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Fulltext Transcriptome analysis of Streptococcus pneumoniae treated with the designed antimicrobial peptides, DM3

Le CF, Gudimella R, Razali R, Manikam R, Sekaran SD

Sci Rep, 2016 05 26;6:26828.
PMID: 27225022 DOI: 10.1038/srep26828

In our previous studies, we generated a short 13 amino acid antimicrobial peptide (AMP), DM3, showing potent antipneumococcal activity in vitro and in vivo. Here we analyse the underlying mechanisms of action using Next-Generation transcriptome sequencing of penicillin (PEN)-resistant and PEN-susceptible pneumococci treated with DM3, PEN, and combination of DM3 and PEN (DM3PEN). DM3 induced differential expression in cell wall and cell membrane structural and transmembrane processes. Notably, DM3 altered the expression of competence-induction pathways by upregulating CelA, CelB, and CglA while downregulating Ccs16, ComF, and Ccs4 proteins. Capsular polysaccharide subunits were downregulated in DM3-treated cells, however, it was upregulated in PEN- and DM3PEN-treated groups. Additionally, DM3 altered the amino acids biosynthesis pathways, particularly targeting ribosomal rRNA subunits. Downregulation of cationic AMPs resistance pathway suggests that DM3 treatment could autoenhance pneumococci susceptibility to DM3. Gene enrichment analysis showed that unlike PEN and DM3PEN, DM3 treatment exerted no effect on DNA-binding RNA polymerase activity but observed downregulation of RpoD and RNA polymerase sigma factor. In contrast to DM3, DM3PEN altered the regulation of multiple purine/pyrimidine biosynthesis and metabolic pathways. Future studies based on in vitro experiments are proposed to investigate the key pathways leading to pneumococcal cell death caused by DM3.
Fulltext Transcripts and MicroRNAs Responding to Salt Stress in Musa acuminata Colla (AAA Group) cv. Berangan Roots

Lee WS, Gudimella R, Wong GR, Tammi MT, Khalid N, Harikrishna JA

PLoS One, 2015;10(5):e0127526.
PMID: 25993649 DOI: 10.1371/journal.pone.0127526

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana.
Fulltext Identifying protein biomarkers in predicting disease severity of dengue virus infection using immune-related protein microarray

Soe HJ, Yong YK, Al-Obaidi MMJ, Raju CS, Gudimella R, Manikam R, et al.

Medicine (Baltimore), 2018 Feb;97(5):e9713.
PMID: 29384851 DOI: 10.1097/MD.0000000000009713

Dengue virus is one of the most widespread flaviviruses that re-emerged throughout recent decades. The progression from mild dengue to severe dengue (SD) with the complications such as vascular leakage and hemorrhage increases the fatality rate of dengue. The pathophysiology of SD is not entirely clear. To investigate potential biomarkers that are suggestive of pathogenesis of SD, a small panel of serum samples selected from 1 healthy individual, 2 dengue patients without warning signs (DWS-), 2 dengue patients with warning signs (DWS+), and 5 patients with SD were subjected to a pilot analysis using Sengenics Immunome protein array. The overall fold changes of protein expressions and clustering heat map revealed that PFKFB4, TPM1, PDCL3, and PTPN20A were elevated among patients with SD. Differential expression analysis identified that 29 proteins were differentially elevated greater than 2-fold in SD groups than DWS- and DWS+. From the 29 candidate proteins, pathways enrichment analysis also identified insulin signaling and cytoskeleton pathways were involved in SD, suggesting that the insulin pathway may play a pivotal role in the pathogenesis of SD.
Fulltext Comparative genomic analysis of ten clinical Streptococcus pneumoniae collected from a Malaysian hospital reveal 31 new unique drug-resistant SNPs using whole genome sequencing

Jindal HM, Ramanathan B, Le CF, Gudimella R, Razali R, Manikam R, et al.

J Biomed Sci, 2018 Feb 15;25(1):15.
PMID: 29448938 DOI: 10.1186/s12929-018-0414-8

BACKGROUND: Streptococcus pneumoniae or pneumococcus is a leading cause of morbidity and mortality worldwide, specifically in relation to community-acquired pneumonia. Due to the overuse of antibiotics, S. pneumoniae has developed a high degree of resistance to a wide range of antibacterial drugs.

METHODS: In this study, whole genome sequencing (WGS) was performed for 10 clinical strains of S. pneumoniae with different levels of sensitivity to standard antibiotics. The main objective was to investigate genetic changes associated with antibiotic resistance in S. pneumoniae.

RESULTS: Our results showed that resistant isolates contain a higher number of non-synonymous single nucleotide polymorphisms (SNPs) as compared to susceptible isolates. We were able to identify SNPs that alter a single amino acid in many genes involved in virulence and capsular polysaccharide synthesis. In addition, 90 SNPs were only presented in the resistant isolates, and 31 SNPs were unique and had not been previously reported, suggesting that these unique SNPs could play a key role in altering the level of resistance to different antibiotics.

CONCLUSION: Whole genome sequencing is a powerful tool for comparing the full genome of multiple isolates, especially those closely related, and for analysing the variations found within antibiotic resistance genes that lead to differences in antibiotic sensitivity. We were able to identify specific mutations within virulence genes related to resistant isolates. These findings could provide insights into understanding the role of single nucleotide mutants in conferring drug resistance.

Study site: University Malaya Medical Centre (UMMC)
Fulltext Molecular characterization and comparative sequence analysis of defense-related gene, Oryza rufipogon receptor-like protein kinase 1

Law YS, Gudimella R, Song BK, Ratnam W, Harikrishna JA

Int J Mol Sci, 2012;13(7):9343-9362.
PMID: 22942769 DOI: 10.3390/ijms13079343

Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression.
Fulltext Augmentation of Autoantibodies by Helicobacter pylori in Parkinson's Disease Patients May Be Linked to Greater Severity

Suwarnalata G, Tan AH, Isa H, Gudimella R, Anwar A, Loke MF, et al.

PLoS One, 2016;11(4):e0153725.
PMID: 27100827 DOI: 10.1371/journal.pone.0153725

Parkinson's disease (PD) is the second most common chronic and progressive neurodegenerative disorder. Its etiology remains elusive and at present only symptomatic treatments exists. Helicobacter pylori chronically colonizes the gastric mucosa of more than half of the global human population. Interestingly, H. pylori positivity has been found to be associated with greater of PD motor severity. In order to investigate the underlying cause of this association, the Sengenics Immunome protein array, which enables simultaneous screening for autoantibodies against 1636 human proteins, was used to screen the serum of 30 H. pylori-seropositive PD patients (case) and 30 age- and gender-matched H. pylori-seronegative PD patients (control) in this study. In total, 13 significant autoantibodies were identified and ranked, with 8 up-regulated and 5 down-regulated in the case group. Among autoantibodies found to be elevated in H. pylori-seropositive PD were included antibodies that recognize Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initiation factor 4A3 (eIFA3). The presence of elevated autoantibodies against proteins essential for normal neurological functions suggest that immunomodulatory properties of H. pylori may explain the association between H. pylori positivity and greater PD motor severity.
Fulltext Deciphering the Draft Genome of Toxoplasma gondii RH Strain

Lau YL, Lee WC, Gudimella R, Zhang G, Ching XT, Razali R, et al.

PLoS One, 2016;11(6):e0157901.
PMID: 27355363 DOI: 10.1371/journal.pone.0157901

Toxoplasmosis is a widespread parasitic infection by Toxoplasma gondii, a parasite with at least three distinct clonal lineages. This article reports the whole genome sequencing and de novo assembly of T. gondii RH (type I representative strain), as well as genome-wide comparison across major T. gondii lineages. Genomic DNA was extracted from tachyzoites of T. gondii RH strain and its identity was verified by PCR and LAMP. Subsequently, whole genome sequencing was performed, followed by sequence filtering, genome assembly, gene annotation assignments, clustering of gene orthologs and phylogenetic tree construction. Genome comparison was done with the already archived genomes of T. gondii. From this study, the genome size of T. gondii RH strain was found to be 69.35Mb, with a mean GC content of 52%. The genome shares high similarity to the archived genomes of T. gondii GT1, ME49 and VEG strains. Nevertheless, 111 genes were found to be unique to T. gondii RH strain. Importantly, unique genes annotated to functions that are potentially critical for T. gondii virulence were found, which may explain the unique phenotypes of this particular strain. This report complements the genomic archive of T. gondii. Data obtained from this study contribute to better understanding of T. gondii and serve as a reference for future studies on this parasite.
Fulltext Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa

Ramanathan B, Jindal HM, Le CF, Gudimella R, Anwar A, Razali R, et al.

PLoS One, 2017;12(8):e0182524.
PMID: 28797043 DOI: 10.1371/journal.pone.0182524

Rapid progress in next generation sequencing and allied computational tools have aided in identification of single nucleotide variants in genomes of several organisms. In the present study, we have investigated single nucleotide polymorphism (SNP) in ten multi-antibiotic resistant Pseudomonas aeruginosa clinical isolates. All the draft genomes were submitted to Rapid Annotations using Subsystems Technology (RAST) web server and the predicted protein sequences were used for comparison. Non-synonymous single nucleotide polymorphism (nsSNP) found in the clinical isolates compared to the reference genome (PAO1), and the comparison of nsSNPs between antibiotic resistant and susceptible clinical isolates revealed insights into the genome variation. These nsSNPs identified in the multi-drug resistant clinical isolates were found to be altering a single amino acid in several antibiotic resistant genes. We found mutations in genes encoding efflux pump systems, cell wall, DNA replication and genes involved in repair mechanism. In addition, nucleotide deletions in the genome and mutations leading to generation of stop codons were also observed in the antibiotic resistant clinical isolates. Next generation sequencing is a powerful tool to compare the whole genomes and analyse the single base pair variations found within the antibiotic resistant genes. We identified specific mutations within antibiotic resistant genes compared to the susceptible strain of the same bacterial species and these findings may provide insights to understand the role of single nucleotide variants in antibiotic resistance.
Fulltext Genome wide profiling in oral squamous cell carcinoma identifies a four genetic marker signature of prognostic significance

Vincent-Chong VK, Salahshourifar I, Woo KM, Anwar A, Razali R, Gudimella R, et al.

PLoS One, 2017;12(4):e0174865.
PMID: 28384287 DOI: 10.1371/journal.pone.0174865

BACKGROUND: Cancers of the oral cavity are primarily oral squamous cell carcinomas (OSCCs). Many of the OSCCs present at late stages with an exceptionally poor prognosis. A probable limitation in management of patients with OSCC lies in the insufficient knowledge pertaining to the linkage between copy number alterations in OSCC and oral tumourigenesis thereby resulting in an inability to deliver targeted therapy.
OBJECTIVES: The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes.
MATERIALS AND METHODS: Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software.
RESULTS: Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC.
CONCLUSION: Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways.
Exosome autoantibody biomarkers for detection of lung cancer

Thuya WL, Peyper JM, Myen TT, Anuar ND, Anwar A, Gudimella R, et al.

Mil Med Res, 2024 Nov 18;11(1):72.
PMID: 39558443 DOI: 10.1186/s40779-024-00575-y