PI3K Pathway Inhibition with NVP-BEZ235 Hinders Glycolytic Metabolism in Glioblastoma Multiforme Cells

Glioblastoma (GBM) is the most lethal primary brain cancer that lacks effective molecular targeted therapies. The PI3K/AKT/mTOR pathway is activated in 90% of all Glioblastoma multiforme (GBM) tumors. To gain insight into the impact of the PI3K pathway on GBM metabolism, we treated U87MG GBM cells with NVP-BEZ235 (PI3K and mTOR a dual inhibitor) and identified differentially expressed genes with RNA-seq analysis. RNA-seq identified 7803 differentially regulated genes in response to NVP-BEZ235. Gene Set Enrichment Analysis (GSEA) identified two glycolysis-related gene sets that were significantly enriched (p \u3c 0.05) in control samples compared to NVP-BEZ235-treated samples. We validated the inhibition of glycolytic genes by NVP-BEZ235 and examined the impact of the FOXO1 inhibitor (AS1842856) on these genes in a set of GBM cell lines. FOXO1 inhibition alone was associated with reduced LDHA expression, but not ENO1 or PKM2. Bioinformatics analyses revealed that PI3K-impacted glycolytic genes were over-expressed and co-expressed in GBM clinical samples. The elevated expression of PI3K-impacted glycolytic genes was associated with poor prognosis in GBM based on Kaplan–Meier survival analyses. Our results suggest novel insights into hallmark metabolic reprogramming associated with the PI3K-mTOR dual inhibition

Pathogenicity of multiple Providencia species (Enterobacteriales: Morganellaceae) to the mass-reared Mexican fruit fly (Diptera: Tephritidae)

Mexican fruit fly (Anastrepha ludens (Loew)) (Diptera: Tephritidae) represents a major threat to fruit production in the Western Hemisphere. Sterile insect technique is used to suppress and eradicate wild populations. Success of this control method necessitates weekly production of hundreds of millions of flies, their sterilization by irradiation, and their aerial release. Diet needed to produce large fly numbers are conducive to the spread of bacteria. Pathogenic bacteria were isolated from 3 rearing facilities and from multiple sources: eggs, larvae, pupae and spent diet, and were found to include some isolates identified to the genus Providencia (Enterobacteriales: Morganellaceae). We identified 41 Providencia isolates and tested their pathogenicity to A. ludens. Based on 16s rRNA sequences, 3 groups were clustered into several species of Providencia with varying capacities to affect the Mexican fruit fly production. Isolates putatively identified as P. alcalifaciens/P. rustigianii were all pathogenic causing larval and pupal yield reduction of 46–64% and 37–57%, respectively. Among them, Providencia isolate 3006 was the most pathogenic reducing larval and pupae yield by 73 and 81%, respectively. Isolates identified as P. sneebia were not pathogenic. The final cluster, P. rettgeri/P. vermicola, were variable in pathogenicity with 3 isolates yielding like the control and the rest causing larval and pupal yield reduction of 26–53% and 23–51%, respectively. Isolates putatively identified as P. alcalifaciens/P. rustigianii were more virulent than P. rettgeri/P. vermicola. Accurate identification of species is needed to diagnose and monitor pathogenic versus nonpathogenic Providencia strains

Use of ITS‑1 to identify Bactrocera dorsalis and Bactrocera occipitalis (Diptera: Tephritidae): a case study using flies trapped in California from 2008 to 2018

Molecular methods are necessary to diagnose immature life stages of the agricultural pest fruit fly Bactrocera dorsalis (Hendel), and are useful to corroborate identifications based on adults because morphological variation within the species can overlap with congeners. DNA sequencing of the nuclear ribosomal internal transcribed spacer 1 (ITS‑1) has been adopted by the International Plant Protection Convention as an internationally accepted method to distinguish between the 2 pestiferous fruit fly species Bactrocera dorsalis and Bactrocera carambolae (Drew & Hancock). Reported ITS‑1 sequences also are distinct and diagnostically informative to distinguish several other Bactrocera species related to B. dorsalis. In this study, we applied DNA sequencing of ITS‑1 to a collection of 513 adult flies trapped in California, USA, in the yr 2008 to 2018. Internal transcribed spacer 1 sequences were successfully recovered from 504 (98%) of these flies. One fly had an ITS‑1 sequence that matched B. occipitalis (Bezzi) records. Re-examination of that fly using cytochrome c oxidase I, elongation factor 1‑alpha, and morphology supports it as the second record of B. occipitalis trapped in California. The other 503 flies had ITS‑1 sequences consistent with B. dorsalis. Six unique ITS‑1 sequences (or DNA types) were observed in the collection of 503 B. dorsalis. Three of the ITS‑1 sequences (types A, B, and C) were present in 84% of the 503 flies and match ITS‑1 records reported in prior publications on B. dorsalis. The other 3 sequences (types D, E, and F) observed in 4% of the 503 B. dorsalis have not been reported in publications. Ambiguous nucleotides were observed from 12% of the 503 B. dorsalis flies, precluding designation of a sequence type. Including the 3 new types from the current study, a total of 15 unique ITS‑1 sequences now are known for B. dorsalis. The study, therefore, documents additional intraspecific variation of ITS‑1 that aids in future applications for species identification. - Los métodos moleculares son necesarios para diagnosticar los estadios de vida inmaduras de la plaga agrícola mosca de la fruta Bactrocera dorsalis (Hendel) y son útiles para corroborar identificaciones basadas en adultos por la variación morfológica dentro de la especie puede superponerse con congéneres. La secuenciación del ADN del espaciador transcrito interno ribosómico nuclear 1 (ITS-1) ha sido adoptada por la Convención Internacional de Protección Fitosanitaria como un método aceptado internacionalmente para distinguir entre las dos especies de moscas de la fruta, Bactrocera dorsalis y Bactrocera carambolae (Drew & Hancock). Las secuencias de ITS-1 notificadas también son distintas y proporcionan información diagnóstica para distinguir varias otras especies de Bactrocera relacionadas con B. dorsalis. En este estudio, aplicamos la secuenciación de ADN de ITS-1 a una colección de 513 moscas adultas atrapadas en California, EE. UU. desde el 2008 hasta el 2018. Se recuperaron las secuencias espaciadoras transcritas internas1 con éxito de 504 (98%) de estas moscas. Una mosca tenía una secuencia ITS-1 que coincidía con los registros de B. occipitalis (Bezzi). El reexamen de esa mosca usando la citocromo c oxidasa I, el factor de elongación 1-alfa y la morfología lo respalda como el segundo registro de B. occipitalis atrapada en California. Las otras 503 moscas tenían secuencias de ITS-1 compatibles con B. dorsalis. Se observaron seis secuencias únicas de ITS-1 (o tipos de ADN) en la colección de 503 B. dorsalis. Tres de las secuencias de ITS-1 (tipos A, B, y C) estaban presentes en el 84% de las 503 moscas y coinciden con los registros de ITS-1 informados en publicaciones anteriores sobre B. dorsalis. Las otras 3 secuencias (tipos D, E, y F) observadas en el 4% de las 503 B. dorsalis no han sido reportadas en publicaciones. Se observaron nucleótidos ambiguos en el 12% de las 503 moscas B. dorsalis, lo que excluye la designación de un tipo de secuencia. Incluyendo los 3 nuevos tipos del estudio actual, ahora se conocen un total de 15 secuencias ITS-1 únicas para B. dorsalis. Por lo tanto, el estudio documenta una variación intraespecífica adicional de ITS-1 que ayuda en futuras aplicaciones para la identificación de especies

Morganella morganii (Enterobacteriales: Enterobacteriaceae) is a lethal pathogen of Mexican fruit fly (Diptera: Tephritidae) larvae

