RNA‐Seq bulked segregant analysis enables the identification of high‐resolution genetic markers for breeding in hexaploid wheat

The identification of genetic markers linked to genes of agronomic importance is a major aim of crop research and breeding programmes. Here, we identify markers for Yr15, a major disease resistance gene for wheat yellow rust, using a segregating F₂ population. After phenotyping, we implemented RNA sequencing (RNA‐Seq) of bulked pools to identify single‐nucleotide polymorphisms (SNP) associated with Yr15. Over 27 000 genes with SNPs were identified between the parents, and then classified based on the results from the sequenced bulks. We calculated the bulk frequency ratio (BFR) of SNPs between resistant and susceptible bulks, selecting those showing sixfold enrichment/depletion in the corresponding bulks (BFR > 6). Using additional filtering criteria, we reduced the number of genes with a putative SNP to 175. The 35 SNPs with the highest BFR values were converted into genome‐specific KASP assays using an automated bioinformatics pipeline (PolyMarker) which circumvents the limitations associated with the polyploid wheat genome. Twenty‐eight assays were polymorphic of which 22 (63%) mapped in the same linkage group as Yr15. Using these markers, we mapped Yr15 to a 0.77‐cM interval. The three most closely linked SNPs were tested across varieties and breeding lines representing UK elite germplasm. Two flanking markers were diagnostic in over 99% of lines tested, thus providing a reliable haplotype for marker‐assisted selection in these breeding programmes. Our results demonstrate that the proposed methodology can be applied in polyploid F₂ populations to generate high‐resolution genetic maps across target intervals.     

Clinical and radiologic rebound after discontinuation of natalizumab therapy in a highly active multiple sclerosis patient was not halted by dimethyl-fumarate: a case report

Background

The evidence on the use of the oral dimethyl-fumarate after the discontinuation of treatment with natalizumab in people with Multiple Sclerosis is still little. Natalizumab discontinuation may induce the recurrence or rebound of the clinical and neuroradiological disease activity. Currently no therapeutic approach has been established to abolish disease reactivation and rebound after natalizumab interruption.

Case Presentation

We describe a case of a 21-year-old woman affected from a highly active relapsing-remitting Multiple Sclerosis who developed a clinical and radiological rebound 5 months after the last infusion of natalizumab, while she was being treated with dimethyl-fumarate 240 mg twice daily. She had received a bridge “therapy” with Cyclophosphamide before staring dimethyl-fumarate.

Conclusion

We report on this case to stimulate further research to establish whether new current and future drugs available for multiple sclerosis are able to halt the disease rebound after the natalizumab interruption.

     

Are atopy and eosinophilic bronchial inflammation associated with relapsing forms of chronic rhinosinusitis with nasal polyps?

Background

The aetiopathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) is still unknown. The role of atopy and the concept of united airways in such patients are still a matter of debate. In this pilot study we aimed at evaluating the degree of eosinophilic inflammation and the frequency of atopy in a cohort of CRSwNP patients candidate for Functional Endoscopic Sinus Surgery (FESS) and assessing the association between these factors and relapsing forms of CRSwNP.

Methods

30 patients (18 men, 12 women) with CRSwNP eligible for FESS were evaluated before and after surgery. Preoperative investigation included: history of previous relapse after FESS, clinical and laboratory allergologic assessment, spirometry, methacholine challenge, blood eosinophilia and determination of the fraction of nitric oxide in exhaled air (FeNO). Nasal fibroendoscopy, spirometry and FeNO determination were also assessed prospectively at 3 and 27 months post-FESS.

