Search results for: clones

Authors: Suwattana Visetnan, Premruethai Supungul, Sureerat Tang, Ikuo Hirono, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. Infection by yellow head virus (YHV) activates the Imd pathway. To investigate the expression of genes involved in YHV infection and under the influence of PmRelish regulation, RNA interference and suppression subtractive hybridization (SSH) are employed. The genes in forward library expressed in shrimp after YHV infection and under the activity of PmRelish were obtained by subtracting the cDNAs from YHV-infected and PmRelish-knockdown shrimp with cDNAs from YHV-infected shrimp. Opposite subtraction gave a reverse library whereby an alternative set of genes under YHV infection and no PmRelish expression was obtained. Sequencing of 252 and 99 cDNA clones from the respective forward and reverse libraries were done and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors. The expression of antimicrobial protein genes, ALFPm3, crustinPm1, penaeidin3 and penaeidin5 were tested under PmRelish silencing and Gram-negative bacterium V. harveyi infection. Together with the results previously reported, the expression of penaeidin5 and also penaeidin3 but not ALFPm3 and crustinPm1 were under the regulation of PmRelish in the Imd pathway.

Keywords: relish, yellow head virus, penaeus monodon, antimicrobial proteins

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Authors: Chieh-Chung Tsou, Chun-Min Lo, Yeh-Shiu Chu

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Cell’s mechanical forces provide important physical cues in regulation of proper cellular functions, such as cell differentiation, proliferation and migration. It is believed that adhesive forces generated by cell-cell interaction are able to transmit to the interior of cell through filamentous cortical cytoskeleton. Prominent among other membrane receptors, Cadherins are prototypical adhesive molecules able to generate remarkable forces to regulate intercellular adhesion. However, the mechanistic steps of mechano-transduction in Cadherin-mediated adhesion remain very controversial. We are interested in understanding how Cadherin protein complexes enable force generation and transmission at cell-cell contact in the initial stage of intercellular adhesion. For providing a better control of time, space, and substrate stiffness, in this study, a combination of protein micropattern, micropipette manipulation, and traction force microscopy is used. Pair micropattern with different forms confines cell spreading area and the gaps in pairs varied from 2 to 8 microns are applied for monitoring the forces that cell pairs generated, measured by traction force microscopy. Moreover, cell clones obtained from epithelial cells undergone genome editing are used to score the importance for known components of Cadherin complexes in force generation. We believe that our results from this combinatory mechanobiological method will provide deep insights on understanding the biophysical principle governing mechano- transduction of Cadherin-mediated intercellular adhesion.

Keywords: cadherin, intercellular adhesion, protein micropattern, traction force microscopy

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Authors: H. Hanine, H. H'ssaini, M. Ibno Alaoui, A. Nablousi, H. Zahir, S. Ennahli, H. Latrache, H. Zine Abidine

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Biochemical and pomological variability among 14 cultivars of Prunus dulcis planted in a germoplasm collection site in Morocco were evaluated. Almond samples from six local and eight foreign cultivars (France, Italy, Spain, and USA) were characterized. Biochemical and pomological data revealed significant genetic variability among the 14 cultivars; local cultivars exhibited higher total polyphenol content. Oil content ranged from 35 to 57% among cultivars; both Texas and Toundout genotypes recorded the highest oil content. Total protein concentration from select cultivars ranged from 50 mg/g in Ferraduel to 105 mg/g in Rizlane1 cultivars. Antioxidant activity of almond samples was examined by a DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging assay; the antioxidant activity varied significantly within the cultivars, with IC50 (the half-maximal inhibitory concentration) values ranging from 2.25 to 20 mg/ml. Autochthonous cultivars originated from the Oujda region exhibited higher tegument total polyphenol and amino acid content compared to others. The genotype Rizlane2 recorded the highest flavonoid content. Pomological traits revealed a large variability within the almond germplasms. The hierarchical clustering analysis of all the data regarding pomological traits distinguished two groups with some particular genotypes as distinct cultivars, and groups of cultivars as polyclone varieties. These results strongly exhibit a potential use of Moroccan-originated almonds as potential clones for future selection due to their nutritional values and pomological traits compared to well-established cultivars.

Keywords: antioxidant activity, DDPH, Moroccan almonds, Prunus dulcis

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Authors: Paromik Bhattacharyya, Vijay Kumar, Johannes Van Staden

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Micropropagation is an important tool for the conservation of threatened and commercially important plant species of which orchids deserve special attention. Ansellia africana is one such medicinally important orchid species having much commercial significance. Thus, development of regeneration protocols for producing clonally stable regenerates using axillary buds is of much importance. However, for large-scale micropropagation to become not only successful but also acceptable by end-users, somaclonal variations occurring in the plantlets need to be eliminated. In the light of the various factors (genotype, ploidy level, in vitro culture age, explant and culture type, etc.) that may account for the somaclonal variations of divergent genetic changes at the cellular and molecular levels, genetic analysis of micropropagated plants using a multidisciplinary approach is of utmost importance. In the present study, the clonal integrity of the long term micropropagated A. africana plants were assessed using advanced molecular marker system i.e. Start Codon Targeted Polymorphism (SCoT). Our studies recorded a clonally stable regeneration protocol for A. africana with a very high degree of clonal fidelity amongst the regenerates. The results obtained from these molecular analyses could help in modifying the regeneration protocols for obtaining clonally stable true to type plantlets for sustainable commercial use.

Keywords: medicinal orchid micropropagation, start codon targeted polymorphism (SCoT), RAP), traditional African pharmacopoeia, genetic fidelity

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Authors: Kien Xuan Ngo

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Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for corynespora leaf fall (CLF) disease in rubber trees. Currently, the molecular mechanism of the cytotoxicity of Cas isoforms (i.e., Cas1, Cas2) on rubber leaves and its host selectivity have not been fully elucidated. This study analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Using high-speed atomic force microscopy and confocal microscopy, this study reveals that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more susceptible to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. In summary, This study unveils that (i) Cas1 and Cas2 directly damage and cause necrosis in the leaves of specific rubber clones; (ii) Cas1 and Cas2 can form biofilm-like structures on specific lipid membranes (negative phospholipids, glycerolipids, and sterols). The biofilm-like formation of Cas toxin plays an important role in selective disruption on lipid membranes; (iii) Vulnerability of the specific cytoplasmic membranes to the selective Cas toxin is the most remarkable feature of cytotoxicity of Cas toxin on plant cells. Finally, researcher’s exploration is crucial to understand the basic molecular mechanism underlying the host-selective toxic interaction of Cas toxin with cytoplasmic membranes in plant cells.

Keywords: cassiicolin, corynespora leaf fall disease, high-speed AFM, giant liposome vesicles

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Authors: M. L. Mangena, N. Mokoena, K. Rashamuse, M. G. Tlou

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Tertiary alcohol ester (TAE) hydrolases are a group of esterases (EC 3.1.1.-) that catalyze the kinetic resolution of TAEs and as a result, they are sought-after for the production of optically pure tertiary alcohols (TAs) which are useful as building blocks for number biologically active compounds. What sets these enzymes apart is, the presence of a GGG(A)X-motif in the active site which appears to be the main reason behind their activity towards the sterically demanding TAEs. The genome of Pseudomonas syringae pv. maculicola (Psm) comprises a multitude of genes that encode esterases. We therefore, hypothesize that some of these genes encode TAE hydrolases. In this study, Psm was screened for TAE hydrolase activity using the linalyl acetate (LA) plate assay and a positive reaction was observed. As a result, the genome of Psm was screened for esterases with a GGG(A)X-motif using the motif search tool and two potential TAE hydrolase genes (PsmEST1 and 2, 1100 and 1000bp, respectively) were identified, PsmEST1 was amplified by PCR and the gene sequenced for confirmation. Analysis of the sequence data with the SingnalP 4.1 server revealed that the protein comprises a signal peptide (22 amino acid residues) on the N-terminus. Primers specific for the gene encoding the mature protein (without the signal peptide) were designed such that they contain NdeI and XhoI restriction sites for directional cloning of the PCR products into pET28a. The gene was expressed in E. coli JM109 (DE3) and the clones screened for TAE hydrolase activity using the LA plate assay. A positive clone was selected, overexpressed and the protein purified using nickel affinity chromatography. The activity of the esterase towards LA was confirmed using thin layer chromatography.

Keywords: hydrolases, tertiary alcohol esters, tertiary alcohols, screening, Pseudomonas syringae pv., maculicola genome, esterase activity, linalyl acetate

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Authors: Z. Nourmohammadi, F. Farahani, M. Shaker

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Aloe is an important commercial crop available in a wide range of species and varieties in international markets. The applications of this plant have been recorded in the ancient cultures of India, Egypt, Greece, Rome and China. Aloe has been used for centuries and is currently being actively studied for medicinal purposes. Aloe is propagated through lateral buds, which is slow, very expensive and low income practice. Nowadays, it has been cultured by in vitro propagation for rapid multiplication of plants, genetic improvement of crops, obtaining disease-free clones and for progressive valuable germplasm. The present study focused on the influence of different phytohormones on rapid in vitro propagation of Aloe plants. We also investigated the effect of gamma radiation on biochemical characters as well as genetic changes. Shoot tip of 2-3 cm were collected from offshoot of Aloe barbadensis and Aloe littoralis, and were inoculated with MS medium containing various concentrations of BA (0.5, 1, 2 mg/l), IAA (0.5, 1 mg/l). The best treatment for a highest shoot number and bud proliferation was MS medium containing 2 mg/l BAP and 0.5 mg/l IAA in A. barbadensis and A. littoralis. Maximum percentage of proliferated shoot buds (90% and 95%) from a single explant were obtained in MS medium after 4-5 weeks of the second and the first subcultures, respectively. Different genome sizes were also indicated among treatments and subcultures. The mixoploids identified in flow cytometery histograms in different treatments. The effect of gamma radiation on A. littoralis showed that by increasing the dose of gamma radiation, amounts of chlorophyll A, B, carotenoids, total protein content and superoxide dismutase were significantly increased compared to control plants. Genetic variation analysis also revealed significant genetic differences between control and gamma radiation treated regenerated plants by AMOVA test. Higher genetic heterozygocity was observed in radiation treated plants. Our findings may provide useful method for improving of Aloe plant proliferation with increasing of useful material such as antioxidant enzymes.

Keywords: aloe, antioxidant enzyme, micropropagation, gamma radiation, genetic variation

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Authors: Hassan Mahmoudzadeh

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The bacterium of Agrobacterium vitis causes crown and root gall disease, an important disease of grapevine, Vitis vinifera L. Also, Phylloxera is one of the most important pests in viticulture. Grapevine rootstocks were developed to provide increased resistance to soil-borne pests and diseases, but rootstock effects on some traits remain unclear. The interaction between rootstock, scion and environment can induce different responses to the grapevine physiology. 'Sultsna' (Vitis vinifera L.) is one of the most valuable raisin grape cultivars in Iran. Thus, the aim of this study was to determine the rootstock effect on the growth characteristics and yield components and quality of 'Sultana' grapevine grown in the Urmia viticulture region. The experimental design was completely randomized blocks, with four treatments, four replicates and 10 vines per plot. The results show that all variables evaluated were significantly affected by the rootstock. The Sultana/110R and Sultana/Nazmieh were among other combinations influenced by the year and had a higher significant yield/vine (13.25 and 12.14, respectively). Indeed, they were higher than that of Sultana/5BB (10.56 kg/vine) and Sultana/Spota (10.25 kg/vine). The number of clusters per burst bud and per vine and the weight of clusters were affected by the rootstock as well. Pruning weight/vine, yield/pruning weight, leaf area/vine and leaf area index are variables related to the physiology of grapevine, which was also affected by the rootstocks. In general, rootstocks had adapted well to the environment where the experiment was carried out, giving vigor and high yield to Sultana grapevine, which means that they may be used by grape growers in this region. In sum, the study found the best rootstocks for 'Sultana' to be Nazmieh and 110R in terms of root and shoot growth. However, the choice of the right rootstock depends on various aspects, such as those related to soil characteristics, climate conditions, grape varieties, and even clones, and production purposes.

