A cost-effective strategy for marker assisted selection (MAS) in apple (M. pumila Mill.): the experience from the Fondazione Edmund Mach programme for resistance and quality traits

7III International Symposium on Molecular Markers in Horticulture, Riva del Garda (TN), Italy, September 25-27, 2013partially_openInternationalMarker assisted selection (MAS) permits the selection of plants carrying genes that control interesting agronomic traits through the screening of associated markers; this allows the breeders to retain only germplasm conferring desirable traits from an early stage. Usually MAS involves the screening of hundreds or thousands of seedlings and thus the main challenge faced is the development of a pipeline that starts with a “lab friendly” and unambiguous seedling disposition in greenhouse, allows the rapid collection of samples avoiding cross contamination, implements a fast, cost-effective, high-throughput method of DNA extraction to be used for a small number of PCRs, and utilizes robust, reliable, and easy to interpret molecular markers. In this study ~6,500 seedlings from 24 apple crosses were genotyped in order to select seedlings conferring resistance traits, fruit quality traits or both. Lab work was performed using the KAPA 3G Plant PCR kit. The main problems encountered were associated with dirty extractions; however, following optimization less than 10% of the seedlings had to be resampled and in total about 70% of the samples tested carried desirable traitspartially_openBanchi, E.; Poles, L.; Magnago, P.; Pindo, M.; Costa, F.; Velasco, R.; Sargent, D.J.Banchi, E.; Poles, L.; Magnago, P.; Pindo, M.; Costa, F.; Velasco, R.; Sargent, D.J

Foregut microbiome in development of esophageal adenocarcinoma

Esophageal adenocarcinoma (EA), the type of cancer linked to heartburn due to gastroesophageal reflux diseases (GERD), has increased six fold in the past 30 years. This cannot currently be explained by the usual environmental or by host genetic factors. EA is the end result of a sequence of GERD-related diseases, preceded by reflux esophagitis (RE) and Barrett’s esophagus (BE). Preliminary studies by Pei and colleagues at NYU on elderly male veterans identified two types of microbiotas in the esophagus. Patients who carry the type II microbiota are >15 fold likely to have esophagitis and BE than those harboring the type I microbiota. In a small scale study, we also found that 3 of 3 cases of EA harbored the type II biota. The findings have opened a new approach to understanding the recent surge in the incidence of EA. 

Our long-term goal is to identify the cause of GERD sequence. The hypothesis to be tested is that changes in the foregut microbiome are associated with EA and its precursors, RE and BE in GERD sequence. We will conduct a case control study to demonstrate the microbiome disease association in every stage of GERD sequence, as well as analyze the trend in changes in the microbiome along disease progression toward EA, by two specific aims. Aim 1 is to conduct a comprehensive population survey of the foregut microbiome and demonstrate its association with GERD sequence. Furthermore, spatial relationship between the esophageal microbiota and upstream (mouth) and downstream (stomach) foregut microbiotas as well as temporal stability of the microbiome-disease association will also be examined. Aim 2 is to define the distal esophageal metagenome and demonstrate its association with GERD sequence. Detailed analyses will include pathway-disease and gene-disease associations. Archaea, fungi and viruses, if identified, also will be correlated with the diseases. A significant association between the foregut microbiome and GERD sequence, if demonstrated, will be the first step for eventually testing whether an abnormal microbiome is required for the development of the sequence of phenotypic changes toward EA. If EA and its precursors represent a microecological disease, treating the cause of GERD might become possible, for example, by normalizing the microbiota through use of antibiotics, probiotics, or prebiotics. Causative therapy of GERD could prevent its progression and reverse the current trend of increasing incidence of EA

Prolonged post-faint hypotension can be reversed by dynamic tension

A severe variant of vasovagal syncope, observed during tilt tests and blood donation has recently been termed “prolonged post-faint hypotension” (PPFH). A 49-year-old male with a life-long history of severe fainting attacks underwent head-up tilt for 20 min, and developed syncope 2 min after nitroglycerine spray. He was unconscious for 40 s and asystolic for 22 s. For the first 2 min of recovery, BP and HR remained low (65/45 mmHg and 40 beats/min) despite passive leg-raising. Blood pressure (and symptoms) only improved following active bilateral leg flexion and extension (“dynamic tension”). During PPFH, when vagal activity is extreme, patients may require central stimulation as well as correction of venous return

ParadisEO-MOEO: A Software Framework for Evolutionary Multi-Objective Optimization

This chapter presents ParadisEO-MOEO, a white-box object-oriented software framework dedicated to the flexible design of metaheuristics for multi-objective optimization. This paradigm-free software proposes a unified view for major evolutionary multi-objective metaheuristics. It embeds some features and techniques for multi-objective resolution and aims to provide a set of classes allowing to ease and speed up the development of computationally efficient programs. It is based on a clear conceptual distinction between the solution methods and the problems they are intended to solve. This separation confers a maximum design and code reuse. This general-purpose framework provides a broad range of fitness assignment strategies, the most common diversity preservation mechanisms, some elitistrelated features as well as statistical tools. Furthermore, a number of state-of-the-art search methods, including NSGA-II, SPEA2 and IBEA, have been implemented in a user-friendly way, based on the fine-grained ParadisEO-MOEO components

Identification and validation of a QTL influencing bitter pit symptoms in apple (Malus x domestica)

Bitter pit is one of the most economically important physiological disorders affecting apple fruit production, causing soft discrete pitting of the cortical flesh of the apple fruits which renders them unmarketable. The disorder is heritable; however, the environment and cultural practices play a major role in expression of symptoms. Bitter pit has been shown to be controllable to a certain extent using calcium sprays and dips; however, their use does not entirely prevent the incidence of the disorder. Previously, bitter pit has been shown to be controlled by two dominant genes, and markers on linkage group 16 of the apple genome were identified that were significantly associated with the expression of bitter pit symptoms in a genome-wide association study. In this investigation, we identified a major QTL for bitter pit defined by two microsatellite (SSR) markers. The association of the SSRs with the bitter pit locus, and their ability to predict severe symptom expression, was confirmed through screening of individuals with stable phenotypic expression from an additional mapping progeny. The data generated in this current study suggest a two gene model could account for the control of bitter pit symptom expression; however, only one of the loci was detectable, most likely due to dominance of alleles carried by both parents of the mapping progeny used. The SSR markers identified are cost-effective, robust and multi-allelic and thus should prove useful for the identification of seedlings with resistance to bitter pit using marker-assisted selection in apple breeding programs

Immunity to HIV-1 Is Influenced by Continued Natural Exposure to Exogenous Virus

Unprotected sexual intercourse between individuals who are both infected with HIV-1 can lead to exposure to their partner's virus, and potentially to super-infection. However, the immunological consequences of continued exposure to HIV-1 by individuals already infected, has to our knowledge never been reported. We measured T cell responses in 49 HIV-1 infected individuals who were on antiretroviral therapy with suppressed viral loads. All the individuals were in a long-term sexual partnership with another HIV-1 infected individual, who was either also on HAART and suppressing their viral loads, or viremic (>9000 copies/ml). T cell responses to HIV-1 epitopes were measured directly ex-vivo by the IFN-γ enzyme linked immuno-spot assay and by cytokine flow cytometry. Sexual exposure data was generated from questionnaires given to both individuals within each partnership. Individuals who continued to have regular sexual contact with a HIV-1 infected viremic partner had significantly higher frequencies of HIV-1-specific T cell responses, compared to individuals with aviremic partners. Strikingly, the magnitude of the HIV-1-specific T cell response correlated strongly with the level and route of exposure. Responses consisted of both CD4+ and CD8+ T cell subsets. Longitudinally, decreases in exposure were mirrored by a lower T cell response. However, no evidence for systemic super-infection was found in any of the individuals. Continued sexual exposure to exogenous HIV-1 was associated with increased HIV-1-specific T cell responses, in the absence of systemic super-infection, and correlated with the level and type of exposure

BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation

Daily Sampling of an HIV-1 Patient with Slowly Progressing Disease Displays Persistence of Multiple env Subpopulations Consistent with Neutrality

The molecular evolution of HIV-1 is characterized by frequent substitutions, indels and recombination events. In addition, a HIV-1 population may adapt through frequency changes of its variants. To reveal such population dynamics we analyzed HIV-1 subpopulation frequencies in an untreated patient with stable, low plasma HIV-1 RNA levels and close to normal CD4+ T-cell levels. The patient was intensively sampled during a 32-day period as well as approximately 1.5 years before and after this period (days −664, 1, 2, 3, 11, 18, 25, 32 and 522). 77 sequences of HIV-1 env (approximately 3100 nucleotides) were obtained from plasma by limiting dilution with 7–11 sequences per time point, except day −664. Phylogenetic analysis using maximum likelihood methods showed that the sequences clustered in six distinct subpopulations. We devised a method that took into account the relatively coarse sampling of the population. Data from days 1 through 32 were consistent with constant within-patient subpopulation frequencies. However, over longer time periods, i.e. between days 1…32 and 522, there were significant changes in subpopulation frequencies, which were consistent with evolutionarily neutral fluctuations. We found no clear signal of natural selection within the subpopulations over the study period, but positive selection was evident on the long branches that connected the subpopulations, which corresponds to >3 years as the subpopulations already were established when we started the study. Thus, selective forces may have been involved when the subpopulations were established. Genetic drift within subpopulations caused by de novo substitutions could be resolved after approximately one month. Overall, we conclude that subpopulation frequencies within this patient changed significantly over a time period of 1.5 years, but that this does not imply directional or balancing selection. We show that the short-term evolution we study here is likely representative for many patients of slow and normal disease progression

Isolation and Characterization of Intestinal Epithelial Cells from Normal and SIV-Infected Rhesus Macaques

Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function