Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W (N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78+/-4 vs. 88+/-2%, respectively, P<0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic (P<0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals (P<0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased (P<0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.

Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity

Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.Laboratory Investigation advance online publication, 24 November 2014; doi:10.1038/labinvest.2014.141.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

INTRODUCTION Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. RESULTS For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient's bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n=88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n=91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n=91), which reflect the total neoplastic burden, revealed four patient groups with different survival. CONCLUSION AND PERSPECTIVE Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

Epithelial NAIPs protect against colonic tumorigenesis.

NLR family apoptosis inhibitory proteins (NAIPs) belong to both the Nod-like receptor (NLR) and the inhibitor of apoptosis (IAP) families. NAIPs are known to form an inflammasome with NLRC4, but other in vivo functions remain unexplored. Using mice deficient for all NAIP paralogs (Naip1-6(Δ/Δ)), we show that NAIPs are key regulators of colorectal tumorigenesis. Naip1-6(Δ/Δ) mice developed increased colorectal tumors, in an epithelial-intrinsic manner, in a model of colitis-associated cancer. Increased tumorigenesis, however, was not driven by an exacerbated inflammatory response. Instead, Naip1-6(Δ/Δ) mice were protected from severe colitis and displayed increased antiapoptotic and proliferation-related gene expression. Naip1-6(Δ/Δ) mice also displayed increased tumorigenesis in an inflammation-independent model of colorectal cancer. Moreover, Naip1-6(Δ/Δ) mice, but not Nlrc4-null mice, displayed hyper-activation of STAT3 and failed to activate p53 18 h after carcinogen exposure. This suggests that NAIPs protect against tumor initiation in the colon by promoting the removal of carcinogen-elicited epithelium, likely in a NLRC4 inflammasome-independent manner. Collectively, we demonstrate a novel epithelial-intrinsic function of NAIPs in protecting the colonic epithelium against tumorigenesis.

“But look: we have sat here too long”: Hilary Mantel’s tightrope walk of present-tense usage

Even after Hilary Mantel has won the Man Booker prize two times in a row with Wolf Hall and Bring up the Bodies, her novelistic account of the life of Thomas Cromwell, her intriguing decision to write these historical novels in the present tense gave cause to surprisingly little extended comment beyond a perfunctory nod to its evocation of immediacy. This presents not only a lacunae in the discussion about Mantel’s novels, but is also symptomatic for a change in the contemporary critical evaluation of present-tense narration in general. If present-tense narration once used to be a marker for experimental daring and might even have implied a certain hostility towards fictionality, Mantel’s novels give ample evidence that literary sensibilities have changed. In order to understand the scope and nature of this change, my paper puts Mantel’s use of the present tense in the context of both the historical development of present-tense usage and the ample contemporary landscape of present-tense narration. This allows me to show that the complexities of present-tense usage belie a reduction of its effect to an evocation of immediacy. Rather, I argue, Mantel uses it for a delicate tightrope walk between proximity and distance, history and fiction, authenticity and imagination.

PROSTATE CANCER STEM CELLS: DRUG TOLERANCE, EXPERIMENTAL RECONSTITUTION AND CASTRATION RESISTANCE

Prostate cancer (PCa) is one of the leading malignancies affecting men in the Western world. Although tremendous effort has been made towards understanding PCa development and developing clinical treatments in the past decades, the exact mechanisms of PCa are still not clearly understood. Emerging evidence has postulated that a population of stem cell-like cells inside a tumor, termed ‘cancer stem cells (CSCs)’, may be the cells responsible for tumor initiation, progression, recurrence, metastasis and therapy resistance. Like CSC studies in other cancer types, it has been reported that PCa also contains CSCs. However, there remain several unresolved questions that need to be clarified. First, the relationship between prostate CSCs (PCSCs) and therapy resistance (chemo- and radio-) is not known. Herein, we have found that not all CSCs are drug-tolerant, and not all drug-tolerant cells are CSCs. Second, whether primary human PCa (HPCa) actually contain PCSCs remains unclear, due to the well-known fact that we have yet to establish a reliable assay system that can reproducibly and faithfully reconstitute tumor regeneration from single HPCa cells. Herein, after utilizing more than 114 HPCa samples we have provided evidence that immortalized bone marrow-derived stromal cells (Hs5) can help dissociated HPCa cells generate undifferentiated tumors in immunodeficient NOD/SCID-IL2Rγ-/- mice, and the undifferentiated PCa cells seem to have a survival advantage to generate tumors. Third, the evolution of PCa from androgen dependent to the lethally castration resistant (CRPC) stage remains enigmatic, and the cells responsible for CRPC development have not been identified. Herein, we have found a putative cell population, ALDH+CD44+α2β1+ PCa cells that may represent a cell-of-origin for CRPC. Taken together, our work has improved our understanding of PCSC properties, possibly highlighting a potential therapeutic target for CRPC.

AMS radiocarbon dates and pollen analysis of ODP Hole 175-1078C

ODP Site 1078 situated under the coast of Angola provides the first record of the vegetation history for Angola. The upper 11 m of the core covers the past 30 thousand years, which has been analysed palynologically in decadal to centennial resolution. Alkenone sea surface temperature estimates were analysed in centennial resolution. We studied sea surface temperatures and vegetation development during full glacial, deglacial, and interglacial conditions. During the glacial the vegetation in Angola was very open consisting of grass and heath lands, deserts and semi-deserts, which suggests a cool and dry climate. A change to warmer and more humid conditions is indicated by forest expansion starting in step with the earliest temperature rise in Antarctica, 22 thousand years ago. We infer that around the period of Heinrich Event 1, a northward excursion of the Angola Benguela Front and the Congo Air Boundary resulted in cool sea surface temperatures but rain forest remained present in the northern lowlands of Angola. Rain forest and dry forest area increase 15 thousand years ago. During the Holocene, dry forests and Miombo woodlands expanded. Also in Angola globally recognised climate changes at 8 thousand and 4 thousand years ago had an impact on the vegetation. During the past 2 thousand years, savannah vegetation became dominant.

Distribution of pollen and spores in sediments of ODP Hole 175-1078C

The distribution of pollen in marine sediments is used to record vegetation change on the continent. Generally, a good latitudinal correspondence exists between the distribution patterns of pollen in the marine surface sediments and the occurrence of the source plants on the adjacent continent. To investigate land-sea interactions during deglaciation, we compare proxies for continental (pollen assemblages) and marine conditions (alkenone-derived sea surface temperatures) of two high-resolution, radiocarbon-dated sedimentary records from the tropical southeast Atlantic. The southern site is located West of the Cunene River mouth; the northern site is located West of the Angolan Huambe Mountains. It is inferred that the vegetation in Angola developed from Afroalpine and open savannah during the last Glacial maximum (LGM) via Afromontane Podocarpus forest during Heinrich Event 1 (H1), to an early increase of lowland forest after 14.5 ka. The vegetation record indicates dry and cold conditions during the LGM, cool and wet conditions during H1 and a gradual rise in temperature starting well before the Younger Dryas (YD) period. Terrestrial and oceanic climate developments seem largely running parallel, in contrast to the situation ca. 5° further South, where marine and terrestrial developments diverge during the YD. The cool and wet conditions in tropical West Africa, South of the equator, during H1 suggest that low-latitude insolation variation is more important than the slowdown of the thermohaline circulation for the climate in tropical Africa.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns.

The Pangenome of the Plant-Selected Rhizobium leguminosarum bv viciae Sub-popuations

Rhizobium leguminosarum bv viciae (Rlv) is a bacterium able to establish effective symbioses with four different legume genera: Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants have previously shown that, given a choice, different plants prefer specific genotypes of rhizobia, which are adapted to the host (1, 2). In previous work we have performed a Pool-Seq analysis bases on pooled DNA samples from Rlv nodule isolates obtained from Pisum sativum, Lens culinaris, Vicia fava and V. sativa plants, used as rhizobial traps. This experiment allowed us to test the host preference hypothesis: different plant hosts select specific sub-populations of rhizobia from the available population present in a given soil. We have observed that plant-selected sub-populations are different at the single nucleotide polymorphism (SNP) level. We have selected individual isolates from each sub-population (9 fava-bean isolates, 14 pea isolates 9 vetch isolates and 9 lentil isolates) and sequenced their genomes at draft level (ca. 30x, 90 contigs). Genomic analyses have been carried out using J-species and CMG-Biotools. All the isolates had similar genome size (7.5 Mb) and number of genes (7,300). The resulting Average Nucleotide Identity (ANIm) tree showed that Rhizobium leguminosarum bv viciae is a highly diverse group. Each plant-selected subpopulation showed a closed pangenome and core genomes of similar size (11,500 and 4,800 genes, respectively). The addition of all four sub-population results in a larger, closed pangenome of 19,040 genes and a core genome of similar size (4,392 genes). Each sub-population contains a characteristic set of genes but no universal, plant-specific genes were found. The core genome obtained from all four sub-populations is probably a representative core genome for Rhizobium leguminosarum, given that the reference genome (Rhizobium leguminosarum bv. viciae strain 3841) contains most of the core genome. We have also analyzed the symbiotic cluster (nod), and different nod cluster genotypes were found in each sub-population. Supported by MINECO (Consolider-Ingenio 2010, MICROGEN Project, CSD2009-00006).

Transfer of human serum IgG to nonobese diabetic Igμnull mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren’s syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren’s syndrome. NOD.Igμnull mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igμnull mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab′)2 fragments from parental NOD mice or human primary Sjögren’s syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren’s syndrome.

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

Ethylene-mediated phenotypic plasticity in root nodule development on Sesbania rostrata

Leguminous plants in symbiosis with rhizobia form either indeterminate nodules with a persistent meristem or determinate nodules with a transient meristematic region. Sesbania rostrata was thought to possess determinate stem and root nodules. However, the nature of nodule development is hybrid, and the early stages resemble those of indeterminate nodules. Here we show that, depending on the environmental conditions, mature root nodules can be of the indeterminate type. In situ hybridizations with molecular markers for plant cell division, as well as the patterns of bacterial nod and nif gene expression, confirmed the indeterminate nature of 30-day-old functional root nodules. Experimental data provide evidence that the switch in nodule type is mediated by the plant hormone ethylene.