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Abstract Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.

Abstract Background Single-cell sequencing techniques are revolutionizing every field of biology by providing the ability to measure the abundance of biological molecules at a single-cell resolution. Although single-cell sequencing approaches have been developed for several molecular modalities, single-cell transcriptome sequencing is the most prevalent and widely applied technique. SPLiT-seq (split-pool ligation-based transcriptome sequencing) is one of these single-cell transcriptome techniques that applies a unique combinatorial-barcoding approach by splitting and pooling cells into multi-well plates containing barcodes. This unique approach required the development of dedicated computational tools to preprocess the data and extract the count matrices. Here we compare eight bioinformatic pipelines (alevin-fry splitp, LR-splitpipe, SCSit, splitpipe, splitpipeline, SPLiTseq-demultiplex, STARsolo and zUMI) that have been developed to process SPLiT-seq data. We provide an overview of the tools, their computational performance, functionality and impact on downstream processing of the single-cell data, which vary greatly depending on the tool used. Results We show that STARsolo, splitpipe and alevin-fry splitp can all handle large amount of data within reasonable time. In contrast, the other five pipelines are slow when handling large datasets. When using smaller dataset, cell barcode results are similar with the exception of SPLiTseq-demultiplex and splitpipeline. LR-splitpipe that is originally designed for processing long-read sequencing data is the slowest of all pipelines. Alevin-fry produced different down-stream results that are difficult to interpret. STARsolo functions nearly identical to splitpipe and produce results that are highly similar to each other. However, STARsolo lacks the function to collapse random hexamer reads for which some additional coding is required. Conclusion Our comprehensive comparative analysis aids users in selecting the most suitable analysis tool for efficient SPLiT-seq data processing, while also detailing the specific prerequisites for each of these pipelines. From the available pipelines, we recommend splitpipe or STARSolo for SPLiT-seq data analysis.

Abstract Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

ObjectiveMesothelin (MSLN) is an attractive target for anticancer therapeutics and bioimaging reagents that utilize antibodies. This study was aimed at developing a novel human anti-MSLN single-domain antibody that exclusively binds to the membrane-attached MSLN using transgenic mice generating human heavy-chain-only antibodies (HCAbs) and exploring the resulting HCAbs as imaging tools.MethodsWe introduced a doxycycline-inducible human MSLN gene in genetically modified mice expressing human HCAbs. This new method of non-invasive immunization by antigen induction results in MSLN antigen production in its native conformation on the cell surface. Screening of 2,000 HCAbs from the resulting immune library yielded numerous binders, from which we chose 19G6 as the lead antibody. This antibody was 111Indium radiolabeled and tested in a xenotransplantation tumor model with OVCAR-3 cells.ResultsThe 19G6 antibody shows nanomolar affinity toward membrane-bound MSLN and does not recognize soluble MSLN. The human MSLN-positive tumors were visualized in an in vivo mouse model. The non-labeled antibody prevented binding when provided in excess, showing tumor specificity.Conclusion19G6 with a human Fc is a promising tumor-cell tracer in vivo. This HCAb can also be engineered into a smaller and shorter-lived tracer (only the VH domain) or combined with other target-binding domains to form multispecific modalities for tumor immunotherapy.

Snakebite envenoming is a priority Neglected Tropical Disease that causes an estimated 81,000–135,000 fatalities each year. The development of a new generation of safer, affordable, and accessible antivenom therapies is urgently needed. With this goal in mind, rigorous characterisation of the specific toxins in snake venom is key to generating novel therapies for snakebite. Monoclonal antibodies directed against venom toxins are emerging as potentially strong candidates in the development of new snakebite diagnostics and treatment. Venoms comprise many different toxins of which several are responsible for their pathological effects. Due to the large variability of venoms within and between species, formulations of combinations of human antibodies are proposed as the next generation antivenoms. Here a high-throughput screening method employing antibody-based ligand fishing of venom toxins in 384 filter-well plate format has been developed to determine the antibody target/s The approach uses Protein G beads for antibody capture followed by exposure to a full venom or purified toxins to bind their respective ligand toxin(s). This is followed by a washing/centrifugation step to remove non-binding toxins and an in-well tryptic digest. Finally, peptides from each well are analysed by nanoLC-MS/MS and subsequent Mascot database searching to identify the bound toxin/s for each antibody under investigation. The approach was successfully validated to rapidly screen antibodies sourced from hybridomas, derived from venom-immunised mice expressing either regular human antibodies or heavy-chain-only human antibodies (HCAbs).

