Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

<p>Abstract</p> <p>Background</p> <p>Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including <it>Coffea arabica</it>.</p> <p>Results</p> <p>We identified the conditions required for successful long-term proliferation of embryogenic cultures in <it>C. arabica </it>and designed a highly efficient and reliable <it>Agrobacterium tumefaciens</it>-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD₆₀₀= 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.</p> <p>Conclusion</p> <p>Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make <it>Agrobacterium</it>-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.</p

Detection of Xylella fastidiosa in coffee germplasm

A bactéria Xylella fastidiosa possui uma ampla gama de plantas hospedeiras que inclui espécies de pelo menos 28 famílias de mono e dicotiledôneas. Em cafeeiro (Coffea spp.), a ocorrência dessa bactéria foi relatada previamente em cultivares da espécie Coffea arabica. Estudos foram realizados para determinar a presença de X. fastidiosa em diferentes espécies e híbridos interespecíficos de cafeeiro. As amostragens foram realizadas em dois anos consecutivos. As espécies de cafeeiro examinadas foram: C. kapakata, C. canephora, C. racemosa, C. arabica, C. dewevrei, C. stenophylla e C. eugenioides. Também foram incluídos neste estudo híbridos interespecíficos de C. arabica: C. arabica x C. dewevrei, C. arabica x C. eugenioides, C. arabica x C. racemosa e C. arabica x C. robusta. Foram coletadas amostras de ramos plagiotrópicos de diferentes plantas para cada espécie e híbrido. A detecção de X. fastidiosa nas amostras foi realizada utilizando os testes serológicos de DAS-ELISA e imunofluorescência indireta. A bactéria foi detectada nas sete espécies e nos quatro híbridos de cafeeiro examinados. Entretanto, as plantas aparentemente não apresentavam sintomas de infecção por X. fastidiosa. A espécie C. arabica apresentou a maior proporção de amostras positivas e maiores valores de absorbância no teste de DAS-ELISA. Em contraste, as espécies C. racemosa e C. dewevrei foram as que apresentaram menores proporções de amostras positivas para presença de X. fastidiosa, como também menores valores de absorbância no teste de DAS-ELISA.The bacterium Xylella fastidiosa has a large host range, including species of 28 different mono and dicotiledoneous plant families. On coffee (Coffea spp.), X. fastidiosa was previously reported only on the species Coffea arabica. Studies were carried out to determine the presence of X. fastidiosa in different species and interspecific hybrids of coffee. The species of coffee included in this study were the following: C. kapakata, C. canephora, C. racemosa, C. arabica, C. dewevrei, C. stenophylla and C. eugenioides. The interespecific hybrids of C. arabica examined were the following: C. arabica x C. dewevrei, C. arabica x C. eugenioides, C. arabica x C. racemosa and C. arabica x C. robusta. Samples were collected from plagiotropic branches of different plants for each species or hybrid. Four replicates were examined for each coffee accession examined. Detection of X. fastidiosa in the samples was determined by DAS-ELISA and indirect immunofluorescence. The bacterium was detected in all seven species and four hybrids of coffee studied. However, the plants did not show any symptom of infection by X. fastidiosa. The species C. arabica showed the highest proportion of positive samples and the largest absorbance values in the DAS-ELISA test. In contrast, the species C. racemosa and C. dewevrei showed the lowest proportion of positive samples for the presence of X. fastidiosa, as well as, the lowest absorbance values in the DAS-ELISA test

Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.Peer ReviewedPostprint (published version

Evidence for the formation of comet 67P/Churyumov-Gerasimenko through gravitational collapse of a bound clump of pebbles

The processes that led to the formation of the planetary bodies in the Solar System are still not fully understood. Using the results obtained with the comprehensive suite of instruments on-board ESA’s Rosetta mission, we present evidence that comet 67P/Churyumov-Gerasimenko likely formed through the gentle gravitational collapse of a bound clump of mm-sized dust aggregates (“pebbles”), intermixed with microscopic ice particles. This formation scenario leads to a cometary make-up that is simultaneously compatible with the global porosity, homogeneity, tensile strength, thermal inertia, vertical temperature profiles, sizes and porosities of emitted dust, and the steep increase in water-vapour production rate with decreasing heliocentric distance, measured by the instruments on-board the Rosetta spacecraft and the Philae lander. Our findings suggest that the pebbles observed to be abundant in protoplanetary discs around young stars provide the building material for comets and other minor bodies

Future perspectives in melanoma research. Meeting report from the “Melanoma Bridge. Napoli, December 2nd-4th 2012”

Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma. After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma. The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma. The third “Melanoma Research: “A bridge from Naples to the World” meeting, shortened as “Bridge Melanoma Meeting” took place in Naples, December 2 to 4th, 2012. The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma. This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients

Organization and molecular evolution of a disease-resistance gene cluster in coffee trees

<p>Abstract</p> <p>Background</p> <p>Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (<it>Coffea arabica</it>), a region spanning the <it>S</it>_<it>H</it><it>3 </it>locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the <it>S</it>_<it>H</it><it>3 </it>locus.</p> <p>Results</p> <p>Sequence analysis of the <it>S</it>_<it>H</it><it>3 </it>region in three coffee genomes, E^aand C^asubgenomes from the allotetraploid <it>C. arabica </it>and C^cgenome from the diploid <it>C. canephora</it>, revealed the presence of 5, 3 and 4 R genes in E^a, C^a, and C^cgenomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the <it>S</it>_<it>H</it><it>3 </it>locus in <it>C. arabica</it>. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the <it>S</it>_<it>H</it><it>3</it>-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the <it>S</it>_<it>H</it><it>3 </it>locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of <it>C. arabica</it>. Significant positive selection was detected in the solvent-exposed residues of the <it>S</it>_<it>H</it><it>3</it>-CNL copies.</p> <p>Conclusion</p> <p>The ancestral <it>S</it>_<it>H</it><it>3</it>-CNL copy was inserted in the <it>S</it>_<it>H</it><it>3 </it>locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the <it>S</it>_<it>H</it><it>3</it>-CNL copies predates the divergence between <it>Coffea </it>species. The <it>S</it>_<it>H</it><it>3</it>-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the <it>S</it>_<it>H</it><it>3 </it>locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of <it>S</it>_<it>H</it><it>3</it>-CNL in coffee trees.</p

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival