The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.publishedVersionPeer reviewe

Monoclonal antibodies to the recombinant nucleocapsid protein of a groundnut bud necrosis virus infecting tomato in Karnataka and their use in profiling the epitopes of Indian tospovirus isolates

A tospovirus infecting tomato in the fields of Karnataka, India, was propagated in greenhouse-grown Nicotiana benthamiana plants by mechanical inoculation. The viral RNA was extracted from purified virus and used for amplification of N and NSs genes by RT– PCR using appropriate primers. The N and NSs PCR products were cloned into a pRSET vector and sequenced. The N gene of tomato tospovirus showed 98% identity with that of Groundnut bud necrosis virus (GBNV), alternate name Peanut bud necrosis virus (PBNV). Interestingly, though the virus was isolated from tomato plants, it showed only 82% identity with the N gene of GBNV-To isolate from Taiwan. The NSs gene of the virus under study showed 98% identity with GBNV. These results suggest that the tomato tospovirus in Karnataka is a strain of GBNV and is henceforth designated as GBNV-To (K). The N gene was overexpressed in Escherichia coli and the recombinant N protein was purified using Ni–NTA agarose affinity chromatography. The purified protein was used for the generation of poly- and monoclonal antibodies (mAbs). The polyclonal antiserum thus obtained had a dilution end-point >1 : 32,000 and nine unique mAbs were also obtained. These mAbs were used for epitope profiling of the tospovirus isolates from South India and for developing detection methods. The results showed that there are distinct GBNV strains in South India. A simple dot-blot assay was developed for detection of GBNV from infected field samples

Monoclonal antibodies to gonadotropin-releasing hormone (GnRH) inhibit binding of the hormone to its receptor

Monoclonal antibodies (MAbs) specific to gonadotropin-releasing hormone (GnRH) were obtained using different strategies of conjugation of the peptide to carrier protein and immunization. Of several antibodies obtained, two, namely F1D3C5 and E2D2 bound GnRH in solution phase. Though the epitopes corresponding to the two overlapped, there was a one amino acid shift in the core epitope. These two antibodies were characterized with respect to inhibition of GnRH induced responses in rat pituitary cultures and α -T3.1 mouse gonadotrope cell line

Monoclonal antibodies to the recombinant nucleocapsid protein of a groundnut bud necrosis virus infecting tomato in Karnataka and their use in profiling the epitopes of Indian tospovirus isolates

A tospovirus infecting tomato in the fields of Karnataka, India, was propagated in greenhouse-grown Nicotiana benthamiana plants by mechanical inoculation. The viral RNA was extracted from purified virus and used for amplification of N and NSs genes by RT- PCR using appropriate primers. The N and NSs PCR products were cloned into a pRSET vector and sequenced. The N gene of tomato tospovirus showed 98% identity with that of Groundnut bud necrosis virus (GBNV), alternate name Peanut bud necrosis virus (PBNV). Interestingly, though the virus was isolated from tomato plants, it showed only 82% identity with the N gene of GBNV-To isolate from Taiwan. The NSs gene of the virus under study showed 98% identity with GBNV. These results suggest that the tomato tospovirus in Karnataka is a strain of GBNV and is henceforth designated as GBNV-To (K). The N gene was overexpressed in Escherichia coli and the recombinant N protein was purified using Ni-NTA agarose affinity chromatography. The purified protein was used for the generation of poly- and monoclonal antibodies (mAbs). The polyclonal antiserum thus obtained had a dilution end-point > 1: 32,000 and nine unique mAbs were also obtained. These mAbs were used for epitope profiling of the tospovirus isolates from South India and for developing detection methods. The results showed that there are distinct GBNV strains in South India. A simple dot-blot assay was developed for detection of GBNV from infected field samples

Production of monoclonal antibodies to Tomato leaf curl Bangalore virus

Purified Tomato leaf curl Bangalore virus (ToLCBV) was injected into mice and the splenocytes were used for establishing hybridoma lines. Initial screening of culture supernatants showed that 13 lines produced antibody, and after further screening four produced functional monoclonal antibodies. Upon characterisation, these were found to be of low affinity, probably due to host protein contamination and poor yield of native virus in the original preparations. In order to circumvent these problems, the coat protein of ToLCBV was over-expressed in Escherichia coli. Fusion experiments using recombinant coat protein as antigen yielded two primary hybridoma clones G11 and E4 that exhibited good affinity of binding to the antigen. Sub-cloning yielded four monoclonal antibodies G11E7E7, G11E7G12, E4E2 and E4G6. G11E7E7 and G11E7G12 successfully detected ToLCBV in infected leaf extracts of tomato and Nicotiana benthamiana, viruliferous whiteflies and weed samples. These monoclonal antibodies could also detect other type III geminiviruses such as Pumpkin yellow vein mosaic virus and Bhendi yellow vein mosaic virus. Thus these monoclonal antibodies can be used for testing field-collected samples

Proinflammatory microenvironment promotes lymphoma progression in mice with high megakaryocyte and TPO levels

•Increased levels of interleukin 1 were found in the bone marrow fluid of TpoTg mice, whereas levels were lowered in Mpl−/− mice.•A proinflammatory microenvironment promoted Eμ-myc lymphoma progression in TpoTg mice with high megakaryocyte and thrombopoietin levels. [Display omitted] Platelets have been shown to enhance the survival of lymphoma cell lines. However, it remains unclear whether they play a role in lymphoma. Here, we investigated the potential role of platelets and/or megakaryocytes in the progression of Eμ-myc lymphoma. Eμ-myc tumor cells were transplanted into recipient wild-type (WT) control, Mpl−/−, or TpoTg mice, which exhibited normal, low, and high platelet and megakaryocyte counts, respectively. TpoTg mice that underwent transplantation exhibited enhanced lymphoma progression with increased white blood cell (WBC) counts, spleen and lymph node weights, and enhanced liver infiltration when compared with WT mice. Conversely, tumor-bearing Mpl−/− mice had reduced WBC counts, lymph node weights, and less liver infiltration than WT mice. Using an Mpl-deficient thrombocytopenic immunocompromised mouse model, our results were confirmed using the human non-Hodgkin lymphoma GRANTA cell line. Although we found that platelets and platelet-released molecules supported Eμ-myc tumor cell survival in vitro, pharmacological inhibition of platelet function or anticoagulation in WT mice transplanted with Eμ-myc did not improve disease outcome. Furthermore, transient platelet depletion or sustained Bcl-xL–dependent thrombocytopenia did not alter lymphoma progression. Cytokine analysis of the bone marrow fluid microenvironment revealed increased levels of the proinflammatory molecule interleukin 1 in TpoTg mice, whereas these levels were lower in Mpl−/− mice. Moreover, RNA sequencing of blood-resident Eμ-myc lymphoma cells from TpoTg and WT mice after tumor transplantation revealed the upregulation of hallmark gene sets associated with an inflammatory response in TpoTg mice. We propose that the proinflammatory microenvironment in TpoTg mice promotes lymphoma progression

A role for pericytes as microenvironmental regulators of human skin tissue regeneration

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin α5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)