Keywords: grafting, vineyards, grapevine, succeptability

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Authors: R. Hasterok, A. Betekhtin, N. Borowska, A. Braszewska-Zalewska, E. Breda, K. Chwialkowska, R. Gorkiewicz, D. Idziak, J. Kwasniewska, M. Kwasniewski, D. Siwinska, A. Wiszynska, E. Wolny

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In contrast to animals, the organisation of plant genomes at the cytomolecular level is still relatively poorly studied and understood. However, the Brachypodium genus in general and B. distachyon in particular represent exceptionally good model systems for such study. This is due not only to their highly desirable ‘model’ biological features, such as small nuclear genome, low chromosome number and complex phylogenetic relations, but also to the rapidly and continuously growing repertoire of experimental tools, such as large collections of accessions, WGS information, large insert (BAC) libraries of genomic DNA, etc. Advanced cytomolecular techniques, such as fluorescence in situ hybridisation (FISH) with evermore sophisticated probes, empowered by cutting-edge microscope and digital image acquisition and processing systems, offer unprecedented insight into chromatin organisation at various phases of the cell cycle. A good example is chromosome painting which uses pools of chromosome-specific BAC clones, and enables the tracking of individual chromosomes not only during cell division but also during interphase. This presentation outlines the present status of molecular cytogenetic analyses of plant genome structure, dynamics and evolution using B. distachyon and some of its relatives. The current projects focus on important scientific questions, such as: What mechanisms shape the karyotypes? Is the distribution of individual chromosomes within an interphase nucleus determined? Are there hot spots of structural rearrangement in Brachypodium chromosomes? Which epigenetic processes play a crucial role in B. distachyon embryo development and selective silencing of rRNA genes in Brachypodium allopolyploids? The authors acknowledge financial support from the Polish National Science Centre (grants no. 2012/04/A/NZ3/00572 and 2011/01/B/NZ3/00177)

Keywords: Brachypodium, B. distachyon, chromosome, FISH, molecular cytogenetics, nucleus, plant genome organisation

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Authors: Daniel Aberdam

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Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.

Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy

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Authors: Young Ah Kim, Hyunsoo Kim, Eun-Jeong Yoon, Young Hee Seo, Kyungwon Lee

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Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from food animals are considered as a reservoir for transmission of ESBL genes to human. The aim of this study is to assess the prevalence and molecular epidemiology of ESBL-producing E. coli colonization in pigs, farm workers, and farm environments to elucidate the transmission of multidrug-resistant clones from animal to human. Nineteen pig farms were enrolled across the country in Korea from August to December 2017. ESBL-producing E. coli isolates were detected in 190 pigs, 38 farm workers, and 112 sites of farm environments using ChromID ESBL (bioMerieux, Marcy l'Etoile, France), directly (stool or perirectal swab) or after enrichment (sewage). Antimicrobial susceptibility tests were done with disk diffusion methods and blaTEM, blaSHV, and blaCTX-M were detected with PCR and sequencing. The genomes of the four CTX-M-55-producing E. coli isolates from various sources in one farm were entirely sequenced to assess the relatedness of the strains. Whole genome sequencing (WGS) was performed with PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA). ESBL genotypes were 85 CTX-M-1 group (one CTX-M-3, 23 CTX-M-15, one CTX-M-28, 59 CTX-M-55, one CTX-M-69) and 60 CTX-M-9 group (41 CTX-M-14, one CTX-M-17, one CTX-M-27, 13 CTX-M-65, 4 CTX-M-102) in total 145 isolates. The rectal colonization rates were 53.2% (101/190) in pigs and 39.5% (15/38) in farm workers. In WGS, sequence types (STs) were determined as ST69 (E. coli PJFH115 isolate from a human carrier), ST457 (two E. coli isolates PJFE101 recovered from a fence and PJFA1104 from a pig) and ST5899 (E. coli PJFA173 isolate from the other pig). The four plasmids encoding CTX-M-55 (88,456 to 149, 674 base pair), whether it belonged to IncFIB or IncFIC-IncFIB type, shared IncF backbone furnishing the conjugal elements, suggesting of genes originated from same ancestor. In conclusion, the prevalence of ESBL-producing E. coli in swine farms was surprisingly high, and many of them shared common ESBL genotypes of clinical isolates such as CTX-M-14, 15, and 55 in Korea. It could spread by horizontal transfer between isolates from different reservoirs (human-animal-environment).

Keywords: Escherichia coli, extended-spectrum β-lactamase, prevalence, whole genome sequencing

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Authors: Anil Kumar, Diwakar Aggarwal, M. Sudhakara Reddy

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The wood of Eucalyptus, Populus and bamboos are some important species used as raw material for the manufacture of pulp. However, higher levels of lignin pose a problem during Kraft pulping and yield of pulp is also lower. In order to increase the yield of pulp per unit wood and reduce the use of chemicals during kraft pulping it is important to reduce the lignin content and/or increase cellulose content in wood. Cellulose biosynthesis in wood takes place by the coordinated action of many enzymes. The two important enzymes are KORRIGAN and SUCROSE SYNTHASE. KORRIGAN (Endo-1,4--glucanase) is implicated in the process of editing growing cellulose chains and improvement of the crystallinity of produced cellulose, whereas SUCROSE SYNTHASE is involved in providing substrate (UDP-glucose) for growing cellulose chains. The present study was aimed at the cloning, characterization and overexpression of these genes in Eucalyptus and Populus. An efficient shoot organogenesis protocol from leaf explants taken from micro shoots of the species has been developed. Agrobacterium mediated genetic transformation using Agrobacterium tumefaciens strain EHA105 and LBA4404 harboring binary vector pBI121 was achieved. Both the genes were cloned from cDNA library of Populus deltoides. These were subsequently characterized using various bioinformatics tools. The cloned genes were then inserted into pBI121 under the CaMV35S promotors replacing GUS gene. The constructs were then mobilized into above strains of Agrobacterium and used for the transformation work. Subsequently, genetic transformation of these clones with target genes following already developed protocol is in progress. Four transgenic lines of Eucalyptus tereticornis overexpressing Korrigan gene under the strong constitutive promoters CaMV35S have been developed, which are being further evaluated. Work on development of more transgenic lines overexpressing these genes in Populus and Eucalyptus is also in progress. This presentation will focus on important developments in this direction.

Keywords: Eucalyptus tereticornis, genetic transformation, Kraft pulping Populus deltoides

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Authors: Ramesh Joshi, Nisha Khatik

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Murraya koenigii (L.) Spreng locally known as “Curry patta” or “Meetha neem” belonging to the family Rutaceae that grows wildly in Southern Asia. Its aromatic leaves are commonly used as the raw material for traditional medicinal formulations in India. The leaves contain essential oil and also used as a condiment. Several monomeric and binary carbazol alkaloids present in the various plant parts. These alkaloids have been reported to possess anti-microbial, mosquitocidal, topo-isomerase inhibition and antioxidant properties. Some of the alkaloids reported in this plant have showed anti carcinogenic and anti-diabetic properties. The conventional method of propagation of this tree is limited to seeds only, which retain their viability for only a short period. Hence, a biotechnological approach might have an advantage edging over traditional breeding as well as the genetic improvement of M. koenigii within a short period. The development of a reproducible regeneration protocol is the prerequisite for ex situ conservation and micropropagation. An efficient protocol for high frequency regeneration of in vitro plants of Murraya koenigii via different explants such as- nodal segments, intermodal segments, leaf, root segments, hypocotyle, cotyledons and cotyledonary node explants is described. In the present investigation, assessment of clonal fidelity in the micropropagated plantlets of Murraya koenigii was attempted using RAPD and ISSR markers at different pathways of plant tissue culture technique. About 20 ISSR and 40 RAPD primers were used for all the samples. Genomic DNA was extracted by CTAB method. ISSR primer were found to be more suitable as compared to RAPD for the analysis of clonal fidelity of M. koenigii. The amplifications however, were finally performed using RAPD, ISSR markers owing to their better performance in terms of generation of amplification products. In RAPD primer maximum 75% polymorphism was recorded in OPU-2 series which exhibited out of 04 scorable bands, three bands were polymorphic with a band range of size 600-1500 bp. In ISSR primers the UBC 857 showed 50% polymorphism with 02 band were polymorphic of band range size between 400-1000 bp.

Keywords: genetic fidelity, Murraya koenigii, aromatic plants, ISSR primers

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Authors: Walter Vera-Ortega, Idoia Busnadiego, Sam J. Wilson

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Introduction: Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the acquired immunodeficiency syndrome (AIDS), a leading cause of death worldwide infecting millions of people each year. Despite intensive research in vaccine development, therapies against HIV-1 infection are not curative, and the huge genetic variability of HIV-1 challenges to drug development. Current animal models for HIV-1 research present important limitations, impairing the progress of in vivo approaches. Macaques require a CD8+ depletion to progress to AIDS, and the maintenance cost is high. Mice are a cheaper alternative but need to be 'humanized,' and breeding is not possible. The development of an HIV-1 clone able to replicate in mice is a challenging proposal. The lack of human co-factors in mice impedes the function of the HIV-1 accessory proteins, Tat and Rev, hampering HIV-1 replication. However, Tat and Rev function can be replaced by constitutive/chimeric promoters, codon-optimized proteins and the constitutive transport element (CTE), generating a novel HIV-1 clone able to replicate in mice without disrupting the amino acid sequence of the virus. By minimally manipulating the genomic 'identity' of the virus, we propose the generation of an HIV-1 clone able to replicate in mice to assist in antiviral drug development. Methods: i) Plasmid construction: The chimeric promoters and CTE copies were cloned by PCR using lentiviral vectors as templates (pCGSW and pSIV-MPCG). Tat mutants were generated from replication competent HIV-1 plasmids (NHG and NL4-3). ii) Infectivity assays: Retroviral vectors were generated by transfection of human 293T cells and murine NIH 3T3 cells. Virus titre was determined by flow cytometry measuring GFP expression. Human B-cells (AA-2) and Hela cells (TZMbl) were used for infectivity assays. iii) Protein analysis: Tat protein expression was determined by TZMbl assay and HIV-1 capsid by western blot. Results: We have determined that NIH 3T3 cells are able to generate HIV-1 particles. However, they are not infectious, and further analysis needs to be performed. Codon-optimized HIV-1 constructs are efficiently made in 293T cells in a Tat and Rev independent manner and capable of packaging a competent genome in trans. CSGW is capable of generating infectious particles in the absence of Tat and Rev in human cells when 4 copies of the CTE are placed preceding the 3’LTR. HIV-1 Tat mutant clones encoding different promoters are functional during the first cycle of replication when Tat is added in trans. Conclusion: Our findings suggest that the development of an HIV-1 Tat-Rev independent clone is challenging but achievable aim. However, further investigations need to be developed prior presenting our HIV-1 clone as a candidate model for research.

Keywords: codon-optimized, constitutive transport element, HIV-1, long terminal repeats, research model

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Authors: Maddalena Arigoni, Maria Luisa Ratto, Raffaele A. Calogero, Luca Alessandri

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The presence of tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Understanding the causes and progression of cancer requires research efforts aimed at characterizing heterogeneity, which can be facilitated by evolving single-cell sequencing technologies. However, analyzing single-cell data necessitates computational methods that often lack objective validation. Therefore, the establishment of benchmarking datasets is necessary to provide a controlled environment for validating bioinformatics tools in the field of single-cell oncology. Benchmarking bioinformatics tools for single-cell experiments can be costly due to the high expense involved. Therefore, datasets used for benchmarking are typically sourced from publicly available experiments, which often lack a comprehensive cell annotation. This limitation can affect the accuracy and effectiveness of such experiments as benchmarking tools. To address this issue, we introduce omics benchmark experiments designed to evaluate bioinformatics tools to depict the heterogeneity in single-cell tumor experiments. We conducted single-cell RNA sequencing on six lung cancer tumor cell lines that display resistant clones upon treatment of EGFR mutated tumors and are characterized by driver genes, namely ROS1, ALK, HER2, MET, KRAS, and BRAF. These driver genes are associated with downstream networks controlled by EGFR mutations, such as JAK-STAT, PI3K-AKT-mTOR, and MEK-ERK. The experiment also featured an EGFR-mutated cell line. Using 10XGenomics platform with cellplex technology, we analyzed the seven cell lines together with a pseudo-immunological microenvironment consisting of PBMC cells labeled with the Biolegend TotalSeq™-B Human Universal Cocktail (CITEseq). This technology allowed for independent labeling of each cell line and single-cell analysis of the pooled seven cell lines and the pseudo-microenvironment. The data generated from the aforementioned experiments are available as part of an online tool, which allows users to define cell heterogeneity and generates count tables as an output. The tool provides the cell line derivation for each cell and cell annotations for the pseudo-microenvironment based on CITEseq data by an experienced immunologist. Additionally, we created a range of pseudo-tumor tissues using different ratios of the aforementioned cells embedded in matrigel. These tissues were analyzed using 10XGenomics (FFPE samples) and Curio Bioscience (fresh frozen samples) platforms for spatial transcriptomics, further expanding the scope of our benchmark experiments. The benchmark experiments we conducted provide a unique opportunity to evaluate the performance of bioinformatics tools for detecting and characterizing tumor heterogeneity at the single-cell level. Overall, our experiments provide a controlled and standardized environment for assessing the accuracy and robustness of bioinformatics tools for studying tumor heterogeneity at the single-cell level, which can ultimately lead to more precise and effective cancer diagnosis and treatment.

Keywords: single cell omics, benchmark, spatial transcriptomics, CITEseq

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Authors: Adenike Adesanya, Nonthaphat Wong, Xiang-Yun Lan, Shea Ping Yip, Chien-Ling Huang

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Background: The most challenging mutation of the oncokinase BCR-ABL protein T315I, which is commonly known as the “gatekeeper” mutation and is notorious for its strong resistance to almost all tyrosine kinase inhibitors (TKIs), especially imatinib. Therefore, this study aims to identify T315I-dependent downstream microRNA (miRNA) pathways associated with drug resistance in chronic myeloid leukemia (CML) for prognostic and therapeutic purposes. Methods: T315I-carrying K562 cell clones (K562-T315I) were generated by the CRISPR-Cas9 system. Imatinib-treated K562-T315I cells were subjected to small RNA library preparation and next-generation sequencing. Putative lncRNA-miRNA-mRNA networks were analyzed with (i) DESeq2 to extract differentially expressed miRNAs, using Padj value of 0.05 as cut-off, (ii) STarMir to obtain potential miRNA response element (MRE) binding sites of selected miRNAs on lncRNA H19, (iii) miRDB, miRTarbase, and TargetScan to predict mRNA targets of selected miRNAs, (iv) IntaRNA to obtain putative interactions between H19 and the predicted mRNAs, (v) Cytoscape to visualize putative networks, and (vi) several pathway analysis platforms – Enrichr, PANTHER and ShinyGO for pathway enrichment analysis. Moreover, mitochondria isolation and transcript quantification were adopted to determine the new mechanism involved in T315I-mediated resistance of CML treatment. Results: Verification of the CRISPR-mediated mutagenesis with digital droplet PCR detected the mutation abundance of ≥80%. Further validation showed the viability of ≥90% by cell viability assay, and intense phosphorylated CRKL protein band being detected with no observable change for BCR-ABL and c-ABL protein expressions by Western blot. As reported by several investigations into hematological malignancies, we determined a 7-fold increase of H19 expression in K562-T315I cells. After imatinib treatment, a 9-fold increment was observed. DESeq2 revealed 171 miRNAs were differentially expressed K562-T315I, 112 out of these miRNAs were identified to have MRE binding regions on H19, and 26 out of the 112 miRNAs were significantly downregulated. Adopting the seed-sequence analysis of these identified miRNAs, we obtained 167 mRNAs. 6 hub miRNAs (hsa-let-7b-5p, hsa-let-7e-5p, hsa-miR-125a-5p, hsa-miR-129-5p, and hsa-miR-372-3p) and 25 predicted genes were identified after constructing hub miRNA-target gene network. These targets demonstrated putative interactions with H19 lncRNA and were mostly enriched in pathways related to cell proliferation, senescence, gene silencing, and pluripotency of stem cells. Further experimental findings have also shown the up-regulation of mitochondrial transcript and lncRNA MALAT1 contributing to the lncRNA-miRNA-mRNA networks induced by BCR-ABL T315I mutation. Conclusions: Our results have indicated that lncRNA-miRNA regulators play a crucial role not only in leukemogenesis but also in drug resistance, considering the significant dysregulation and interactions in the K562-T315I cell model generated by CRISPR-Cas9. In silico analysis has further shown that lncRNAs H19 and MALAT1 bear several complementary miRNA sites. This implies that they could serve as a sponge, hence sequestering the activity of the target miRNAs.