Tephritid pests, such as the Mexican fruit fly, Anastrepha ludens (Loew), represent a major threat to fruit production worldwide. In order to control these pests, sterile insect technique is used to suppress and eradicate wild populations. For this control method to be successful, hundreds of millions of flies must be produced weekly in mass rearing facilities. The large quantity of artificial diet and close proximity of flies at various life stages allows bacteria from family Enterobacteriaceae, Bacillaceae, Pseudomonadaceae, and others to multiply and spread more easily. In this study, bacteria with a possible pathogenic effect were isolated from Mexican fruit fly eggs and dead Mexican fruit fly larvae. Two strains of bacteria associated with dead and dying larvae were identified using the 16S rRNA sequence as a species of Morganella. Further sequencing of multiple genes and the entire genomes identified both strains as Morganella morganii. Pathogenicity tests were completed to assess this bacterium as a Mexican fruit fly pathogen. Several measures of pathogenicity including effects on larval and pupal weight, adult percent emergence, and flight ability were measured for the 2 strains of Morganella compared against a control. In all cases, the presence of the Morganella strains significantly reduced all quality control measurements compared to the control. Also, at 105 colony forming units per ml or higher levels of inoculum, the presence of Morganella resulted in 100% mortality of larvae. This study illustrates that Morganella morganii is an extremely lethal pathogen of mass reared Mexican fruit flies. - Las moscas tefritidas como la mosca Mexicana de la fruta (Anastrepha ludens Loew), representan un peligro para la producción de la fruta a nivel mundial. Para controlar estas plagas, la técnica del insecto estéril es usada para suprimir y erradicar poblaciones naturales. Para que este método de control tenga éxito, cientos de millones de moscas deben ser producidos en plantas de crianza masiva. La gran cantidad de dieta artificial y la cercanía de moscas en diversos estadios de desarrollo permiten que las bacterias perteneciendo a la familia Entobacteriacea, Baciliiacea, Pseudomonadacea, y otros se multipliquen y diseminen rápidamente. En este estudio, bacterias con posibilidad de ser patogénicas fueron aislados de huevos y larvas muertas de la mosca Mexicana de la fruta. Dos cepas de bacteria aisladas de larvas muertas o moribundas fueron identificados usando secuencias de ARN ribosoma 16S como una especie de Morganella. Adicionalmente, la realización de secuencias de múltiples genes y el genoma entero reveló que las dos cepas de bacteria fueron identificados como Morganella morganii. Pruebas de patogenicidad fueron completadas para determinar la patogenicidad de Morganella hacia la mosca Mexicana de la fruta. Diferentes variables de patogenicidad como el efecto sobre el peso de las larvas y pupas, porcentaje de emergencia de moscas adultas y habilidad de vuelo fueron evaluadas para las cepas de dos Morganella y comparados contra el control. En todos los casos, la presencia de cepas de Morganella redujeron significativamente las medidas de control de calidad comparado con el control. También, al 105 de unidades formadoras de colonias, la presencia de Morganella resultó en una mortalidad del 100% de larvas. Este estudio ilustra que Morganella morganii es un patógeno extremadamente letal para la mosca Mexicana de la fruta

NVP-BEZ235 or JAKi Treatment leads to decreased survival of examined GBM and BBC cells

Cancer cells almost universally harbor constitutively active Phosphatidylinositol-3 Kinase (PI3K) Pathway ac-tivity via mutation of key signaling components and/or epigenetic mechanisms. Scores of PI3K Pathway in-hibitors are currently under investigation as putative chemotherapeutics. However, feedback and stem cell mechanisms induced by PI3K Pathway inhibition can lead to reduced treatment efficacy. To address therapeutic barriers, we examined whether JAKi would reduce stem gene expression in a setting of PI3K Pathway inhibition in order to improve treatment efficacy. We targeted the PI3K Pathway with NVP-BEZ235 (dual PI3K and mTOR inhibitor) in combination with the Janus Kinase inhibitor JAKi in glioblastoma (GBM) and basal-like breast cancer (BBC) cell lines. We examined growth, gene expression, and apoptosis in cells treated with NVP-BEZ235 and/or JAKi. Growth and recovery assays showed no significant impact of dual treatment with NVP-BEZ235/ JAKi compared to NVP-BEZ235 treatment alone. Gene expression and flow cytometry revealed that single and dual treatments induced apoptosis. Stem gene expression was retained in dual NVP-BEZ235/JAKi treatment samples. Future in vivo studies may give further insight into the impact of combined NVP-BEZ235/JAKi treat-ment in GBM and BBC

A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Dwm) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Dwm required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Dwm were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted q0) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Dwm, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Dwm with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Dwm below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

Background Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. Results We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. Conclusions Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications)

The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines

Background: The PI3K pathway controls diverse cellular processes including growth, survival, metabolism, and apoptosis. Nuclear FOXO factors were observed in cancers that harbor constitutively active PI3K pathway output and stem signatures. FOXO1 and FOXO3 were previously published to induce stem genes such as OCT4 in embryonic stem cells. Here, we investigated FOXO-driven stem gene expression in U87MG glioblastoma cells. Methods: PI3K-activated cancer cell lines were investigated for changes in gene expression, signal transduction, and clonogenicity under conditions with FOXO3 disruption or exogenous expression. The impact of PI3K pathway inhibition on stem gene expression was examined in a set of glioblastoma cell lines. Results: We found that CRISPR-Cas9-mediated FOXO3 disruption in U87MG cells caused decreased OCT4 and SOX2 gene expression, STAT3 phosphorylation on tyrosine 705 and clonogenicity. FOXO3 over expression led to increased OCT4 in numerous glioblastoma cancer cell lines. Strikingly, treatment of glioblastoma cells with NVP-BEZ235 (a dual inhibitor of PI3K and mTOR), which activates FOXO factors, led to robust increases OCT4 gene expression. Direct FOXO factor recruitment to the OCT4 promoter was detected by chromatin immunoprecipitation analyses using U87MG extracts. Discussion: We show for the first time that FOXO transcription factors promote stem gene expression glioblastoma cells. Treatment with PI3K inhibitor NVP-BEZ235 led to dramatic increases in stem genes in a set of glioblastoma cell lines. Conclusion: Given that, PI3K inhibitors are actively investigated as targeted cancer therapies, the FOXO-mediated induction of stem genes observed in this study highlights a potential hazard to PI3K inhibition. Understanding the molecular underpinnings of stem signatures in cancer will allow refinements to therapeutic strategies. Targeting FOXO factors to reduce stem cell characteristics in concert with PI3K inhibition may prove therapeutically efficacious