Results

18/30 subjects were atopic, 6/18 (33 %) were monosensitized, 16/30 (53 %) were asthmatics and 10/30 (33 %) had non steroidalantinflammatory drugs (NSAIDs) hypersensitivity. Twenty-one patients (70 %) were classified as relapsers, 15/18 (83 %) among atopics, 6/12 (50 %) among non atopics (p = 0.05). Among patients with NSAIDs hypersensitivity, 9/10 (90 %) were relapsers. The median IgE concentration was 161.5 UI/mL in relapsers and 79 UI/mL in non-relapsers (ns). The mean FeNO decreased after FESS (43.1–26.6 ppb) in 84 % of patients, but this effect disappeared over time (FeNO = 37.7 ppb at 27 months). Higher levels of FeNO pre-FESS were detected in atopics, and in particular in relapsing ones (median 51.1 ppb vs 22.1, ns). Higher levels of FeNO pre-FESS were detected in asthmatic patients, especially in those who relapsed (median: 67 vs 64.85 ppb in non-relapsed patients, ns). The Tiffeneau Index (FEV1/FVC) was significantly lower in asthmatic relapsers than in non relapsers asthmatics (94.7 ± 11.1 versus 105 ± 5.9—p = 0.04). Patients with asthma and atopy had a major risk of relapse (p = 0.05).

Conclusion

In our pilot study, atopy, severe asthma, bronchial inflammation, NSAIDs hypersensitivity and high level of total IgE are possible useful prognostic factors for the proneness to relapse after FESS. The role of allergy in CRSwNP pathogenesis should consequently be given deeper consideration. Allergen specific immunotherapy, combined with anti-IgE therapy, may have an immunomodulatory effect preventing polyps relapse and need to be investigated.

Electronic supplementary material

The online version of this article (doi:10.1186/s12948-015-0026-8) contains supplementary material, which is available to authorized users.     

Cardiac surgery in 260 octogenarians: a case series

Background

The elderly undergo cardiac surgery more and more frequently, often present multiple comorbidities, assume chronic therapies, and present a unique physiology. Aim of our study was to analyze the experience of a referral cardiac surgery center with all types of cardiac surgery interventions performed in patients ≥80 years old over a six years’ period.

Methods

A retrospective observational study performed in a university hospital. 260 patients were included in the study (3.5% of the patients undergoing cardiac surgery in the study period).

Results

Mean age was 82 ± 1.8 years. Eighty-five percent of patients underwent elective surgery, 15% unplanned surgery and 4.2% redo surgery. Intervention for aortic valve pathology and coronary artery bypass grafting were performed in 51% and 46% of the patients, respectively. Interventions involving the mitral valve were the 26% of the total, those on the tricuspid valve were 13% and those on the ascending aortic arch the 9.6%. Postoperative low output syndrome was identified in 44 patients (17%). Mortality was 3.9% and most of the patients (91%) were discharged from hospital in good clinical conditions. Hospital mortality was lower in planned vs unplanned surgery: 3.8% vs 14% respectively. Chronic obstructive pulmonary disease (OR 9.106, CI 2.275 – 36.450) was the unique independent predictor of mortality.

Conclusions

Clinicians should be aware that cardiac surgery can be safely performed at all ages, that risk stratification is mandatory and that hemodynamic treatment to avoid complications is expected.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2253-15-15) contains supplementary material, which is available to authorized users.     

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples

To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan–Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan–Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.Triple-negative breast cancers (TNBC)¹ are defined by a lack of expression of estrogen (ER), progesterone (PR), and HER2/neu receptors and account for about 15% of all breast cancers. This subtype is associated with poor prognosis (1) in terms of distant free survival (DFS) and overall survival (OS), and to date, there is no clinically available targeted therapy for patients diagnosed with TNBC. Because of the absence of specific treatment guidelines for this group of patients, TNBC are managed with standard adjuvant chemotherapy (2), which, however, seems to be less effective in those cancers. In order to improve survival, it is important to determine new specific-targeted treatment.A proteomic analysis has several inherent advantages over a genomic approach in that measured mRNA levels do not necessarily correlate to corresponding protein levels. In addition, protein detection is probably also more reflective of the tumor microenvironment. Several proteomic studies have been conducted on TNBC (3–5), but no proteomic study was conducted on large cohorts including the clinical outcome of the patients, except a recent comparative proteome analysis that identified a 11-protein signature for aggressive TNBC in a large cohort of 93 microdissected tumors (6). Although microdissection was necessary to elucidate the contribution of TNBC cells, it did not reflect the tumor with its microenvironment that is increasingly described as fundamental to explain the tumor outcome. Thus, it is now recognized that carcinomas derive from phenomena that occur in tissues, not in individual cancer cells. From this perspective, the microenvironment becomes an integral, essential part of the tumor (7, 8). In this context, taking into account the tumor microenvironment, we investigated a cohort of 83 TNBC samples without microdissection by a quantitative proteomic approach using iTRAQ labeling. Based on clinical data, we established a protein signature of the most aggressive tumors. From these differentially expressed proteins, some of them appeared to be potential therapeutic targets.     

Forskolin Suppresses Delayed-Rectifier K+ Currents and Enhances Spike Frequency-Dependent Adaptation of Sympathetic Neurons

In signal transduction research natural or synthetic molecules are commonly used to target a great variety of signaling proteins. For instance, forskolin, a diterpene activator of adenylate cyclase, has been widely used in cellular preparations to increase the intracellular cAMP level. However, it has been shown that forskolin directly inhibits some cloned K⁺ channels, which in excitable cells set up the resting membrane potential, the shape of action potential and regulate repetitive firing. Despite the growing evidence indicating that K⁺ channels are blocked by forskolin, there are no studies yet assessing the impact of this mechanism of action on neuron excitability and firing patterns. In sympathetic neurons, we find that forskolin and its derivative 1,9-Dideoxyforskolin, reversibly suppress the delayed rectifier K⁺ current (I_KV). Besides, forskolin reduced the spike afterhyperpolarization and enhanced the spike frequency-dependent adaptation. Given that I_KV is mostly generated by Kv2.1 channels, HEK-293 cells were transfected with cDNA encoding for the Kv2.1 α subunit, to characterize the mechanism of forskolin action. Both drugs reversible suppressed the Kv2.1-mediated K⁺ currents. Forskolin inhibited Kv2.1 currents and I_KV with an IC₅₀ of ~32 μM and ~24 µM, respectively. Besides, the drug induced an apparent current inactivation and slowed-down current deactivation. We suggest that forskolin reduces the excitability of sympathetic neurons by enhancing the spike frequency-dependent adaptation, partially through a direct block of their native Kv2.1 channels.     

Long-Term Asymmetric Hearing Affects Cochlear Implantation Outcomes Differently in Adults with Pre- and Postlingual Hearing Loss

In many countries, a single cochlear implant is offered as a treatment for a bilateral hearing loss. In cases where there is asymmetry in the amount of sound deprivation between the ears, there is a dilemma in choosing which ear should be implanted. In many clinics, the choice of ear has been guided by an assumption that the reorganisation of the auditory pathways caused by longer duration of deafness in one ear is associated with poorer implantation outcomes for that ear. This assumption, however, is mainly derived from studies of early childhood deafness. This study compared outcomes following implantation of the better or poorer ear in cases of long-term hearing asymmetries. Audiological records of 146 adults with bilateral hearing loss using a single hearing aid were reviewed. The unaided ear had 15 to 72 years of unaided severe to profound hearing loss before unilateral cochlear implantation. 98 received the implant in their long-term sound-deprived ear. A multiple regression analysis was conducted to assess the relative contribution of potential predictors to speech recognition performance after implantation. Duration of bilateral significant hearing loss and the presence of a prelingual hearing loss explained the majority of variance in speech recognition performance following cochlear implantation. For participants with postlingual hearing loss, similar outcomes were obtained by implanting either ear. With prelingual hearing loss, poorer outcomes were obtained when implanting the long-term sound-deprived ear, but the duration of the sound deprivation in the implanted ear did not reliably predict outcomes. Contrary to an apparent clinical consensus, duration of sound deprivation in one ear has limited value in predicting speech recognition outcomes of cochlear implantation in that ear. Outcomes of cochlear implantation are more closely related to the period of time for which the brain is deprived of auditory stimulation from both ears.