Protocol for assessing bacterial wilt resistance in greenhouse and field conditions. International cooperators’ guide

This protocol is an updated version of “Assessing potato clone field resistance to bacterial wilt” issued in The International Cooperators’ Guide (CIP 2007). The first edition of the protocol presented a standard procedure for field assessment of resistance to bacterial wilt for documenting levels of resistance of advanced potato germplasm. This second edition has included a standardized procedure for greenhouse screening of potato seedlings for bacterial wilt resistance useful for perform genetic studies, parental selection or identification of new sources of resistance in accessions of wild species propagated or maintained as true see

Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities

New synchronization method for <i>Plasmodium falciparum</i>

&lt;b&gt;Background&lt;/b&gt;: Plasmodium falciparum is usually asynchronous during in vitro culture. Although various synchronization methods are available, they are not able to narrow the range of ages of parasites. A newly developed method is described that allows synchronization of parasites to produce cultures with an age range as low as 30 minutes. &lt;b&gt;Methods&lt;/b&gt;: Trophozoites and schizonts are enriched using Plasmion. The enriched late stage parasites are immobilized as a monolayer onto plastic Petri dishes using concanavalin A. Uninfected erythrocytes are placed onto the monolayer for a limited time period, during which time schizonts on the monolayer rupture and the released merozoites invade the fresh erythrocytes. The overlay is then taken off into a culture flask, resulting in a highly synchronized population of parasites. &lt;b&gt;Results&lt;/b&gt;: Plasmion treatment results in a 10- to 13-fold enrichment of late stage parasites. The monolayer method results in highly synchronized cultures of parasites where invasion has occurred within a very limited time window, which can be as low as 30 minutes. The method is simple, requiring no specialized equipment and relatively cheap reagents. &lt;b&gt;Conclusions&lt;/b&gt;: The new method for parasite synchronization results in highly synchronized populations of parasites, which will be useful for studies of the parasite asexual cell cycle

Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment

Tumour-associated microglia/macrophages (TAM) are the most numerous non-neoplastic populations in the tumour microenvironment in glioblastoma multiforme (GBM), the most common malignant brain tumour in adulthood. The mTOR pathway, an important regulator of cell survival/proliferation, is upregulated in GBM, but little is known about the potential role of this pathway in TAM. Here, we show that GBM-initiating cells induce mTOR signalling in the microglia but not bone marrow-derived macrophages in both in vitro and in vivo GBM mouse models. mTOR-dependent regulation of STAT3 and NF-jB activity promotes an immunosuppressive microglial phenotype. This hinders effector T-cell infiltration, proliferation and immune reactivity, thereby contributing to tumour immune evasion and promoting tumour growth in mouse models. The translational value of our results is demonstrated in whole transcriptome datasets of human GBM and in a novel in vitro model, whereby expandedpotential stem cells (EPSC)-derived microglia-like cells are conditioned by syngeneic patient-derived GBM-initiating cells. These results raise the possibility that microglia could be the primary target of mTOR inhibition, rather than the intrinsic tumour cells in GB

Calcitonin gene-related peptide promotes cellular changes in trigeminal neurons and glia implicated in peripheral and central sensitization

<p>Abstract</p> <p>Background</p> <p>Calcitonin gene-related peptide (CGRP), a neuropeptide released from trigeminal nerves, is implicated in the underlying pathology of temporomandibular joint disorder (TMD). Elevated levels of CGRP in the joint capsule correlate with inflammation and pain. CGRP mediates neurogenic inflammation in peripheral tissues by increasing blood flow, recruiting immune cells, and activating sensory neurons. The goal of this study was to investigate the capability of CGRP to promote peripheral and central sensitization in a model of TMD.</p> <p>Results</p> <p>Temporal changes in protein expression in trigeminal ganglia and spinal trigeminal nucleus were determined by immunohistochemistry following injection of CGRP in the temporomandibular joint (TMJ) capsule of male Sprague-Dawley rats. CGRP stimulated expression of the active forms of the MAP kinases p38 and ERK, and PKA in trigeminal ganglia at 2 and 24 hours. CGRP also caused a sustained increase in the expression of c-Fos neurons in the spinal trigeminal nucleus. In contrast, levels of P2X₃in spinal neurons were only significantly elevated at 2 hours in response to CGRP. In addition, CGRP stimulated expression of GFAP in astrocytes and OX-42 in microglia at 2 and 24 hours post injection.</p> <p>Conclusions</p> <p>Our results demonstrate that an elevated level of CGRP in the joint, which is associated with TMD, stimulate neuronal and glial expression of proteins implicated in the development of peripheral and central sensitization. Based on our findings, we propose that inhibition of CGRP-mediated activation of trigeminal neurons and glial cells with selective non-peptide CGRP receptor antagonists would be beneficial in the treatment of TMD.</p

