between the various lineages of cells forming an organ at different stages, describing the topological rearrangements tissues undergo during morphogenesis, and characterizing the interactions between cells in different structures. However, despite the great expectations raised in the field of regenerative medicine, only limited progress has been made in using regenerative therapy to repair the cardiovascular system. Recent research has highlighted the role of the epicardium during cardiac regeneration, but it is still unclear whether it is important for molecular signaling or acts as a source of progenitor cells during this process. Consequently, increasing knowledge about the origin, diversification and potential of epicardial cells during development and homeostasis and under pathological conditions is of fundamental importance both for basic research and for the development of effective cellular therapies. The aims of this article were to provide a general overview of the classical techniques used for tracing cell lineages, including their potential and limitations, and to describe novel techniques for studying the origin and differentiation of the epicardium and its role in cardiac regeneration.

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Determination of the Myogenic Potential of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells

Human embryonic stem cells (hESCs) have the potential to differentiate to all adult somatic cells. This property makes hESCs a very promising area of research for the treatment of disorders in which specific cell populations need to be restored. Despite this potential, research that focuses on producing mesodermally derived cell populations from hESCs is decidedly limited, notwithstanding the prevalence of disorders involving mesodermal tissues for which treatment options are limited. Skeletal muscle myoblasts are derivatives of mesodermal cells and are characterized by the expression of the MyoD gene. These cells are difficult to obtain from hESCs in a reproducible and efficient manner. Recent developments in the field have showed some success in obtaining myogenic cells from hESCs through a mesenchymal stem cell (MSC)-like intermediate population. MSCs, which are an adult stem cell population typically derived from the bone marrow, are capable of generating multiple cell types including skeletal muscle. The aim of this study was to develop an efficient method that derives myoblasts from an MSC-like intermediate. To accomplish this goal, we first set out to isolate and expand the MSC-like intermediate from hESCs differentiated in vitro. Difficulties in reproducing published cell-differentiation methodologies, which represent a significant and familiar challenge in hESC research, are highlighted in this report.

Elucidating the IGFBP2 signaling pathway in glioma development and progression

Diffuse gliomas are highly lethal central nervous system malignancies which, unfortunately, are the most common primary brain tumor and also the least responsive to the very few therapeutic modalities currently available to treat them. IGFBP2 is a newly recognized oncogene that is operative in multiple cancer types, including glioma, and shows promise for a targeted therapeutic approach. Elevated IGFBP2 expression is present in high-grade glioma and correlates with poor survival. We have previously demonstrated that IGFBP2 induces glioma development and progression in a spontaneous glioma mouse model, which highlighted its significance and potential for future therapy. However, we did not yet know the key physiological pathways associated with this newly characterized oncogene. We first evaluated human glioma genomics data harnessed from the publicly available Rembrandt source to identify major pathways associated with IGFBP2 expression. Integrin and ILK, among other cell migration and invasion-related pathways, were the most prominently associated. We confirmed that these pathways are regulated by IGFBP2 in glioma cells lines, and demonstrated that 1) IGFBP2 activates integrin α5β1, leading to the activation of key pathways important in glioma; 2) IGFBP2 mediates cell migration pathways through ILK; and 3) IGFBP2 activates NF-kB via an integrin α5 interaction. We then sought to determine whether this was a physiologically active signaling pathway in vivo by assessing its ability to induce glioma progression in the RCAS/tv-a spontaneous glioma mouse model. We found that ILK is a key downstream mediator of IGFBP2 that is required for the induction of glioma progression. Most significantly, a genetic therapeutic approach revealed that perturbation of any point in the pathway thwarted tumor progression, providing strong evidence that targeting the key players could potentially produce a significant benefit for human glioma patients. The elucidation of this signaling pathway is a critical step, since efforts to create a small molecule drug targeting IGFBP2 have so far not been successful, but a number of inhibitors of the other pathway constituents, including ILK, integrin and NF-kB, have been developed.

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.

