Displaying publications 21 - 40 of 70 in total

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  1. Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library
    Tan HY, Sieo CC, Lee CM, Abdullah N, Liang JB, Ho YW
    J Microbiol, 2011 Jun;49(3):492-8.
    PMID: 21717338 DOI: 10.1007/s12275-011-0319-7
    Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.
    Matched MeSH terms: Gene Library*
  2. Effect of condensed tannins on bovine rumen protist diversity based on 18S rRNA gene sequences
    Tan HY, Sieo CC, Abdullah N, Liang JB, Huang XD, Ho YW
    J. Eukaryot. Microbiol., 2013 Jan-Feb;60(1):98-100.
    PMID: 23205499 DOI: 10.1111/jeu.12011
    Molecular diversity of protists from bovine rumen fluid incubated with condensed tannins of Leucaena leucocephala hybrid-Rendang at 20 mg/500 mg dry matter (treatment) or without condensed tannins (control) was investigated using 18S rRNA gene library. Clones from the control library were distributed within nine genera, but clones from the condensed tannin treatment clone library were related to only six genera. Diversity estimators such as abundance-based coverage estimation and Chao1 showed significant differences between the two libraries, although no differences were found based on Shannon-Weaver index and Libshuff.
    Matched MeSH terms: Gene Library
  3. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus
    Tang TH, Polacek N, Zywicki M, Huber H, Brugger K, Garrett R, et al.
    Mol Microbiol, 2005 Jan;55(2):469-81.
    PMID: 15659164
    By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense-box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion, this is the first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression.
    Matched MeSH terms: Gene Library
  4. Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
    Teo MYM, Ng JJC, Fong JY, Hwang JS, Song AA, Lim RLH, et al.
    PeerJ, 2021;9:e11063.
    PMID: 33959410 DOI: 10.7717/peerj.11063
    Background: KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins.

    Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.

    Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.

    Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

    Matched MeSH terms: Gene Library
  5. Source Determination of Red Gel Pen Inks using Raman Spectroscopy and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy combined with Pearson's Product Moment Correlation Coefficients and Principal Component Analysis
    Mohamad Asri MN, Mat Desa WNS, Ismail D
    J Forensic Sci, 2018 Jan;63(1):285-291.
    PMID: 28480527 DOI: 10.1111/1556-4029.13522
    The potential combination of two nondestructive techniques, that is, Raman spectroscopy (RS) and attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy with Pearson's product moment correlation (PPMC) coefficient (r) and principal component analysis (PCA) to determine the actual source of red gel pen ink used to write a simulated threatening note, was examined. Eighteen (18) red gel pens purchased from Japan and Malaysia from November to December 2014 where one of the pens was used to write a simulated threatening note were analyzed using RS and ATR-FTIR spectroscopy, respectively. The spectra of all the red gel pen inks including the ink deposited on the simulated threatening note gathered from the RS and ATR-FTIR analyses were subjected to PPMC coefficient (r) calculation and principal component analysis (PCA). The coefficients r = 0.9985 and r = 0.9912 for pairwise combination of RS and ATR-FTIR spectra respectively and similarities in terms of PC1 and PC2 scores of one of the inks to the ink deposited on the simulated threatening note substantiated the feasibility of combining RS and ATR-FTIR spectroscopy with PPMC coefficient (r) and PCA for successful source determination of red gel pen inks. The development of pigment spectral library had allowed the ink deposited on the threatening note to be identified as XSL Poppy Red (CI Pigment Red 112).
    Matched MeSH terms: Gene Library
  6. Fulltext Regulation of terpenoid biosynthesis by miRNA in Persicaria minor induced by Fusarium oxysporum
    Samad AFA, Rahnamaie-Tajadod R, Sajad M, Jani J, Murad AMA, Noor NM, et al.
    BMC Genomics, 2019 07 16;20(1):586.
    PMID: 31311515 DOI: 10.1186/s12864-019-5954-0
    BACKGROUND: Persicaria minor (kesum) is an herbaceous plant with a high level of secondary metabolite compounds, particularly terpenoids. These terpenoid compounds have well-established roles in the pharmaceutical and food industries. Although the terpenoids of P. minor have been studied thoroughly, the involvement of microRNA (miRNA) in terpenoid regulation remains poorly understood and needs to be explored. In this study, P. minor plants were inoculated with the pathogenic fungus Fusarium oxysporum for terpenoid induction.

    RESULT: SPME GC-MS analysis showed the highest terpenoid accumulation on the 6th day post-inoculation (dpi) compared to the other treatment time points (0 dpi, 3 dpi, and 9 dpi). Among the increased terpenoid compounds, α-cedrene, valencene and β-bisabolene were prominent. P. minor inoculated for 6 days was selected for miRNA library construction using next generation sequencing. Differential gene expression analysis showed that 58 miRNAs belonging to 30 families had significantly altered regulation.
    Among these 58 differentially expressed genes (DEGs), 27 [corrected] miRNAs were upregulated, whereas 31 [corrected] miRNAs were downregulated. Two putative novel pre-miRNAs were identified and validated through reverse transcriptase PCR. Prediction of target transcripts potentially involved in the mevalonate pathway (MVA) was carried out by psRobot software, resulting in four miRNAs: pmi-miR530, pmi-miR6173, pmi-miR6300 and a novel miRNA, pmi-Nov_13. In addition, two miRNAs, miR396a and miR398f/g, were predicted to have their target transcripts in the non-mevalonate pathway (MEP). In addition, a novel miRNA, pmi-Nov_12, was identified to have a target gene involved in green leaf volatile (GLV) biosynthesis. RT-qPCR analysis showed that pmi-miR6173, pmi-miR6300 and pmi-nov_13 were downregulated, while miR396a and miR398f/g were upregulated. Pmi-miR530 showed upregulation at 9 dpi, and dynamic expression was observed for pmi-nov_12. Pmi-6300 and pmi-miR396a cleavage sites were detected through degradome sequence analysis. Furthermore, the relationship between miRNA metabolites and mRNA metabolites was validated using correlation analysis.

    CONCLUSION: Our findings suggest that six studied miRNAs post-transcriptionally regulate terpenoid biosynthesis in P. minor. This regulatory behaviour of miRNAs has potential as a genetic tool to regulate terpenoid biosynthesis in P. minor.

    Matched MeSH terms: Gene Library
  7. Fulltext Data on degradome sequencing and analysis from mock-inoculated and Fusarium oxysporum treated leaves samples in Persicaria minor
    Samad AFA, Sajad M, Jani J, Murad AMA, Ismail I
    Data Brief, 2018 Oct;20:555-557.
    PMID: 30197911 DOI: 10.1016/j.dib.2018.08.034
    Degradome sequencing referred as parallel analysis of RNA ends (PARE) by modifying 5'-rapid amplification of cDNA ends (RACE) with deep sequencing method. Deep sequencing of 5' products allow the determination of cleavage sites through the mapping of degradome fragments against small RNAs (miRNA or siRNA) on a large scale. Here, we carried out degradome sequencing in medicinal plant, Persicaria minor, to identify cleavage sites in small RNA libraries in control (mock-inoculated) and Fusarium oxysporum treated plants. The degradome library consisted of both control and treated samples which were pooled together during library preparation and named as D4. The D4 dataset have been deposited at GenBank under accession number SRX3921398, https://www.ncbi.nlm.nih.gov/sra/SRX3921398.
    Matched MeSH terms: Gene Library
  8. A survey of genes in Eimeria tenella merozoites by EST sequencing
    Wan KL, Chong SP, Ng ST, Shirley MW, Tomley FM, Jangi MS
    Int J Parasitol, 1999 Dec;29(12):1885-92.
    PMID: 10961844
    A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.
    Matched MeSH terms: Gene Library
  9. Fulltext Towards complete and error-free genome assemblies of all vertebrate species
    Rhie A, McCarthy SA, Fedrigo O, Damas J, Formenti G, Koren S, et al.
    Nature, 2021 Apr;592(7856):737-746.
    PMID: 33911273 DOI: 10.1038/s41586-021-03451-0
    High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.
    Matched MeSH terms: Gene Library
  10. Production cross-sections of radionuclides from α-induced reactions on natural copper up to 50MeV
    Usman AR, Khandaker MU, Haba H, Otuka N, Murakami M, Komori Y
    Appl Radiat Isot, 2016 08;114:104-13.
    PMID: 27227905 DOI: 10.1016/j.apradiso.2016.04.027
    The excitation functions were measured for the (nat)Cu(α,x)(66,67)Ga,(65)Zn,(57,58,60)Co reactions in the energy range of 16.5 -50MeV. A conventional stacked-foil activation technique combined with HPGe γ-ray spectrometry was employed to determine cross-sections. The measured cross-sections were critically compared with relevant previous experimental data and also with the evaluated data in the TENDL-2014 library. Present results confirmed some of the previous experimental data, whereas only a partial agreement was found with the evaluated data. The measured data are useful for reducing the existing discrepancies in the literature, to improve the nuclear reaction model codes, and to enrich the experimental database towards various applications.
    Matched MeSH terms: Gene Library
  11. Isolation and cloning of microRNAs from recalcitrant plant tissues with small amounts of total RNA: a step-by step approach
    Yew CW, Kumar SV
    Mol Biol Rep, 2012 Feb;39(2):1783-90.
    PMID: 21625851 DOI: 10.1007/s11033-011-0919-7
    MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
    Matched MeSH terms: Gene Library*
  12. Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus)
    Yusuf NH, Ong WD, Redwan RM, Latip MA, Kumar SV
    Gene, 2015 Oct 15;571(1):71-80.
    PMID: 26115767 DOI: 10.1016/j.gene.2015.06.050
    MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant.
    Matched MeSH terms: Gene Library
  13. Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae: A Step Towards Heterologous Production of Pyrethrins in Fungi
    Mohamed ME, Pahirulzaman KA, Lazarus CM
    Mol Biotechnol, 2016 Mar;58(3):172-8.
    PMID: 26718544 DOI: 10.1007/s12033-015-9911-0
    Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.
    Matched MeSH terms: Gene Library
  14. Fulltext Phosphoinositide-dependent Kinase-1 (PDPK1) regulates serum/glucocorticoid-regulated Kinase 3 (SGK3) for prostate cancer cell survival
    Nalairndran G, Hassan Abdul Razack A, Mai CW, Fei-Lei Chung F, Chan KK, Hii LW, et al.
    J Cell Mol Med, 2020 Oct;24(20):12188-12198.
    PMID: 32926495 DOI: 10.1111/jcmm.15876
    Prostate cancer (PCa) is the most common malignancy and is the second leading cause of cancer among men globally. Using a kinome-wide lentiviral small-hairpin RNA (shRNA) library screen, we identified phosphoinositide-dependent kinase-1 (PDPK1) as a potential mediator of cell survival in PCa cells. We showed that knock-down of endogenous human PDPK1 induced significant tumour-specific cell death in PCa cells (DU145 and PC3) but not in the normal prostate epithelial cells (RWPE-1). Further analyses revealed that PDPK1 mediates cancer cell survival predominantly via activation of serum/glucocorticoid-regulated kinase 3 (SGK3). Knock-down of endogenous PDPK1 in DU145 and PC3 cells significantly reduced SGK3 phosphorylation while ectopic expression of a constitutively active SGK3 completely abrogated the apoptosis induced by PDPK1. In contrast, no such effect was observed in SGK1 and AKT phosphorylation following PDPK1 knock-down. Importantly, PDPK1 inhibitors (GSK2334470 and BX-795) significantly reduced tumour-specific cell growth and synergized docetaxel sensitivity in PCa cells. In summary, our results demonstrated that PDPK1 mediates PCa cells' survival through SGK3 signalling and suggest that inactivation of this PDPK1-SGK3 axis may potentially serve as a novel therapeutic intervention for future treatment of PCa.