Population Genomic Analysis of a Bacterial Plant Pathogen: Novel Insight into the Origin of Pierce's Disease of Grapevine in the U.S.

Invasive diseases present an increasing problem worldwide; however, genomic techniques are now available to investigate the timing and geographical origin of such introductions. We employed genomic techniques to demonstrate that the bacterial pathogen causing Pierce's disease of grapevine (PD) is not native to the US as previously assumed, but descended from a single genotype introduced from Central America. PD has posed a serious threat to the US wine industry ever since its first outbreak in Anaheim, California in the 1880s and continues to inhibit grape cultivation in a large area of the country. It is caused by infection of xylem vessels by the bacterium Xylella fastidiosa subsp. fastidiosa, a genetically distinct subspecies at least 15,000 years old. We present five independent kinds of evidence that strongly support our invasion hypothesis: 1) a genome-wide lack of genetic variability in X. fastidiosa subsp. fastidiosa found in the US, consistent with a recent common ancestor; 2) evidence for historical allopatry of the North American subspecies X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa; 3) evidence that X. fastidiosa subsp. fastidiosa evolved in a more tropical climate than X. fastidiosa subsp. multiplex; 4) much greater genetic variability in the proposed source population in Central America, variation within which the US genotypes are phylogenetically nested; and 5) the circumstantial evidence of importation of known hosts (coffee plants) from Central America directly into southern California just prior to the first known outbreak of the disease. The lack of genetic variation in X. fastidiosa subsp. fastidiosa in the US suggests that preventing additional introductions is important since new genetic variation may undermine PD control measures, or may lead to infection of other crop plants through the creation of novel genotypes via inter-subspecific recombination. In general, geographically mixing of previously isolated subspecies should be avoided

The Complex Biogeography of the Plant Pathogen Xylella fastidiosa: Genetic Evidence of Introductions and Subspecific Introgression in Central America

Datos y artículo incluido por Lisela Moreira Carmona, responsable de depósitos de publicaciones del área de Patógenos y Plagas del CIBCMThe bacterium Xylella fastidiosa is a plant pathogen with a history of economically damaging introductions of subspecies to regions where its other subspecies are native. Genetic evidence is presented demonstrating the introduction of two new taxa into Central America and their introgression into the native subspecies, X. fastidiosa subsp. fastidiosa. The data are from 10 genetic outliers detected by multilocus sequence typing (MLST) of isolates from Costa Rica. Six (five from oleander, one from coffee) defined a new sequence type (ST53) that carried alleles at six of the eight loci sequenced (five of the seven MLST loci) diagnostic of the South American subspecies Xylella fastidiosa subsp. pauca which causes two economically damaging plant diseases, citrus variegated chlorosis and coffee leaf scorch. The two remaining loci of ST53 carried alleles from what appears to be a new South American form of X. fastidiosa. Four isolates, classified as X. fastidiosa subsp. fastidiosa, showed a low level of introgression of non-native DNA. One grapevine isolate showed introgression of an allele from X. fastidiosa subsp. pauca while the other three (from citrus and coffee) showed introgression of an allele with similar ancestry to the alleles of unknown origin in ST53. The presence of X. fastidiosa subsp. pauca in Central America is troubling given its disease potential, and establishes another route for the introduction of this economically damaging subspecies into the US or elsewhere, a threat potentially compounded by the presence of a previously unknown form of X. fastidiosa.Universidad de Costa Rica/[801-B2-516]/UCR/Costa RicaUniversidad de Costa Rica/[801-A1-801]/UCR/Costa RicaInternational Foundation for Science/[grant C/5152-1]/IFS/SueciaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM