One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes

A chemokine network of T cell exhaustion and metabolic reprogramming in renal cell carcinoma

Renal cell carcinoma (RCC) is frequently infiltrated by immune cells, a process which is governed by chemokines. CD8+ T cells in the RCC tumor microenvironment (TME) may be exhausted which most likely influence therapy response and survival. The aim of this study was to evaluate chemokine-driven T cell recruitment, T cell exhaustion in the RCC TME, as well as metabolic processes leading to their functional anergy in RCC. Eight publicly available bulk RCC transcriptome collectives (n=1819) and a single cell RNAseq dataset (n=12) were analyzed. Immunodeconvolution, semi-supervised clustering, gene set variation analysis and Monte Carlo-based modeling of metabolic reaction activity were employed. Among 28 chemokine genes available, CXCL9/10/11/CXCR3, CXCL13/CXCR5 and XCL1/XCR1 mRNA expression were significantly increased in RCC compared to normal kidney tissue and also strongly associated with tumor-infiltrating effector memory and central memory CD8+ T cells in all investigated collectives. M1 TAMs, T cells, NK cells as well as tumor cells were identified as the major sources of these chemokines, whereas T cells, B cells and dendritic cells were found to predominantly express the cognate receptors. The cluster of RCCs characterized by high chemokine expression and high CD8+ T cell infiltration displayed a strong activation of IFN/JAK/STAT signaling with elevated expression of multiple T cell exhaustion-associated transcripts. Chemokinehigh RCCs were characterized by metabolic reprogramming, in particular by downregulated OXPHOS and increased IDO1-mediated tryptophan degradation. None of the investigated chemokine genes was significantly associated with survival or response to immunotherapy. We propose a chemokine network that mediates CD8+ T cell recruitment and identify T cell exhaustion, altered energy metabolism and high IDO1 activity as key mechanisms of their suppression. Concomitant targeting of exhaustion pathways and metabolism may pose an effective approach to RCC therapy

Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design

Wolbachia in the flesh: symbiont intensities in germ-line and somatic tissues challenge the conventional view of Wolbachia transmission routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought

An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroups

The Y chromosome is paternally inherited and therefore serves as an evolutionary marker of patrilineal descent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be represented as a monophyletic phylogenetic tree in which the branches (haplogroups) are defined by at least one SNP. Previous human population genetics research has produced a wealth of knowledge about the worldwide distribution of Y-SNP haplogroups. Here, we apply previous and very recent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplex genotyping assays that allow for the hierarchical detection of 28 Y-SNPs defining the major worldwide Y haplogroups. PCR amplicons were kept small to make the method sensitive and thereby applicable to DNA of limited amount and/or quality such as in forensic settings. These Y-SNP assays thus form a valuable tool for researchers in the fields of forensic genetics and genetic anthropology to infer a man's patrilineal bio-geographic ancestry from DNA

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1

Aberrant transforming growth factor–β (TGF-β) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-β signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-β signaling activity and that stromal cellconditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-β–induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment

A Nonsynonymous Change in Adhesion G Protein–Coupled Receptor L3 Associated With Risk for Equine Degenerative Myeloencephalopathy in the Caspian Horse

Equine degenerative myeloencephalopathy (EDM), a neurological disease of young horses, causes progressive development of symmetric ataxia predominantly in the pelvic limbs. Equine degenerative myeloencephalopathy is likely inherited and with no known treatment affected horses frequently need euthanasia. Alpha-tocopherol deficiency during early life appears to contribute to the phenotype. This study sought to identify any genetic variants correlated with EDM in Caspian foals. Two half-sibling EDM-diagnosed cases were genotyped at 52,063 loci and evaluated by the Autozygosity by Difference statistic. Additional horses not affected by EDM were used for genetic comparison to identify regions unique to the case phenotype. The associated region on chromosome 3 contains only one gene encoding adhesion G protein–coupled receptor L3 (ADGRL3). Adhesion G protein–coupled receptor L3 is a member of the latrophilin subfamily of G protein–coupled receptors and may contribute to attention deficit/hyperactivity disorder in humans and hyperactive motor function in mice and zebrafish. Analysis of the predicted coding regions for Equine ADGRL3 in affected horses revealed a nonsynonymous single nucleotide polymorphism at Chr3:71,917,591 bp. Caspian and Caspian cross-relatives (n = 81) of the two initial cases and unrelated horses from similar breeds (n = 130, including Arabians, American Miniatures, and Shetlands) possessed this allele at 5% frequency, with no homozygotes observed within the non-Caspian breeds. This study suggests that a polymorphism in ADGRL3 could contribute to a genetic predisposition to Caspian horse EDM

RDML-Ninja and RDMLdb for standardized exchange of qPCR data

Background: The universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that did not meet the expectations of the consortium in the level of support or the use of elements. Results: In the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released (http://sourceforge.net/projects/qpcr-ninja/). RDML-Ninja allows to visualize, edit and validate RDML files and thus clarifies the use of RDML elements. Furthermore RDML-Ninja serves as reference implementation for RDML and enables migration between RDML versions independent of the instrument software. The database RDMLdb will serve as an online repository for RDML files and facilitate the exchange of RDML data (http://www.rdmldb.org). Authors can upload their RDML files and reference them in publications by the unique identifier provided by RDMLdb. The MIQE guidelines propose a rich set of information required to document each qPCR run. RDML provides the vehicle to store and maintain this information and current development aims at further integration of MIQE requirements into the RDML format. Conclusions: The editor RDML-Ninja and the database RDMLdb enable scientists to evaluate and exchange qPCR data in the instrument-independent RDML format. We are confident that this infrastructure will build the foundation for standardized qPCR data exchange among scientists, research groups, and during publication

The functional maturation of M cells is dramatically reduced in the Peyer's patches of aged mice

The transcytosis of antigens across the follicle-associated epithelium (FAE) of Peyer's patches by microfold cells (M cells) is important for the induction of efficient immune responses to mucosal antigens. The mucosal immune response is compromised by ageing, but effects on M cells were unknown. We show that M-cell density in the FAE of aged mice was dramatically reduced. As a consequence, aged Peyer's patches were significantly deficient in their ability to transcytose particulate lumenal antigen across the FAE. Ageing specifically impaired the expression of Spi-B and the downstream functional maturation of M cells. Ageing also dramatically impaired C-C motif chemokine ligand 20 expression by the FAE. As a consequence, fewer B cells were attracted towards the FAE, potentially reducing their ability to promote M-cell maturation. Our study demonstrates that ageing dramatically impedes the functional maturation of M cells, revealing an important ageing-related defect in the mucosal immune system's ability to sample lumenal antigens