    Matched MeSH terms: Gene Library
  15. Phage display antibodies for diagnostic applications
    Hairul Bahara NH, Tye GJ, Choong YS, Ong EB, Ismail A, Lim TS
    Biologicals, 2013 Jul;41(4):209-16.
    PMID: 23647952 DOI: 10.1016/j.biologicals.2013.04.001
    With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.
    Matched MeSH terms: Gene Library*
  16. Fulltext Directed evolution of nucleotide-based libraries using lambda exonuclease
    Lim BN, Choong YS, Ismail A, Glökler J, Konthur Z, Lim TS
    Biotechniques, 2012 Dec;53(6):357-64.
    PMID: 23227986 DOI: 10.2144/000113964
    Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.
    Matched MeSH terms: Gene Library*
  17. Construction of Naive and Immune Human Fab Phage-Display Library
    Omar N, Lim TS
    Methods Mol Biol, 2018;1701:25-44.
    PMID: 29116498 DOI: 10.1007/978-1-4939-7447-4_2
    This protocol describes the processes involved in the generation of human antibody libraries in Fab format. The antibody repertoire is derived from peripheral blood mononucleocytes focusing on different immunoglobulin isotypes. A two-step cloning process was used to generate a diverse human Fab library for subsequent selection by phage display. The method can be applied for the generation of both naive and immune antibody libraries. The naive repertoire allows for the library to be applied for the generation of human monoclonal antibodies against a broad range of target antigens making it a useful resource for antibody generation. However, the immune repertoire will be focused against target antigens from a particular disease. The protocol will focus on the generation of the library including the panning process.
    Matched MeSH terms: Gene Library*
  18. Construction of Naïve and Immune Human Fab Phage Display Library
    Lai JY, Lim TS
    Methods Mol Biol, 2023;2702:39-58.
    PMID: 37679614 DOI: 10.1007/978-1-0716-3381-6_3
    Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in any phage display experiment is the availability of an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature of the library. Naïve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be applied for the construction of either a naïve or immune human antibody library in Fab format followed by the subsequent panning.
    Matched MeSH terms: Gene Library
  19. Fulltext Differential expression patterns of leukaemia associated genes in leukaemia cell lines compared to healthy controls
    Ang Pei-Shen, Rajesh Ramasamy, Noor Hamidah Hussin, Cheong Soon-Keng, Seow Heng-Fong, Maha Abdullah
    Malaysian Journal of Medicine and Health Sciences, 2016;12(1):0-0.
    MyJurnal
    Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may
    have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We
    previously established two subtracted cDNA libraries with differentially expressed genes from an
    acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To
    compare gene expression of the leukaemia associated genes with selected biological characteristics
    in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated
    with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/
    hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin
    remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid
    (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells
    from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell
    cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine
    drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia
    cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation
    (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only
    two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and
    PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell
    lines. No significant difference was observed between myeloid cell lines and healthy controls. This
    may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle.
    G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture
    while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had
    an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1,
    was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast,
    MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed
    more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus,
    B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes.
    Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be
    useful markers to study biological differences including drug resistance between lineages.
    Matched MeSH terms: Gene Library
  20. Fulltext Haplotype divergence and multiple candidate genes at Rphq2, a partial resistance QTL of barley to Puccinia hordei
    Yeo FK, Wang Y, Vozabova T, Huneau C, Leroy P, Chalhoub B, et al.
    Theor Appl Genet, 2016 Feb;129(2):289-304.
    PMID: 26542283 DOI: 10.1007/s00122-015-2627-5
    Rphq2, a minor gene for partial resistance to Puccinia hordei , was physically mapped in a 188 kbp introgression with suppressed recombination between haplotypes of rphq2 and Rphq2 barley cultivars.
    Matched MeSH terms: Gene Library
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