Refractive results after topography-guided anterior lamellar keratoplasty with excimer laser in keratoconus grade III

Objetivo: determinar los resultados refractivos y funcionales en operados de queratoplastia lamelar anterior superficial asistida con láser excimer topográfico en queratocono grado III. Materiales y métodos: estudio experimental, abierto, no controlado, longitudinal &nbsp;y prospectivo, en 35 ojos de 31 pacientes con edad promedio de 35,3 años, seguimiento entre 12 y 51 meses, operados en la Clínica Internacional de Retinosis Pigmentaria “Camilo Cienfuegos”, entre enero de 2006 y diciembre del 2010. Resultados: la agudeza visual sin corrección mejoró de 0,04 a 0,39 y la agudeza visual con corrección de 0,28 a 0,78; el equivalente esférico se redujo de -13,02 a -1,69; la esfera y el cilindro disminuyeron de -10,13D ± 4,73 a -0,44D ± 3,22 y de -5,67D ± 2,32 a -2,52D ± 1,38, respectivamente. La queratometría disminuyó de 56,8D y 51,53D a 45,02 y 42,02 en el posoperatorio. La densidad celular preoperatoria fue de 2313,81 cel/mm2 ± 322,10; varió a 2170 cel/mm2 ± 294,92 en el posoperatorio. El lecho residual estromal fue de 153,22 μ. Discusión: en correlación con la literatura, el queratocono se presenta en la adolescencia y la juventud y conduce al trasplante de córnea con más frecuencia en estas etapas de la vida. La queratometría promedio posoperatoria fue inferior a la reportada por otros investigadores que realizaron estudios con semejante técnica quirúrgica. Conclusiones: la queratoplastia lamelar anterior superficial con láser excimer, puede ser considerada como indicación primaria de tratamiento en el queratocono grado III (MÉD.UIS. 2012;25(3):195-202)Objective: determine refractive and functional results in patients with keratoconus grade III underwent to topography-guided anterior lamellar keratoplasty assisted with excimer laser. Materials and Methods: uncontrolled experimental study, longitudinal and prospective, in 35 eyes of 31 patients with an average age of 35.3 years old, followed between 12 and 51 moths; operated at “Camilo Cienfuegos” International Center of Retinitis Pigmentosa, between January 2006 and December 2010. Results: the uncorrected visual acuity improved from 0.04 to 0.39 and the best corrected visual acuity improved from 0.28 to 0.78; the spherical equivalent was reduced from -13.02 to -1.69D. The sphere and cylinder decreased from -10.13D ± 4.73 to -0.44D ± 3.22 and from -5.67D ± 2.32 to -2.52 ±1.38, respectively. The average of the keratometry decreased from 56.8D and 51.53D to 45.02 and 42.02 in the post-surgery. The cellular density pre-surgery changed from 2313.81 cel/mm2 ± 322.10 to 2170 cel/mm2 ± 294.92 in postoperative. The residual stromal bed was 153.22 μ. Discussion: in correlation with the literature, the keratoconus appears in the adolescence and youth, leading to the corneal transplantation more often in these life stages. The average postsurgical keratometry was inferior to the reported by others investigators who practiced procedures with similar surgical technique. Conclusions: superficial anterior lamellar keratoplasty, assisted with excimer laser, can be considered as primary indication of treatment in keratoconus grade III (MÉD.UIS. 2012;25(3):195-202). &nbsp

Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM