Highest Transformation Frequency Research Articles

Language and cultural creolisation in nigerian script texts

This article deals with the peculiarities of script texts being a part of cinematography – one of modern art form. The scientific problem is determined by the trend towards globalisation of all contemporary art types, on the one hand, and the need to preserve cultural heritage, on the other hand. The aim of the work is to identify the characteristic features of Nigerian English script texts – phenomena created due to creolisation process defined as the process of British English adapting procedure to the realities of local languages and cultures. The Nigerian script text is a creolised type of text with characteristic of unique properties – the presence of detailed instructions in the form of author’s remarks which provide expanded information for subsequent stepwise actions for the director, actors, editors, makeup and costume designers; a double process of creolisation due to the necessary adaptation to numerous local languages and cultures. It is proved that Nigerian script texts, influenced by the process of creolisation, are largely subject to grammatical transformations. The most productive grammatical changes include: the consistent use of nouns and pronouns within the same semantic group; the simplification of the specific verb forms manifested in the use of simple forms and omission of auxiliary verbs in interrogative and negative sentences. High-frequency grammatical transformation processes in Nigerian script texts are explained by the influence of tribal languages and be needs of the Nigerians to preserve the heritage of local cultures.

Agrobacterial Transformation Enhancement by Improved Competent Cell Preparation and Optimized Electroporation.

The introduction of plasmids into Agrobacterium cells is one of the key steps in the Agrobacterium-mediated transformation of plants for gene editing applications. Depending on chromosomal background, some Agrobacterium strains exhibit a very low transformation efficiency, which results in a low number of colonies for subsequent screening and thus limits the potential for automated high-throughput transformation processes, especially with low copy or large plasmids. This study demonstrates improvements of transformation frequency by modifying the competent cell preparation process and optimizing electroporation parameters for two Agrobacterium strains. The competent cell preparation process was modified by prolonging bacterial growth in the log phase and optimizing the endpoint cell density for cell harvest which resulted in a significant cell yield increase and transformation frequency improvement. Optimization of electroporation by fine-tuning the parameters not only resulted in a 30-fold transformation frequency increase but also revealed a strain-dependent requirement for field strength and electric pulse length. To further improve transformation of a recalcitrant strain, different concentrations of dimethyl sulfoxide (DMSO) in recovery medium were examined. The study revealed an important role of DMSO in transformed cell recovery, with 5% DMSO resulting in the highest transformation frequency. The significant improvements in Agrobacterium transformation frequency addressed a critical bottleneck towards establishing a high throughput process.

Fine-tuning CRISPR/Cas9 gene editing in common bean (Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models.

The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success. We initially tested three different transformation methods to induce hairy roots and selected a preferred method suitable for a variety of different common bean genotypes. This method involved inoculating a severed radicle with Rhizobium rhizogenes K599 and was fast, had a high transformation frequency of 42-48%, and resulted in numerous hairy roots. This method was further used for the transformation of explants using R. rhizogenes harboring different CRISPR/Cas9 constructs and evaluated the on-target activity of sgRNAs targeting raffinose family oligosaccharides biosynthetic genes and the impact of different promoters driving Cas9 on the gene editing efficiency. Additionally, we evaluated the reliability of the in silico tools, CRISPOR, CRISPR RGEN, and inDelphi to predict the sgRNA efficiencies and resulting mutations. Our results showed that the hairy root transformation system allows for rapid evaluation of multiple sgRNAs and promoters. We also identified several highly efficient sgRNAs that induced frameshift mutations at rates of up to 70% when a parsley ubiquitin promoter was driving Cas9 expression, providing valuable information for the selection of the most effective sgRNAs and promoters for future transformation experiments. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta results, the Lindel model proved to be the most reliable for P. vulgaris, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. Furthermore, the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants because they provide the possibility of predicting repair outcomes.

Establishment of Agrobacterium rhizogenes-mediated RNAi of Phryma leptostachya accelerates the functional identification of key genes of the furofuran lignan biosynthetic pathway

Phryma leptostachya is a well-known medicinal plant that produces furofuran lignans with various biological activities. However, the biosynthetic pathways of these natural products in P. leptostachya remain unclear. Hairy root cultures provide a novel method for elucidating the biosynthetic pathways of secondary metabolites. Here, we established a hairy root system for P. leptostachya by optimizing five parameters: Agrobacterium strains, explant types, infection time, A. rhizogenes cell density, and acetosyringone (AS) concentration. The highest transformation frequency (74.51 %) was achieved when the stem explants were infected with ATCC15834 (OD600 = 0.8) strain of A. rhizogenes for 30 min on 1/2 MS medium supplemented with 200 µM AS. Based on the optimal protocol, RNAi hairy roots were induced by silencing the cytochrome P450 gene, PlCYP81Q38, which converts (+)-pinoresinol to (+)-sesamin by means of (+)-piperitol in P. leptostachya, to determine its in vivo role in the lignan biosynthetic pathway and verify the availability of this system. PlCYP81Q38 silencing resulted in significant down-regulation of the PlCYP81Q38 gene at the transcriptional level, consistent with a remarkable decrease in the content of leptostachyol acetate and 6-desmethoxy-leptostachyol acetate, demonstrating its significance in the production of lignans. In conclusion, the approaches and results presented here provide a solid foundation for elucidating the role of key genes involved in the lignan biosynthesis pathway in P. leptostachya and for the industrial production of lignan through plant biotechnology.

Stormwater runoff treatment through electrocoagulation: antibiotic resistant bacteria removal and its transmission risks

ABSTRACT Recently, increasing attention has been paid to antibiotic resistant bacteria (ARB) in stormwater runoff. However, there were little data on ARB removal through electrocoagulation (EC) treatment. In this study, batch experiments were conducted to investigate key designs for ARB removal, role of SS, effects of water matrix, and potential risks after EC treatment under the pre-determined conditions. EC treatment with 5 mA/cm2 of current density and 4 cm of inter-electrode distance was optimal with the highest ARB removal (3.04 log reduction for 30 min). The presence of SS significantly improved ARB removal during EC treatment, where ARB removal increased with the increase of SS levels when SS less than 300 mg/L. Large ARB removal was found under particles with size lower than 150 μm with low contribution (less than 10%) of the settlement without EC treatment, implying that the enhancement of ARB adsorption onto small particles could be one of the reasonable approaches for ARB removal through EC treatment. ARB removal increased firstly and then decreased with the increase of pH, while had proportional relationship with conductivity. After the optimal condition, there were weak conjugation transfer but high transformation frequency (5.5 × 10−2 for bla TEM) for target antibiotic resistance genes (ARGs), indicating that there could be still a risk of antibiotic resistance transformation after EC treatment. These suggested that the combination of EC and other technologies (like electrochemical disinfection) should be potential ways to control antibiotic resistance transmission through stormwater runoff.

Recombinant tissue plasminogen activator (rTPA) management for first onset acute ischemic stroke with covid -19 and non-covid -19 patients

ObjectivesCerebrovascular stroke (CVS) is one of the well-known complications of coronavirus-2019 (Covid-19), but less is known about the outcome and safety of thrombolytic therapy in these patients. In this study we compare the efficacy and safety of Tissue plasminogen activator (rTPA) in acute ischemic stroke (AIS) patients with or without Covid-19 infection. Materials and methodsA comparative prospective study in which all patients who presented with AIS and eligible for rTPA were recruited from the emergency department and classified into 2 groups (AIS with Covid-19 infection and AIS without Covid-19 as controls). Demographic data, symptoms of Covid-19, clinical examination, neuroimaging, and laboratory investigations were obtained in each patient. National Institute of Health Stroke Scale (NIHSS) and the Modified Rankin Scale (mRS) were assessed before, immediately after rTPA, and 3 months later. ResultsThere were 22 patients in the COVID-19 group and 25 control patients. Those with COVID-19 were more likely to have a history of smoking and Diabetes Mellitus than controls. On admission, motor symptoms were more severe in patients with COVID-19. COVID-19 patients were more likely to have symptomatic intra-cerebral hemorrhage and radiological hemorrhagic transformation than controls. Onset to door time (ODT) and onset to successful reperfusion time were significantly longer in Covid-19 patients than controls. Clinical improvement and frequency of re-occlusion and recurrent ischemic stroke at 3 months follow-up did not differ between groups, although there was higher number of deaths (27.3%) in the Covid-19 group than controls (16%). ConclusionsUsing rTPA is safe and effective in patients with AIS with or without COVID-19 infection despite the high frequency of hemorrhagic transformation and high number of deaths.

A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger.

For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

Long-term adaptation of ParA, RelE/ParE partition system, replication protein and phage proteins encoding low-cost plasmids ofEscherichiaspecies isolated from diarrheic children of North East India

The prevalent distribution of plasmid-mediated β-lactam resistance is the most pressing global problem in enteric diseases. The current work aims to characterize plasmid-carrying β-lactam resistant Enterobacteriaceae isolates from North East India for horizontal gene transfer (HGT) and plasmid adaptation study. In vitro transconjugation and transformation showed overall high conjugation frequency (4.11×10-1-9.2×10-1) and moderate transformation efficiency/µg DNA (1.02×102 -1×103), and the highest conjugation frequency (9.2×10-1) and transformation efficiency (1×103) for Escherichia species S-10. Intra/intergenus plasmid transformation efficiency was highest for the transformation of Klebsiella pneumoniae S-2 to Shigellaflexneri S-42 (1.3×103) and lowest for Escherichia species S-10 to Escherichia fergusonii S-30 (2×102). In the plasmid stability test, S-10 was detected with the highest plasmid carrying frequency (83.44%) and insignificant segregational loss rate (0.0004) until the 60th day with low plasmid cost on the host. The above findings were also validated by whole-plasmid sequencing of Escherichia species S-10. The genome was identified with two plasmids constituting multiple phage proteins, relaxosomal protein NikA, replication protein RepA, and the plasmid maintenance proteins (ParA, RelE/ParE), thus assisting stable plasmid maintenance. The results thus indicate that the high conjugation ability and low plasmid fitness cost might lead to horizontal gene transfer of the plasmid to the environment due to their prolonged adaptation in nonselective conditions, intensifying the infection's severity.

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

Establishment of Agrobacterium-mediated genetic transformation and application of CRISPR/Cas9 genome-editing system to Brassica rapa var. rapa

BackgroundGenome editing is essential for crop molecular breeding. However, gene editing in turnip (Brassica rapa var. rapa) have not been reported owing to the very low transformation efficiency.ResultsIn this study, we established a transformation procedure involving chemical-inducible activation of the BrrWUSa gene, which resulted in high transformation frequencies of turnip. Estradiol-inducible BrrWUSa transgenic plants were fertile and showed no obvious developmental defects. Furthermore, we used CRISPR/Cas9 gene-editing technology to edit BrrTCP4b and generated 20 BrrTCP4b-edited seedlings with an increase in leaf trichome number.ConclusionThe results demonstrate that BrrWUSa improves the regeneration efficiency in turnip. The transformation procedure represents a promising strategy to improve genetic transformation and for functional characterization of genes in turnip.

Use of the phosphomannose isomerase (PMI) gene for agrobacteriummediated transformation of <i>Prunus domestica</i> L. leaf explants without the use of selective antibiotic resistance genes

We developed an efficient system for agro-bacterial transformation of plum (Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. Th e variety `Startovaya` was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40 %) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoid ing formation of escapes. Integration of the manA and gfp transgenes was confi rmed by PCR and Southern blotting. On the whole, no direct correlation between the fluorescence level and the copy numbers of the transgenes was found in the present study, though the most intensive fluorescence was observed in line #9 with a single-copy insert. The difference of GFP expression level may have been caused by the integration site or by other factors such as DNA methylation and varying copy number. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of variety traits of plum plants.

Identification of pretreatment genomic biomarkers and mechanisms of acquired resistance to first-line osimertinib in advanced EGFR-mutant lung cancers.

9100 Background: Despite its widespread use, few series explore genomic biomarkers that impact progression-free survival (PFS) for first line osimertinib use and how mechanisms of acquired resistance impact post-osimertinib progression survival. Methods: All pts treated with first line osimertinib at Memorial Sloan Kettering Cancer Center were identified; available pre-osimertinib and post-progression tumor samples underwent next-generation sequencing (NGS) using MSK-IMPACT. Real-world (rw)PFS was estimated with Kaplan-Meier methods from start of osimertinib to progression, defined using interpretation of imaging reports. Post-baseline factors were evaluated using time-dependent covariate Cox model. Results: We identified 331 pts, of which 67 had paired tumor samples and 28 had post-progression tumor samples that underwent MSK-IMPACT. Most pts with biopsies were women (68%), never smokers (83%) and did not have baseline brain metastases (56%). With median follow-up of 24 months (mo), median rwPFS was 14 mo (95% confidence interval (CI) 13 -17 mo, n = 331). EGFR driver alterations (n = 40 atypical, n = 108 L858R, n = 182 exon 19 deletion) were associated with distinct rwPFS (median 8 (95% CI 6-12), 14 (12-18) and 16 mo (13-21), respectively, p < 0.001 logrank test). Pts with concurrent pre-treatment TP53 (14 mo,12-17) or TP53/RB1 (12 mo, 9-15) alterations had shorter median rwPFS compared to pts without these alterations (20 mo,16-24, p < 0.001, logrank test). Fifty patients (53%) had an identified mechanism of resistance. Off target mechanisms (n = 9 MET amplification (amp), n = 3 HER2 amp, n = 3 PIK3CA mutations, n = 3 acquired fusions, n = 2 RB1 loss and n = 1 CCND1 amp, MYC amp, CDK4 amp, KRAS G12A, respectively) and histological transformation (n = 7 small cell, n = 5 squamous and n = 2 large cell neuroendocrine) were detected. On target acquired mechanisms were EGFR amplification (n = 7), and G724S (n = 2) and C797S (n = 3) mutations. Pts with an identified mechanism of resistance did not have improved post-progression survival (12 mo HR 1.6, p = 0.09), but receiving next line of therapy based on post-progression tumor biopsy results (including platinum-etoposide for transformation) did improve post-progression survival (12 mo HR 0.4, p = 0.01). Conclusions: Pts with atypical EGFR drivers or concurrent TP53+/- RB1 alterations have significantly shorter rwPFS on first line osimertinib, highlighting need for additional interventions for these patients. Given the high frequency of transformation and improvement in post-progression survival by tailoring next line of therapy to the identified mechanism, pts with EGFR-mutant lung cancer on first line osimertinib may benefit from tissue biopsies at progression. For pts without an identified resistance mechanism by NGS, additional methods of interrogating tumors at progression are needed.

Chitin degradation and its effect on natural transformation: a systematic genetic study in Vibrio parahaemolyticus.

The degradation of polymeric chitin by chitinase liberates soluble N-acetyl glucosamine oligosaccharides (GlcNAcn≥2), a source of nutrition that can also induce a state of natural genetic competence in Vibrio parahaemolyticus. This analysis revealed that among seven predicted chitinases, the synergistic action of VPA0055 (ChiA2), VP0619 (ChiB), and VPA0832 (Cdx) were essential for the robust growth and high transformation frequency on chitin. The endochitinase, ChiA2, and periplasmic chitinase, Cdx were indispensable for chitin degradation. ChiB was not essential for growth on chitin but did have an effect on the rate of chitin degradation. Interestingly, the loss of Cdx drastically reduced growth on insoluble chitin, but growth on soluble GlcNAc5/6 remained same. The utilization of GlcNAc5/6 was only inhibited when there was mutation of Cdx with the other periplasmic chitinases VP0755 and VP2486. This suggests that Cdx might also be involved in extracellular degradation of chitin, in addition to its role as a periplasmic chitinase. Moreover, the periplasmic chitin oligosaccharide-binding protein (CBP) was found to be essential for the efficient utilization of chitin. The CBP was specifically needed for the processing of GlcNAc4-6 during growth on chitin. Overall, this study provides detailed analysis of the machinery behind chitin degradation in V. parahaemolyticus.

Development of methods for regeneration and genetic transformation of the commercial plum variety Startovaya from somatic tissues

An effi cient technique for regeneration of plants from somatic tissues of domestic plum (Prunus domestica L.) varieties, as well as selecting schemes using various reporter (uidA, gfp) and selective marker genes (nptII, hpt) have been developed. During the transformation of somatic tissues of the commercial plum variety, transgenic plants containing both reporter genes and genes of economically valuable traits were obtained. For the fi rst time, the eff ect of calcium pantothenate on the adventive regeneration of plum shoots was investigated. It was shown that the selective antibiotic hygromycin is more eff ective for the selection of plum transformants than kanamycin or geneticin, which was expressed in a higher frequency of transformation — 1.2-2.2 % versus 0.2-0.5 % in selection on kanamycin. When using geneticin, transgenic plants were not obtained. Optimization of conditions made it possible to increase the percentage of regeneration of shoots to 79.4 % for the variety Startovaya and up to 39.6 % for the variety Etude. Analysis of GFP expression in transgenic lines showed that the leader sequences of herbaceous plant species encoding signal peptides can be used to eff ectively cellular compartmentalization of heterologous gene expression products in the plum. Preliminary results also show a higher level of expression of the reticular form of GFP in comparison with the vacuolar and cytosolic forms. Nine transgenic lines containing the insertion of the target bar gene, which gives resistance to phosphinothricin-containing herbicides of the “Basta” type, were obtained, which was confi rmed by the presence in the genome of the sequence of the transferred gene and the resistance of the obtained transgenic plants to 3 times the fi eld concentration of the herbicide “Basta”.

Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18

In addition to traditional methods, advanced biotechnologies, especially CRISPR/Cas9-mediated genome editing, have emerged as effective tools for improving important agronomic traits in rice. However, the critical step for utilizing these systems is to develope an effective system for rice transformation and regeneration. This study was performed to establish procedures for Agrobacterium-mediated transformation and regeneration of rice cultivar Khang Dan 18 (KD18) – a popular indica variety in the North of Vietnam. The tissue cultures procedure with optimized medium compositions showed high frequencies of callus induction and shoot regeneration, at 94.7% and 45.3% respectively. We found that 30 mg/L of hygromycin was an effective concentration for transgenic KD18 selection. Light-yellow friable calli were used as the starting material for transformation mediated by A. tumefaciens strain AGL1 harbouring pHUE411 vector containing gus intron and hygromycin resistance gene (hptII). Important factors related to transformation procedure had been optimized in this study. The high transformation frequency (12.8%) was achieved by using the optimized procedure for KD18 cultivar. In which, bacterial density (OD600), infection time and co-cultivation period were performed at 0.1, 20 minutes and 3 days, respectively. PCR analysis using specific primers and the histological GUS staining demonstrated the presence of hptII and gus genes in transgenic rice lines. This result provides a potential protocol to transfer genes of interest into KD18 as well as other indica rice cultivars.

A simple and efficient protocol for hairy root culture of Arabidopsis thaliana

Hairy root culture (HRC) represent a valuable biotechnological tool for the production of plant secondary metabolites. Secondary metabolome study of Arabidopsis thaliana may help to understand the biological roles of various secondary metabolites present in it. The present work deals with the establishment of Agrobacterium rhizogenes strains transformed HRC of A. thaliana with very high transformation frequency resulting in long term hairy root cultures grown in hormone free media. Optimization of culture medium and standardisation of co-cultivation period are the key role players in obtaining high frequency of hairy roots. Four days of preculture in CIM medium and five min of co-cultivation in the bacterial suspension were found to be optimal conditions for root induction. This protocol could become a powerful tool for transcriptomics, metabolomics and proteomics-based studies for different transgenic root lines of A. thaliana.

Analysis of chromatin accessibility in p53 deficient spermatogonial stem cells for high frequency transformation into pluripotent state

ObjectivesSpermatogonial stem cells (SSCs), the germline stem cells (GSCs) committed to spermatogenesis in niche, can transform into pluripotent state in long‐term culture without introduction of exogenous factors, typically in p53 deficiency condition. As the guardian for genomic stability, p53 is associated with epigenetic alterations during SSCs transformation. However, the mechanism is still unknown, since complicated roles of p53 baffle our understanding of the regulating process.Materials and MethodsThe chromatin accessibility and differentially expressed genes (DEGs) were analysed in p53 +/+ and p53 −/− SSCs using the Assay for Transposase‐Accessible Chromatin with high‐throughput Sequencing (ATAC‐seq) and RNA‐sequencing (RNA‐seq), to explore the connection of p53 and cell fate at chromosomal level.ResultsSeveral transcription factors (TFs), such as CTCF, SMAD3 and SOX2, were predicted as important factors mediating the transformation. Molecular evidence suggested that SMAD3 efficiently promoted pluripotency‐associated gene expression both in fresh and long‐term cultured SSCs. However, p53 knockout (KO) is insufficient to induce SMAD3 expression in SSCs.ConclusionsThese observations indicate that SMAD3 is a key factor for SSCs transformation, and an unknown event is required to activate SMAD3 as the prerequisite for SSCs reprogramming, which may occur in the long‐term culture of SSCs. This study demonstrates the connection of p53 and pluripotency‐associated factors, providing new insight for understanding the mechanisms of SSCs reprogramming and germline tumorigenesis.

Elicitor-induced phenolic acids accumulation in Salvia virgata Jacq. hairy root cultures

Phenolic acids, as the predominant secondary metabolites of Salvia plants, are largely used in pharmaceutical industries. The main aim of the study was to establish hairy root cultures of Salvia virgata Jacq. Also, the effects of methyl jasmonate (22.4 and 11.2 ppm), Ag+ ions (5 and 2.5 ppm) and yeast extract (100 and 50 ppm) were assessed on total phenol, total flavonoid, rosmarinic acid and salvianolic acid A contents in the hairy roots after 1, 3 and 5 days of exposure. Results showed that Agrobacterium rhizogenes strains (A4, ATCC15834, R1000, GM1534 and C58C1) had different potential to induce hairy roots on leaf explants. The transformed roots were molecularly confirmed using rolC gene, and the highest transformation frequency (56%) was obtained by ATCC15834 strain. Among the established hairy root lines, the highest amount of rosmarinic acid (0.45 ± 0.01 mg/g DW) and dry root biomass (2.29 ± 0.04 g) was obtained in AT3, the line which was induced by ATCC15834 strain. The maximum accumulation of total phenol (123.6 ± 0.93 mg GAE/g DW), total flavonoid (5.09 ± 0.07 mg QUE/g DW), rosmarinic acid (18.45 ± 0.8 mg/g DW) and salvianolic acid A (2.11 ± 0.04 mg/g DW) was observed in the hairy roots elicited with 22.4 ppm methyl jasmonate on day three after treatment. The results support that elicitation could be an effective procedure for the improvement of caffeic acid derivatives production in S. virgata hairy root cultures. The results of this study approved that the application of elicitors is an effective procedure for the stimulation of phenolic acids production in the hairy root cultures of Salvia virgata Jacq.

Anti-inflammatory compounds produced in hairy roots culture of Sphaeralcea angustifolia

Sphaeralcea angustifolia (Cav) G. Don is used in traditional Mexican medicine to treat inflammations and gastric disease. Its anti-inflammatory and immunomodulatory activities in mice and rats acute and chronic models have been attributed mainly to scopoletin. Scopoletin reduced joint inflammation, the number of new vessels, production of endogenous angiogenic inducers, and reversed the histopathological alterations in rat adjuvant-induced arthritis. Tomentin and sphaeralcic acid from S. angustifolia cells in suspension proved anti-inflammatory and immunomodulatory activities in mice kaolin/λ-carrageenan-induced arthritis. Transformed roots of S. angustifolia have been proposed as active compounds producers. A high transformation frequency mediated by Agrobacterium rhizogenes ATCC15834/pTDT was obtained from nodal segments (59.5 ± 10.5%, 145 hairy root lines) and leaves (40.0 ± 25, 52 hairy root lines) of 2-month-old plantlets. Among seven lines selected according to their phenotypic characteristics and growth index, the SaTR N7.2 line presented the highest sphaeralcic acid production (17.6 ± 1.72 mg/g DW); this production was 440-fold superior to that reported in S. angustifolia wild plants, and in comparison to cells in suspension of S. angustifolia in MS medium with nitrate restriction this was 263-fold higher when cultured in flasks and 5-fold higher in a stirred-tank type bioreactor. The SaTR N7.2, SaTR N5.1, SaTR N7.1, and SaTR N15.1 lines excreted sphaeralcic acid into the culture medium at similar levels. Genetic transformation of hairy roots was confirmed by amplifying a 490 bp fragment of the rolC gene. After 2 years in culture, S. angustifolia hairy roots are producers of scopoletin and sphaeralcic acid and they can be stressed by nitrate reduction and/or copper increased to stimulate scopoletin and sphaeralcic acid production.

Tumor Growth in the High Frequency Medulloblastoma Mouse Model Ptch1+/-/Tis21KO Has a Specific Activation Signature of the PI3K/AKT/mTOR Pathway and Is Counteracted by the PI3K Inhibitor MEN1611.

We have previously generated a mouse model (Ptch1+/−/Tis21KO), which displays high frequency spontaneous medulloblastoma, a pediatric tumor of the cerebellum. Early postnatal cerebellar granule cell precursors (GCPs) of this model show, in consequence of the deletion of Tis21, a defect of the Cxcl3-dependent migration. We asked whether this migration defect, which forces GCPs to remain in the proliferative area at the cerebellar surface, would be the only inducer of their high frequency transformation. In this report we show, by further bioinformatic analysis of our microarray data of Ptch1+/−/Tis21KO GCPs, that, in addition to the migration defect, they show activation of the PI3K/AKT/mTOR pathway, as the mRNA levels of several activators of this pathway (e.g., Lars, Rraga, Dgkq, Pdgfd) are up-regulated, while some inhibitors (e.g. Smg1) are down-regulated. No such change is observed in the Ptch1+/− or Tis21KO background alone, indicating a peculiar synergy between these two genotypes. Thus we investigated, by mRNA and protein analysis, the role of PI3K/AKT/mTOR signaling in MBs and in nodules from primary Ptch1+/−/Tis21KO MB allografted in the flanks of immunosuppressed mice. Activation of the PI3K/AKT/mTOR pathway is seen in full-blown Ptch1+/−/Tis21KO MBs, relative to Ptch1+/−/Tis21WT MBs. In Ptch1+/−/Tis21KO MBs we observe that the proliferation of neoplastic GCPs increases while apoptosis decreases, in parallel with hyper-phosphorylation of the mTOR target S6, and, to a lower extent, of AKT. In nodules derived from primary Ptch1+/−/Tis21KO MBs, treatment with MEN1611, a novel PI3K inhibitor, causes a dramatic reduction of tumor growth, inhibiting proliferation and, conversely, increasing apoptosis, also of tumor CD15+ stem cells, responsible for long-term relapses. Additionally, the phosphorylation of AKT, S6 and 4EBP1 was significantly inhibited, indicating inactivation of the PI3K/AKT/mTOR pathway. Thus, PI3K/AKT/mTOR pathway activation contributes to Ptch1+/−/Tis21KO MB development and to high frequency tumorigenesis, observed when the Tis21 gene is down-regulated. MEN1611 could provide a promising therapy for MB, especially for patient with down-regulation of Btg2 (human ortholog of the murine Tis21 gene), which is frequently deregulated in Shh-type MBs.