Are tree seed systems for forest landscape restoration fit for purpose? An analysis of four Asian countries

High-quality, suitably adapted tree seed at volume is a key component for the implementation and long-term success of forest landscape restoration (FLR). We analysed the tree seed systems in four Asian countries—the Philippines, Indonesia, Malaysia and India—which have committed to restore in total over 47.5 million hectares of degraded lands by 2030. We assessed the national seed systems using an established indicator framework, literature review and expert surveys and interviews. Additionally, we surveyed 61 FLR practitioners about their challenges in acquiring seed to understand how the strengths and weaknesses identified at the national level were reflected in FLR projects on the ground. Identified key constraints common to the studied countries are (i) a mismatch between the growing demand for priority native species and the limited seed supply in terms of quantity and quality, (ii) lack of effective quality control for seed of native species and (iii) lack of information about the effects of climate change on native species to guide species selection and seed sourcing and to improve the resilience of restored ecosystems. We discuss options to strengthen seed systems for native tree species both in terms of quality and volume to enable them to effectively respond to the national FLR commitments and make recommendations on promising technical solutions

C-Terminal Region of EBNA-2 Determines the Superior Transforming Ability of Type 1 Epstein-Barr Virus by Enhanced Gene Regulation of LMP-1 and CXCR7

Type 1 Epstein-Barr virus (EBV) strains immortalize B lymphocytes in vitro much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. Here we demonstrate that the greater transforming activity of type 1 EBV correlates with a stronger and more rapid induction of the viral oncogene LMP-1 and the cell gene CXCR7 (which are both required for proliferation of EBV-LCLs) during infection of primary B cells with recombinant viruses. Surprisingly, although the major sequence differences between type 1 and type 2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1 EBNA-2 to induce proliferation of EBV-infected lymphoblasts is mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus of type 1 EBNA-2 into the type 2 protein is sufficient to confer a type 1 growth phenotype and type 1 expression levels of LMP-1 and CXCR7 in an EREB2.5 cell growth assay. Within this region, the RG, CR7 and TAD domains are the minimum type 1 sequences required. Sequencing the C-terminus of EBNA-2 from additional EBV isolates showed high sequence identity within type 1 isolates or within type 2 isolates, indicating that the functional differences mapped are typical of EBV type sequences. The results indicate that the C-terminus of EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell proliferation, through mechanisms that include higher induction of genes (LMP-1 and CXCR7) required for proliferation and survival of EBV-LCLs

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies

Epstein - Barr virus Encoded EBER RNAs

Epstein-Barr virus (EBV) establishes a lifelong latent infection in 95% of the world’s population and is associated with several human cancers. Some of the most abundant viral products in latent infection are the functional EBV-encoded RNAs EBER1 and EBER2. The aim of this thesis was the identification of distinct functions of EBER1 and EBER2 during EBV infection. Epstein-Barr virus bacterial artificial chromosomes with deletion of either EBER1 or EBER2 and corresponding revertant viral genomes were constructed to analyse broad range effects of EBER1 or EBER2 on host cell gene expression. The resulting recombinant viruses were used to infect primary B lymphocytes and establish lymphoblastoid cell lines (LCLs). Microarray expression profiling revealed clear changes in host cell gene expression correlating with EBER expression and significant differences between gene sets regulated with EBER1 and EBER2. Functions of EBER target genes include membrane signalling, regulation of apoptosis and the interferon response. In additional studies, the interaction of EBER1 with ribosomal protein L22 (RPL22), a component of the large ribosomal subunit, was demonstrated in LCL extracts. Using recombinant viruses and EBER expression vectors, the nuclear redistribution of RPL22 by EBER1 was investigated. The delocalisation of RPL22 from nucleoli into the nucleoplasm upon EBV infection was demonstrated in HEK 293, nasopharyngeal carcinoma and gastric carcinoma-derived cell lines. EBER1 was identified as the only viral component necessary for the delocalisation of RPL22. In contrast to the cancer-derived cell lines, LCLs showed a predominantly cytoplasmic expression of RPL22, which was not significantly changed by EBER1. Subsequently, a possible role of RPL22 and EBER1 in p53-dependent stress responses was explored. The data presented in this thesis provide further understanding of the role of EBERs in EBV infection and several of the EBER-regulated genes might be used as markers to elucidate the mechanism of EBER action

Epstein-Barr virus encoded EBER RNAs

Epstein-Barr virus (EBV) establishes a lifelong latent infection in 95% of the world’s population and is associated with several human cancers. Some of the most abundant viral products in latent infection are the functional EBV-encoded RNAs EBER1 and EBER2. The aim of this thesis was the identification of distinct functions of EBER1 and EBER2 during EBV infection. Epstein-Barr virus bacterial artificial chromosomes with deletion of either EBER1 or EBER2 and corresponding revertant viral genomes were constructed to analyse broad range effects of EBER1 or EBER2 on host cell gene expression. The resulting recombinant viruses were used to infect primary B lymphocytes and establish lymphoblastoid cell lines (LCLs). Microarray expression profiling revealed clear changes in host cell gene expression correlating with EBER expression and significant differences between gene sets regulated with EBER1 and EBER2. Functions of EBER target genes include membrane signalling, regulation of apoptosis and the interferon response. In additional studies, the interaction of EBER1 with ribosomal protein L22 (RPL22), a component of the large ribosomal subunit, was demonstrated in LCL extracts. Using recombinant viruses and EBER expression vectors, the nuclear redistribution of RPL22 by EBER1 was investigated. The delocalisation of RPL22 from nucleoli into the nucleoplasm upon EBV infection was demonstrated in HEK 293, nasopharyngeal carcinoma and gastric carcinoma-derived cell lines. EBER1 was identified as the only viral component necessary for the delocalisation of RPL22. In contrast to the cancer-derived cell lines, LCLs showed a predominantly cytoplasmic expression of RPL22, which was not significantly changed by EBER1. Subsequently, a possible role of RPL22 and EBER1 in p53-dependent stress responses was explored. The data presented in this thesis provide further understanding of the role of EBERs in EBV infection and several of the EBER-regulated genes might be used as markers to elucidate the mechanism of EBER action.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Epstein-barr viruses (EBVs) deficient in EBV-encoded RNAs have higher levels of latent membrane protein 2 RNA expression in lymphoblastoid cell lines and efficiently establish persistent infections in humanized mice

Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system

Cellular Gene Expression That Correlates with EBER Expression in Epstein-Barr Virus-Infected Lymphoblastoid Cell Lines ▿ †

Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs