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Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes

Chuman Y, Nobuhisa I, Ogawa T, Deshimaru M, Chijiwa T, Tan NH, et al.

Toxicon, 2000 Mar;38(3):449-62.
PMID: 10669032

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.

Matched MeSH terms: Gene Library
Identification of chromosome 21 materials using the whole chromosome 21 specific library

Isa MN, Boyd E, Turner TL, Tolmie J, Connor JM

Southeast Asian J. Trop. Med. Public Health, 1995;26 Suppl 1:92-5.
PMID: 8629150

The chromosome in situ suppression hybridization or chromosome painting technic was applied to confirm and eliminate the markers involving chromosome 21 segments using a chromosome 21 DNA library. The library ATCCLL21SNO2 was amplified, directly biotinylated using the polymerase chain reaction. The results demonstrated a translocation of chromosome 21 material on chromosome 2 and X and eliminate the origin of the marker. Thus, the technique provides an important tool to complement the conventional G-banding technic.

Matched MeSH terms: Gene Library
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
Fulltext Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis

Arifin N, Yunus MH, Nolan TJ, Lok JB, Noordin R

Am J Trop Med Hyg, 2018 04;98(4):1165-1170.
PMID: 29436335 DOI: 10.4269/ajtmh.17-0697

Strongyloides stercoralis is a human parasite that can cause a long-term infection. In immunosuppressed patients, strongyloidiasis may be fatal when there is overwhelming autoinfection resulting in the migration of large numbers of larvae through many organs. Definitive diagnosis is still a challenge, and a combination of symptoms, microscopic identification, and serology test results are often used to arrive at a clinical decision. However, intermittent larval excretion, low parasite burden, and occult infections are challenges with parasitological diagnosis of infection with S. stercoralis. Meanwhile, serologic tests using immunoglobulin G and parasite antigen extract have problems of cross-reactivity with other helminthic infections. Recombinant antigen-based serodiagnosis is a good alternative to overcome the laboratory diagnostic issues. Herein, we report on the isolation of cDNA clone encoding an antigen of potential diagnostic value identified from immunoscreening of a S. stercoralis cDNA library. The translated protein had highest similarity to Strongyloides ratti immunoglobulin-binding protein 1. The recombinant antigen produced, rSs1a, was assessed using western blot and enzyme-linked immunosorbent assay. The latter showed 96% diagnostic sensitivity and 93% specificity; thus, rSs1a has good potential for use in serodiagnosis of human strongyloidiasis.

Matched MeSH terms: Gene Library
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
Fulltext DNA-based taxonomy of a mangrove-associated community of fishes in Southeast Asia

Zainal Abidin DH, Mohd Nor SA, Lavoué S, A Rahim M, Jamaludin NA, Mohammed Akib NA

Sci Rep, 2021 Sep 07;11(1):17800.
PMID: 34493747 DOI: 10.1038/s41598-021-97324-1

The Merbok Estuary comprises one of the largest remaining mangrove forests in Peninsular Malaysia. Its value is significant as it provides important services to local and global communities. It also offers a unique opportunity to study the structure and functioning of mangrove ecosystems. However, its biodiversity is still partially inventoried, limiting its research value. A recent checklist based on morphological examination, reported 138 fish species residing, frequenting or subject to entering the Merbok Estuary. In this work, we reassessed the fish diversity of the Merbok Estuary by DNA barcoding 350 specimens assignable to 134 species initially identified based on morphology. Our results consistently revealed the presence of 139 Molecular Operational Taxonomic Units (MOTUs). 123 of them are congruent with morphology-based species delimitation (one species = one MOTU). In two cases, two morphological species share the same MOTU (two species = one MOTU), while we unveiled cryptic diversity (i.e. COI-based genetic variability > 2%) within seven other species (one species = two MOTUs), calling for further taxonomic investigations. This study provides a comprehensive core-list of fish taxa in Merbok Estuary, demonstrating the advantages of combining morphological and molecular evidence to describe diverse but still poorly studied tropical fish communities. It also delivers a large DNA reference collection for brackish fishes occurring in this region which will facilitate further biodiversity-oriented research studies and management activities.

Matched MeSH terms: Gene Library
Next generation sequencing-data analysis for cellulose- and xylan-degrading enzymes from pome metagenome

Farah Fadwa Benbelgacem, Oualid Abdelkader Bellag, Adibah Parman, Ibrahim Ali Noorbatcha, Mohd Noor Mat Isa, Muhammad Alfatih Muddathir Abdelrahim, et al.

Sains Malaysiana, 2018;47:2951-2960.

Metagenomic DNA library from palm oil mill effluent (POME) was constructed and subjected to high-throughput screening
to find genes encoding cellulose- and xylan-degrading enzymes. DNA of 30 positive fosmid clones were sequenced with next
generation sequencing technology and the raw data (short insert-paired) was analyzed with bioinformatic tools. First,
the quality of 64,821,599 reverse and forward sequences of 101 bp length raw data was tested using Fastqc and SOLEXA.
Then, raw data filtering was carried out by trimming low quality values and short reads and the vector sequences were
removed and again the output was checked and the trimming was repeated until a high quality read sets was obtained.
The second step was the de novo assembly of sequences to reconstruct 2900 contigs following de Bruijn graph algorithm.
Pre-assembled contigs were arranged in order, the distances between contigs were identified and oriented with SSPACE,
where 2139 scaffolds have been reconstructed. 16,386 genes have been identified after gene prediction using Prodigal
and putative ID assignment with Blastp vs NR protein. The acceptable strategy to handle metagenomic NGS-data in order
to detect known and potentially unknown genes is presented and we showed the computational efficiency of de Bruijn
graph algorithm of de novo assembly to 21 bioprospect genes encoding cellulose-degrading enzymes and 6 genes
encoding xylan-degrading enzymes of 30.3% to 100% identity percentage.

Matched MeSH terms: Gene Library
Culture-independent-based analyses identified site-specific microbial in two oil-wells from Malaysia

Cheng-Yee Fish-Low, Chee HY, Ainon Hamzah

Sains Malaysiana, 2015;44:1625-1633.

Microbial communities of two oil reservoirs from Malaysia, denoted as Platform Bo and Platform Pe were studied using
culture-independent approach. Environmental DNA was extracted and the universal amplified ribosomal region (UARR)
was target amplified for both prokaryotes and eukaryotes. The amplified products were purified and cloned into pTZ57R/T
vector to construct the 16S/18S rDNA library. Restriction endocucleases HhaI and MspI were used to screen the library.
From that, 125 and 253 recombinant plasmid representative clones from Platform Bo and Platform Pe, respectively, were
sent for DNA sequencing. Twenty-six operational taxonomic units (OTUs) consist of 20 genera detected at Platform Bo
and 17 OTUs consist of 13 genera detected at Platform Pe. Marinobacter and Acinetobacter species co-occurred in both
platforms whereas the rest are site-specific. Gammaproteobacteria accounted for 86.0% of the microbial community in
Platform Bo, where OTUs affiliated to Marinobacter, Pseudomonas and Marinobacterium that were the most abundant. The
major OTUs in the Platform Pe were with affinities to Achromobacter, followed by Stenotrophomonas and Serratia. The
only archaeal isolates were detected in Platform Pe, which affiliated to Thermocladium. The singletons and doubletons
accounted for about 50.0% of the OTU abundance in both platforms, which considerably significant despite their rare
occurrence.

Matched MeSH terms: Gene Library
Identification and characterisation of a copper-inducible metallothionein gene from cockle, Anadara granosa.

Wong KK, Noor-Arniwati Mat-Daud, Roohaida Othman, Zubir Din, Wan KL, Salmijah Surif

Sains Malaysiana, 2009;38.

The cockle, Anadara granosa, was experimentally exposed to low (0.1 mg/L) and sublethal (1.0 mg/L) doses of copper (Cu) for a period of 24 hrs. Significant increase in Cu concentrations in whole tissues and hepatopancreas compared to control animals were observed. In order to study the effect of copper exposure at molecular levels, a subtractive cDNA library was constructed from the hepatopancreas of cockles exposed to 1.0 mg/L Cu. Screening of the subtractive cDNA library using reverse northern analysis resulted in several differentially expressed genes, including one that codes for metallothionein (MT). The complete coding sequence of the MT gene (designated as AnaMT2) reveals an open reading frame of 234 bp in length that encodes a 77 amino acid polypeptide as revealed by the deduced amino acid composition. Although showing similarities with other molluscan MTs, AnaMT2 can be distinguished by its lower glycine and higher asparagine and proline content. Expression analysis of the AnaMT2 by northern analysis indicated higher mRNA level in cockle exposed to 1.0 mg/L Cu and was undetectable in those treated with 0.1 mg/L. This suggests that AnaMT2 represents a primarily inducible MT not highly expressed under basal conditions.

Matched MeSH terms: Gene Library
Optimization of illumina truseq targeted RNA Expression (TREx) library quality

Nadiah Abu, Noraini Nordin, Noorjahan Banu Alitheen, Nadiah Abu, Sheau Wei Tan, Swee Keong Yeap, et al.