Keywords: chronic myeloid leukemia, imatinib resistance, lncRNA-miRNA-mRNA, T315I mutation

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

Abstract:

Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

Procedia PDF Downloads 51

Authors: Jan F. Talts, Amit Saxena, Kåre Engkilde

Abstract:

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most frequent side effects caused by anti-neoplastic agents, with a prevalence from 19 % to 85 %. Clinically, CIPN is a mostly sensory neuropathy leading to pain and to motor and autonomic changes. Due to its high prevalence among cancer patients, CIPN constitutes a major problem for both cancer patients and survivors, especially because currently, there is no single effective method of preventing CIPN. Hearing loss is the most common form of sensory impairment in humans and can be caused by ototoxic chemical compounds such as chemotherapy (platinum-based antineoplastic agents).In rodents, single or repeated cisplatin injections induce peripheral neuropathy and hearing impairment mimicking human disorder, allowing studying the efficacy of new pharmacological candidates in chemotherapy-induced hearing loss and peripheral neuropathy. RNA sequencing data from full term amniotic fluid (TAF) mesenchymal stemcell (MSC) clones was used to identify neural-specific markers present on TAF-MSC. Several prospective neural markers were tested by flow cytometry on cultured TAF-MSC. One of these markers was used for cell-sorting using Tyto MACSQuant cell sorter, and the neural marker positive cell population was expanded for several passages to the final therapeutic product stage. Peripheral neuropathy and hearing loss was induced in mice by administration of cisplatin in three week-long cycles. The efficacy of neural-specific TAF-MSC in treating hearing loss and pain perception was evaluated by administration of three injections of 3 million cells/kg by intravenous route or three injections of 3 million cells/kg by intra-arterial route after each cisplatin cycle treatment. Auditory brainstem responses (ABR) are electric potentials recorded from scalp electrodes, and the first ABR wave represents the summed activity of the auditory nerve fibers contacting the inner hair cells. For ABR studies, mice were anesthetized, then earphones were placed in the left ear of each mouse, an active electrode was placed in the vertex of the skull, a reference electrode under the skin of the mastoid bone, and a ground electrode in the neck skin. The stimuli consisted of tone pips of five frequencies (2, 4, 6, 12, 16, and 24 kHz) at various sound levels (from 0 to 90 dB) ranging to cover the mouse auditory frequency range. The von Frey test was used to assess the onset and maintenance of mechanical allodynia over time. Mice were placed in clear plexiglass cages on an elevated mesh floor and tested after 30 min of habituation. Mechanical paw withdrawal threshold was examined using an electronic von Frey anesthesiometer. Cisplatin groups treated with three injections of 3 million cells/kg by intravenous route and three injections of 3 million cells/kg by intra-arterial route after each cisplatin cycle treatment presented, a significant increase of hearing acuity characterized by a decrease of ABR threshold and a decrease of neuropathic pain characterized by an increase of von Frey paw withdrawal threshold compared to controls only receiving cisplatin. This study shows that treatment with MSCselected for neural specificity presents significant positive efficacy on the chemotherapy-induced neuropathic pain and the chemotherapy-induced hearing loss.

Keywords: mesenchymal stem cell, peripheral neuropathy, amniotic fluid, regenerative medicine

Procedia PDF Downloads 129