Potential REST Targets in Neural Stem Cells and Glioblastoma-Derived Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. The current theory is that these tumors are caused by self-renewing glioblastoma-derived stem cells (GSCs). At the current time, the mechanisms that regulate self-renewal and other oncogenic properties of GSCs remain unknown. Recently, we found transcriptional repressor REST maintains self-renewal in neural stem cells (NSCs) and in GSCs. REST also regulates other oncogenic properties, such as apoptosis, invasion and proliferation. However, the mechanisms by which REST regulates these oncogenic properties are unknown. In an attempt to determine these mechanisms, we performed loss and gain-of-function experiments and genome-wide mRNA expression analysis in GSCs, and we were able to identify REST-regulated genes in GSCs. This was accomplished, after screening concordantly regulated genes in NSCs and GSCs, utilizing two RE1 databases, and setting two-fold expression as filters on the resulting genes. These results received further validation by qRT-PCR. Ingenuity Pathway Analysis (IPA) analysis further revealed the top REST target genes in GSCs were downstream targets of REST and/or involved in other cancers in other cell lines. IPA also revealed that many of the differentially-regulated genes identified in this study are involved in oncogenic properties seen in GBM, and which we believe are related to REST expression.

TRANSCRIPTIONAL REGULATION OF PROFILIN IS REQUIRED FOR DROSOPHILA LARVAL WOUND CLOSURE

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways (the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway) and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound- induced cell migrations. Here we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE Publication No.___________ Lisa Harn-Ging Shiue, B.S. Supervisory Professor: Madeleine Duvic, M.D. Extracorporeal photopheresis (ECP) is an effective, low-risk immunomodulating therapy for leukemic cutaneous T cell lymphoma (L-CTCL) and graft versus host disease (GVHD), but whether the mechanism(s) of action in these two diseases is (are) identical or different is unclear. To determine the effects of ECP in vivo, we studied regulatory T cells (T-regs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) by immunofluorescence flow cytometry in 18 L-CTCL and 11 GVHD patients before and after ECP at Day 2, 1 month, 3 months, and 6 months. In this study, ECP was effective in 12/18 L-CTCL patients with a 66.7% overall response rate (ORR) and 6/11 GVHD patients with a 54.5% ORR. Prior to ECP, the percentages of CD4+Foxp3+ T cells in 9 L-CTCL patients were either lower (L-CTCL-Low, n=2) or higher (L-CTCL-High, n=7) than normal. Five of the 7 GVHD patients had high percentages of CD4+Foxp3+ T cells (GVHD-High). Six of 7 L-CTCL-High patients had >80% CD4+Foxp3+ T cells which were correlated with tumor cells, and were responders. Both L-CTCL-High and GVHD-High patients had decreased percentages of CD4+Foxp3+ and CD4+Foxp3+CD25- T cells after 3 months of treatment. CD4+Foxp3+CD25+ T cells increased in GVHD-High patients but decreased in L-CTCL-High patients after 3 months of ECP. In addition, numbers of CTLs were abnormal. We confirmed that numbers of CTLs were low in L-CTCL patients, but high in GVHD patients prior to ECP. After ECP, CTLs increased after 1 month in 4/6 L-CTCL patients whereas CTLs decreased after 6 months in 3/3 GVHD patients. Myeloid (mDCs) and plasmacytoid DCs (pDCs) were also low at baseline in L-CTCL and GVHD patients confirming the DC defect. After 6 months of ECP, numbers and percentages of mDCs and pDCs increased in L-CTCL and GVHD. MDCs were favorably increased in 8/12 L-CTCL responders whereas pDCs were favorably increased in GVHD patients. These data suggest that ECP is favorably modulating the DC subsets. In L-CTCL patients, the mDCs may orchestrate Th1 cell responses to overcome immune suppression and facilitate disease regression. However, in GVHD patients, ECP is favorably down-regulating the immune system and may be facilitating immune tolerance to auto-or allo-antigens. In both L-CTCL and GVHD patients, DCs are modulated, but the T cell responses orchestrated by the DCs are different, suggesting that ECP modulates depending on the immune milieu. _______________