Sains Malaysiana, 2018;47:303-308.

RNA-seq has become an essential tool in molecular research. Nevertheless, application of RNA-seq was limited by cost and technical difficulties. Illumina has introduced the cost effective and ease to handle Truseq Targeted RNA Sequencing. In this study, we present the requirements and the optimization procedure for this Truseq Targeted RNA sequencing on cell line. Total RNA was recommended as starting materials but it required optimization including additional purification step and adjusting the AMPure beads ratio to eliminate unwanted contaminants. This can be resolved by using PolyA-enriched mRNA as starting material. TREx is a useful assay to evaluate gene expression. Quality library of TREx can be prepared by adding multiple washing steps or changing input sample to mRNA.

Matched MeSH terms: Gene Library
Inhibitors of leishmania mexicana phosphoglycerate mutase identified by virtual screening and verified by inhibition studies

Fazia Adyani Ahmad Fuad, Houston Douglas R, Michels Paul AM, Fothergill-gilmore Linda A, Walkinshaw Malcolm D

Sains Malaysiana, 2016;45:1113-1120.

Cofactor-independent phosphoglycerate mutase has been proposed as a therapeutic target for the treatment of
trypanosomatid diseases. In this paper, we report the identification of compounds that could potentially be developed as
selective inhibitors of cofactor-independent phosphoglycerate mutase from Leishmania mexicana (LmiPGAM). Virtual
screening was used in this search, as well as compounds identified by high-throughput screening. A ligand-based virtual
screen programme, ultra fast shape recognition with atom types (UFSRAT), was used to screen for compounds resembling
the substrate/product, before a structure-based approach was applied using AutoDock 4 and AutoDock Vina in a consensus
docking scheme. In this way eight selected compounds were identified. In addition, three compounds from the Library of
Pharmacologically Active Compounds (LOPAC) were selected from the published results of high-throughput screening of
this library. The inhibitory effects of these compounds were tested at a fixed concentration of 1 mM. The results showed
that seven compounds inhibited LmiPGAM activity and of these, two compounds (one each from high-throughput and
virtual screening) showed substantial inhibition (i.e. 14% and 49% remaining activity, respectively). Taken together, the
findings from this study indicate that these compounds have potential as novel inhibitors that specifically target LmiPGAM.

Matched MeSH terms: Gene Library
Fulltext Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF

Balasubramaniam VR, Hong Wai T, Ario Tejo B, Omar AR, Syed Hassan S

PLoS One, 2013;8(9):e72429.
PMID: 24073193 DOI: 10.1371/journal.pone.0072429

We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

Matched MeSH terms: Gene Library
Fulltext A checklist of the bats of Peninsular Malaysia and progress towards a DNA barcode reference library

Lim VC, Ramli R, Bhassu S, Wilson JJ

PLoS One, 2017;12(7):e0179555.
PMID: 28742835 DOI: 10.1371/journal.pone.0179555

Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD) found that 86 (of the 110) species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs) by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities.

Matched MeSH terms: Gene Library
Kernel-specific cDNA clones encoding three different isoforms of seed storage protein glutelin from oil palm Elaeis guineensis

Cha TS, Habib Shah F

Plant Sci, 2001 Apr;160(5):913-923.
PMID: 11297788

The mRNA differential display method was used to identify and isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the kernel of Elaeis guineensis, var. Tenera. We successfully isolated two cDNA clones, KT7 (312 bp) and KT8 (266 bp). Interestingly, both clones show 79% nucleotide sequence identity to each other. This suggests that both clones encode the isoforms of the same protein. We screened the kernel (15 WAA) cDNA library and isolated the clone pKT7 (587 bp) using KT7 as probe, and isolated another isoform with KT8 probe, which designated as pKT9 (900 bp). Clone pKT9 has 93% nucleotide identity to KT8 and only 46% to pKT7 in their 3'-untranslated region. All three clones displayed significant amino acid sequence identity to seed storage protein glutelin from monocotyledon and globulin from dicotyledon plants. The coding sequence of KT8 (106 bp) shows 76 and 97% identity to pKT9 and pKT7, respectively. Therefore, we suggest that clones KT8 and pKT7 are members of the same subfamily (A), while pKT9 belongs to another subfamily (B) of glutelin multigene families. Southern analysis shows that there are at least four members for the subfamily B. Northern analysis shows that these three members of the glutelin family are co-ordinately expressed and developmentally regulated during the development of the kernel. The transcripts begin to accumulate at 12 WAA, increase in 15 WAA and show a significant reduction at 17 WAA.

Matched MeSH terms: Gene Library
A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase

Shah FH, Cha TS

Plant Sci, 2000 May 29;154(2):153-160.
PMID: 10729614

The differential display method was used to isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the mesocarp of two oil palms, Elaeis oleifera and Elaeis guineensis, Tenera. DNA-free total RNA from mesocarp and kernel of E. guineensis, Tenera and E. oleifera (15 WAA) were used to obtain differential gene expression patterns between these tissues from the two species. In this report, we describe the isolation and characterization of a specific cDNA clone, MO1 (434 bp) which was shown to be mesocarp-specific as well as species-specific for E. oleifera Sequencing of this fragment showed homology to the enzyme sesquiterpene synthase. Its longer cDNA clone, pMO1 (1072 bp), isolated from a 15-week E. oleifera mesocarp cDNA library confirmed that it encodes for sesquiterpene synthase. The complete sequence of 1976 bp was obtained using 5'RACE method. Northern hybridization showed that MO1 and pMO1 mRNA transcripts are highly expressed only in the mesocarp of E. oleifera from 5 to 20 WAA. No expression was detected in the kernel (12-17 WAA) and vegetative tissues of both species nor in the mesocarp of E. guineensis. This is the first communication to document on the isolation and characterisation of a mesocarp-and species-specific cDNA clone from oil palm.

Matched MeSH terms: Gene Library
Fulltext Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

Ho WS, Pang SL, Abdullah J

Physiol Mol Biol Plants, 2014 Jul;20(3):393-7.
PMID: 25049467 DOI: 10.1007/s12298-014-0230-x

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5' EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests.

Matched MeSH terms: Gene Library
Fulltext Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools

Amelia K, Singh J, Shah FH, Bhore SJ

Pharmacognosy Res, 2015 Apr-Jun;7(2):209-12.
PMID: 25829797 DOI: 10.4103/0974-8490.150536

Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM) gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it.

Matched MeSH terms: Gene Library
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
Fulltext DNA barcoding of commercially important reef fishes in Weh Island, Aceh, Indonesia

Fadli N, Mohd Nor SA, Othman AS, Sofyan H, Muchlisin ZA

PeerJ, 2020;8:e9641.
PMID: 32844060 DOI: 10.7717/peerj.9641

Knowledge on the precise identification of fish resources is critical for sustainable fisheries management. This study employs the DNA barcoding approach to generate a molecular taxonomic catalogue of commercially important reef fishes in the waters of Weh Island (Aceh Province), the most northerly inhabited island in the biodiverse Indonesian Archipelago. The waters not only support artisanal fisheries but also a feeder for the industry in the greater island of Aceh. In total, 230 specimens from 72 species belonging to 32 genera and 17 families were DNA barcoded, representing a major segment of the captured reef fish taxa and a quarter of fish species diversity that had previously been recorded. The sequence read lengths were 639 bp revealing 359 conserved sites, 280 variable sites, 269 parsimony informative and 11 singletons. Our molecular findings paralleled the morphological identification with no evidence of cryptic species or new species discovery. This study is a significant contribution to the fisheries statistics of this area, which would facilitate assessment of species catch composition and hence for strategizing management plans. It is an important input to the DNA barcode library of Indonesian marine fishes and to the global DNA barcode entries in general.

Matched MeSH terms: Gene Library
Fulltext A new insight to biomarkers related to resistance in survived-white spot syndrome virus challenged giant tiger shrimp, Penaeus monodon

Mohd Ghani F, Bhassu S

PeerJ, 2019;7:e8107.
PMID: 31875142 DOI: 10.7717/peerj.8107

The emergence of diseases such as white spot disease has become a threat to Penaeus monodon cultivation. Although there have been a few studies utilizing RNA-Seq, the cellular processes of host-virus interaction in this species remain mostly anonymous. In the present study, P. monodon was challenged with WSSV by intramuscular injection and survived for 12 days. The effect of the host gene expression by WSSV infection in the haemocytes, hepatopancreas and muscle of P. monodon was studied using Illumina HiSeq 2000. The RNA-Seq of cDNA libraries was developed from surviving WSSV-challenged shrimp as well as from normal healthy shrimp as control. A comparison of the transcriptome data of the two groups showed 2,644 host genes to be significantly up-regulated and 2,194 genes significantly down-regulated as a result of the infection with WSSV. Among the differentially expressed genes, our study discovered HMGB, TNFSF and c-Jun in P. monodon as new potential candidate genes for further investigation for the development of potential disease resistance markers. Our study also provided significant data on the differential expression of genes in the survived WSSV infected P. monodon that will help to improve understanding of host-virus interactions in this species.

Matched MeSH terms: Gene Library

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