Abstract Our understanding of the organization of the chromatin fiber within the cell nucleus has made great progress in the last few years. High-resolution techniques based on next-generation sequencing as well as optical imaging that can investigate chromatin conformations down to the single cell level have revealed that chromatin structure is highly heterogeneous at the level of the individual allele. While TAD boundaries and enhancer–promoter pairs emerge as hotspots of 3D proximity, the spatiotemporal dynamics of these different types of chromatin contacts remain largely unexplored. Investigation of chromatin contacts in live single cells is necessary to close this knowledge gap and further enhance the current models of 3D genome organization and enhancer–promoter communication. In this review, we first discuss the potential of single locus labeling to study architectural and enhancer–promoter contacts and provide an overview of the available single locus labeling techniques such as FROS, TALE, CRISPR–dCas9 and ANCHOR, and discuss the latest developments and applications of these systems.

Abstract The formation of the synovial joint begins with the visible emergence of a stripe of densely packed mesenchymal cells located between distal ends of the developing skeletal anlagen called the interzone. Recently the transcriptome of the early synovial joint was reported. Knowledge about enhancers would complement these data and lead to a better understanding of the control of gene transcription at the onset of joint development. Using ChIP-sequencing we have mapped the H3-signatures H3K27ac and H3K4me1 to locate regulatory elements specific for the interzone and adjacent phalange, respectively. This one-stage atlas of candidate enhancers (CEs) was used to map the association between these respective joint tissue specific CEs and biological processes. Subsequently, integrative analysis of transcriptomic data and CEs identified new putative regulatory elements of genes expressed in interzone (e.g., GDF5, BMP2 and DACT2) and phalange (e.g., MATN1, HAPLN1 and SNAI1). We also linked such CEs to genes known as crucial in synovial joint hypermobility and osteoarthritis, as well as phalange malformations. These analyses show that the CE atlas can serve as resource for identifying, and as starting point for experimentally validating, putative disease-causing genomic regulatory regions in patients with synovial joint dysfunctions and/or phalange disorders, and enhancer-controlled synovial joint and phalange formation.

Human coronavirus OC43 causes respiratory disease and is maintained in the human population through recurring infections. Here, by extensive structural analyses, the authors provide insights into the binding sites and breadth of neutralizing antibodies against this endemic coronavirus.

Emerging SARS-CoV-2 variants have accrued mutations within the spike protein rendering most therapeutic monoclonal antibodies against COVID-19 ineffective. Hence there is an unmet need for broad-spectrum mAb treatments for COVID-19 that are more resistant to antigenically drifted SARS-CoV-2 variants. Here we describe the design of a biparatopic heavy-chain-only antibody consisting of six antigen binding sites recognizing two distinct epitopes in the spike protein NTD and RBD. The hexavalent antibody showed potent neutralizing activity against SARS-CoV-2 and variants of concern, including the Omicron sub-lineages BA.1, BA.2, BA.4 and BA.5, whereas the parental components had lost Omicron neutralization potency. We demonstrate that the tethered design mitigates the substantial decrease in spike trimer affinity seen for escape mutations for the hexamer components. The hexavalent antibody protected against SARS-CoV-2 infection in a hamster model. This work provides a framework for designing therapeutic antibodies to overcome antibody neutralization escape of emerging SARS-CoV-2 variants.

Modern-day hematopoietic stem cell (HSC) therapies, such as gene therapy, modify autologous HSCs prior to re-infusion into myelo-conditioned patients and hold great promise for treatment of hematological disorders. While this approach has been successful in numerous clinical trials, it relies on transplantation of ex vivo modified patient HSCs, which presents several limitations. It is a costly and time-consuming procedure, which includes only few patients so far, and ex vivo culturing negatively impacts on the viability and stem cell-properties of HSCs. If viral vectors are used, this carries the additional risk of insertional mutagenesis. A therapy delivered to HSCs in vivo, with minimal disturbance of the HSC niche, could offer great opportunities for novel treatments that aim to reverse disease symptoms for hematopoietic disorders and could bring safe, effective and affordable genetic therapies to all parts of the world. However, substantial unmet needs exist with respect to the in vivo delivery of therapeutics to HSCs. In the last decade, in particular with the development of gene editing technologies such as CRISPR/Cas9, nanoparticles (NPs) have become an emerging platform to facilitate the manipulation of cells and organs. By employing surface modification strategies, different types of NPs can be designed to target specific tissues and cell types in vivo. HSCs are particularly difficult to target due to the lack of unique cell surface markers that can be utilized for cell-specific delivery of therapeutics, and their shielded localization in the bone marrow (BM). Recent advances in NP technology and genetic engineering have resulted in the development of advanced nanocarriers that can deliver therapeutics and imaging agents to hematopoietic stem- and progenitor cells (HSPCs) in the BM niche. In this review we provide a comprehensive overview of NP-based approaches targeting HSPCs to control and monitor HSPC activity in vitro and in vivo, and we discuss the potential of NPs for the treatment of malignant and non-malignant hematological disorders, with a specific focus on the delivery of gene editing tools.

Here, the authors report the isolation and characterization of two human monoclonal antibodies (mAbs) from immunized mice with trimeric spike ectodomains of three human betacoronaviruses HCoV-OC43, SARS-CoV and MERSCoV, and show that while exhibiting cross-reactivity, the mAbs only neutralize MERS-CoV but not SARS-CoV nor SARS-CoV-2, likely due to the subtle epitope differences in the spike S2 fusion subunit.

Vaccines and targeted therapeutics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently lacking. Here, the authors report a human monoclonal antibody capable of neutralizing both authentic SARS-CoV and SARS-CoV-2 by targeting a common epitope.

Chromatin domains and loops are important elements of chromatin structure and dynamics, but much remains to be learned about their exact biological role and nature. Topological associated domains and functional loops are key to gene expression and hold the answer to many questions regarding developmental decisions and diseases. Here, we discuss new findings, which have linked chromatin conformation with development, differentiation and diseases and hypothesized on various models while integrating all recent findings on how chromatin architecture affects gene expression during development, evolution and disease.

ABSTRACTThe Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.

Summary: Transcription factors are key players in a broad range of cellular processes such as cell-fate decision. Understanding how they act to control these processes is of critical importance for therapy purposes. FLI-1 controls several hematopoietic lineage differentiation including megakaryopoiesis and erythropoiesis. Its aberrant expression is often observed in cancer and is associated with poor prognosis. We showed that FLI-1 interacts with the LDB1 complex, which also plays critical roles in erythropoiesis and megakaryopoiesis. In this study, we aimed to unravel how FLI-1 and the LDB1 complex act together in murine erythroleukemia cells and in megakaryocyte. Combining omics techniques, we show that FLI-1 enables the recruitment of the LDB1 complex to regulatory sequences of megakaryocytic genes and to enhancers. We show as well for the first time that FLI-1 is able to modulate the 3D chromatin organization by promoting chromatin looping between enhancers and promoters most likely through the LDB1 complex.

The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies.

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies this involves either random pairing of VH and VL domains in combinatorial display libraries, or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single cell sorting, single cell RT-PCR and bulk cloning of isolated natural VH-VL pairs. Heavy chain only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology, for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high affinity influenza A strain X-31 hemagglutinin (HA) specific HCAbs

Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive “hook” bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of β-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs.

Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by ∼6-fold and ∼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by ∼17-fold and ∼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1+, c-Kit+ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy.

β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) ‘responders’ who increase hemoglobin to therapeutic levels; (ii) ‘moderate-responders’ who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) ‘non-responders’ who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it had little impact on cells from responders. Part of the gene program up-regulated by hydroxyurea in non-responders was already highly expressed in responders before hydroxyurea treatment. Baseline HbF expression was low in non-responders, and hydroxyurea treatment induced significant cell death. We conclude that cells from responders have adapted well to constitutive stress conditions and display a propensity to proceed to the erythroid differentiation program.

Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS) or weak transcriptional activators (HIF3α/NEPAS). Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel function of Hif3α beyond the hypoxia response.

The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics.

BackgroundCurrent clinical therapy of non-small cell lung cancer depends on histo-pathological classification. This approach poorly predicts clinical outcome for individual patients. Gene expression profiling holds promise to improve clinical stratification, thus paving the way for individualized therapy.Methodology and principal findingsA genome-wide gene expression analysis was performed on a cohort of 91 patients. We used 91 tumor- and 65 adjacent normal lung tissue samples. We defined sets of predictor genes (probe sets) with the expression profiles. The power of predictor genes was evaluated using an independent cohort of 96 non-small cell lung cancer- and 6 normal lung samples. We identified a tumor signature of 5 genes that aggregates the 156 tumor and normal samples into the expected groups. We also identified a histology signature of 75 genes, which classifies the samples in the major histological subtypes of non-small cell lung cancer. Correlation analysis identified 17 genes which showed the best association with post-surgery survival time. This signature was used for stratification of all patients in two risk groups. Kaplan-Meier survival curves show that the two groups display a significant difference in post-surgery survival time (p = 5.6E-6). The performance of the signatures was validated using a patient cohort of similar size (Duke University, n = 96). Compared to previously published prognostic signatures for NSCLC, the 17 gene signature performed well on these two cohorts.ConclusionsThe gene signatures identified are promising tools for histo-pathological classification of non-small cell lung cancer, and may improve the prediction of clinical outcome.

In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X ratio A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.To obtain more insight in the role of the XratioA ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X ratio A ratios, provides evidence that the X ratio A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X ratio A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X ratio A ratio. This finding supports the presence of an X-encoded activator of the XCI process.

BACKGROUND: Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerotic lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP. METHODS AND FINDINGS: Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR-/-) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt-->LDLR-/- mice (2.3-fold) and even further in the huPLTPtg/tg-->LDLR-/- mice (4.5-fold) compared with the control PLTPwt/wt-->LDLR-/- mice (both P