Molecular and cytogenetic characterization of wild Musa species

All relevant data are within the paper and its Supporting Information files.The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world's largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny

Molecular and cytological characterization of the global Musa germplasm collection provides insights into the treasure of banana diversity

Bananas (Musa spp.) are one of the main fruit crops grown worldwide. With the annual production reaching 144 million tons, their production represents an important contribution to the economies of many countries in Asia, Africa, Latin-America and Pacific Islands. Most importantly, bananas are a staple food for millions of people living in the tropics. Unfortunately, sustainable banana production is endangered by various diseases and pests, and the breeding for resistant cultivars relies on a far too small base of genetic variation. Greater diversity needs to be incorporated in breeding, especially of wild species. Such work requires a large and thoroughly characterized germplasm collection, which also is a safe depository of genetic diversity. The largest ex situ Musa germplasm collection is kept at the International Transit Centre (ITC) in Leuven (Belgium) and currently comprises over 1500 accessions. This report summarizes the results of systematic cytological and molecular characterization of the Musa ITC collection. By December 2015, 630 accessions have been genotyped. The SSR markers confirmed the previous morphological based classification for 84% of ITC accessions analyzed. The remaining 16% of the genotyped entries may need field verification by taxonomist to decide if the unexpected classification by SSR genotyping was correct. The ploidy level estimation complements the molecular data. The genotyping continues for the entire ITC collection, including newly introduced accessions, to assure that the genotype of each accession is known in the largest global Musa gene bank

Characterisation of wild musa accessions recently introduced to the International Gene Bank

Bananas and plantains (Musa spp.) are one of the most important world trade commodities and are a staple food for millions of people in countries of the humid tropics. The production of bananas is, however, threatened by the rapid spread of various diseases and adverse environmental conditions. The Musa genetic diversity, which is of paramount importance for breeding of resistant cultivars, needs to be preserved and better characterised. The world's largest banana and plantain collection is managed by the Bioversity International Transit Centre (ITC) in Belgium and contains more than 1500 accessions maintained in vitro. The collection is being continuously expanded by new accessions representing various edible cultivars, improved materials and wild species from different parts of the world. Recently new germplasm was collected in Indonesia and successively introduced into the international Musa gene bank. The aim of this work was to characterise the genotype of these accessions in order to shed light on their genome structure and to confirm their taxonomic classification. A total of 21 wild Musa accessions were analysed and their nuclear genome size and the genomic distribution of ribosomal RNA genes were determined, showing a high degree of variability in both characters. Genotyping with a set of 19 microsatellite markers identified Musa species that are closely related to the studied accessions and provided data to aid in their classification. Sequence analysis of their internal transcribed spacers ITS1 and ITS2 suggested that some of the accessions are of interspecific hybrid origin and/or represent backcross progenies of interspecific hybrids

Descending serotonergic facilitation and the antinociceptive effects of pregabalin in a rat model of osteoarthritic pain

Background: Descending facilitation, from the brainstem, promotes spinal neuronal hyperexcitability and behavioural hypersensitivity in many chronic pain states. We have previously demonstrated enhanced descending facilitation onto dorsal horn neurones in a neuropathic pain model, and shown this to enable the analgesic effectiveness of gabapentin. Here we have tested if this hypothesis applies to other pain states by using a combination of approaches in a rat model of osteoarthritis (OA) to ascertain if 1) a role for descending 5HT mediated facilitation exists, and 2) if pregabalin (a newer analogue of gabapentin) is an effective antinociceptive agent in this model. Further, quantitative-PCR experiments were undertaken to analyse the alpha(2)delta-1 and 5-HT3A subunit mRNA levels in L3-6 DRG in order to assess whether changes in these molecular substrates have a bearing on the pharmacological effects of ondansetron and pregabalin in OA.Results: Osteoarthritis was induced via intra-articular injection of monosodium iodoacetate (MIA) into the knee joint. Control animals were injected with 0.9% saline. Two weeks later in vivo electrophysiology was performed, comparing the effects of spinal ondansetron (10-100 mu g/50 mu l) or systemic pregabalin (0.3-10 mg/kg) on evoked responses of dorsal horn neurones to electrical, mechanical and thermal stimuli in MIA or control rats. In MIA rats, ondansetron significantly inhibited the evoked responses to both innocuous and noxious natural evoked neuronal responses, whereas only inhibition of noxious evoked responses was seen in controls. Pregabalin significantly inhibited neuronal responses in the MIA rats only; this effect was blocked by a pre-administration of spinal ondansetron. Analysis of alpha(2)delta-1 and 5-HT3A subunit mRNA levels in L3-6 DRG revealed a significant increase in alpha(2)delta-1 levels in ipsilateral L3&4 DRG in MIA rats. 5-HT3A subunit mRNA levels were unchanged.Conclusion: These data suggest descending serotonergic facilitation plays a role in mediating the brush and innocuous mechanical punctate evoked neuronal responses in MIA rats, suggesting an adaptive change in the excitatory serotonergic drive modulating low threshold evoked neuronal responses in MIA-induced OA pain. This alteration in excitatory serotonergic drive, alongside an increase in alpha(2)delta-1 mRNA levels, may underlie pregabalin's state dependent effects in this model of chronic pain

Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations

The effect of mesenchymal stem cell transplantation on the recovery of bladder and hindlimb function after spinal cord contusion in rats

<p>Abstract</p> <p>Background</p> <p>Mesenchymal stem cells are widely used for transplantation into the injured spinal cord in vivo model and for safety, many human clinical trials are continuing to promote improvements of motor and sensory functions after spinal cord injury. Yet the exact mechanism for these improvements remains undefined. Neurogenic bladder following spinal cord injury is the main problem decreasing the quality of life for patients with spinal cord injury, but there are no clear data using stem cell transplantation for the improvement of neurogenic bladder for in vivo studies and the clinical setting.</p> <p>The purpose of this study was to delineate the effect of human mesenchymal stem cell (hMSCs) transplantation on the restoration of neurogenic bladder and impaired hindlimb function after spinal cord contusion of rats and the relationship between neurotrophic factors such as brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and bladder and hindlimb functions.</p> <p>Results</p> <p>Modified moderate contusion injury were performed on the thoracic spinal cord of Sprague-Dawley rats using MASCIS impactor and hMSCs, human fibroblasts or phosphate-buffered saline were transplanted into injured spinal cord 9 days after injury for hMSC and two control groups respectively. Ladder test showed more rapid restoration of hindlimb function in hMSC group than in control group, but Basso, Beattie, and Bresnahan score and coupling score were not different significantly among hMSC and two control groups. Neurogenic bladder was not improved in either group. ED1 positive macrophages were significantly reduced in hMSC group than in two control groups, but ELISA and RT-PCR studies revealed BDNF and NT-3 levels in spinal cord and bladder were not different among hMSC and two control groups regardless the experimental duration.</p> <p>Conclusion</p> <p>hMSC transplantation was effective in reducing inflammatory reaction after spinal cord contusion of rats but not sufficient to recover locomotor and bladder dysfunction. BDNF and NT-3 levels in the spinal cord and bladder were not increased 28 and 56 days after hMSC transplantation.</p

Contrasted Patterns of Selection on MHC-Linked Microsatellites in Natural Populations of the Malagasy Plague Reservoir

Plague (Yersinia pestis infection) is a highly virulent rodent disease that persists in many natural ecosystems. The black rat (Rattus rattus) is the main host involved in the plague focus of the central highlands of Madagascar. Black rat populations from this area are highly resistant to plague, whereas those from areas in which the disease is absent (low altitude zones of Madagascar) are susceptible. Various lines of evidence suggest a role for the Major Histocompatibility Complex (MHC) in plague resistance. We therefore used the MHC region as a candidate for detecting signatures of plague-mediated selection in Malagasy black rats, by comparing population genetic structures for five MHC-linked microsatellites and neutral markers in two sampling designs. We first compared four pairs of populations, each pair including one population from the plague focus and one from the disease-free zone. Plague-mediated selection was expected to result in greater genetic differentiation between the two zones than expected under neutrality and this was observed for one MHC-class I-linked locus (D20Img2). For this marker as well as for four other MHC-linked loci, a geographic pattern of genetic structure was found at local scale within the plague focus. This pattern would be expected if plague selection pressures were spatially variable. Finally, another MHC-class I-linked locus (D20Rat21) showed evidences of balancing selection, but it seems more likely that this selection would be related to unknown pathogens more widely distributed in Madagascar than plague

A simple and robust real-time qPCR method for the detection of PIK3CA mutations

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen breast cancer biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using both Sanger sequencing and deep next generation sequencing methods. The detection sensitivity for PIK3CA E545K mutation was approximately 10%. We propose these methods as simple, fast and inexpensive diagnostic tools to determine PIK3CA mutation status

Mitochondrial ATP synthase: architecture, function and pathology

Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to complex V deficiency and current therapeutic strategies are highlighted. Despite the huge progress in this research field over the past decades, questions remain to be answered regarding the structure of subunits, the function of the rotary nanomotor at a molecular level, and the human complex V assembly process. The elucidation of more nuclear genetic defects will guide physio(patho)logical studies, paving the way for future therapeutic interventions