METHODS AND RESULTS: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants.

METHODS AND RESULTS: Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura × Pisifera (D × P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21 years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279.

METHODS AND RESULTS: Ten primers of each, ISSR and DAMD molecular markers, were used to assess the genetic stability of the eight samples of Ficus carica var. Black Jack. ISSR markers showed 97.87% of monomorphism whereas DAMD markers showed 100% monomorphism. Polymorphism of 2.13% was observed for the UBC840 ISSR-DNA primer which was negated under the genetic similarity index analysis for the eight samples. The findings of this study revealed that ISSR and DAMD markers are efficient in determining the polymorphism and monomorphism percentage among the in vitro and ex vitro samples of Ficus carica var. Black Jack.

METHODOLOGY: In this study, 47 GDM patients and 40 age-matched controls were genotyped for rs10946398 CDKAL1 variant using Tetra primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra ARMS-PCR).

METHODS: We analysed the cancerous and adjacent non-cancerous formalin-fixed paraffin embedded (FFPE) tissue of 83 CRC patients from a single medical centre in Malaysia. TaqMan probe-based qPCR targeting the 16S rRNA gene was used to detect the presence of FN in the extracted FFPE DNA. The differences in FN expression between cancer and non-cancer tissues were evaluated. Association studies between FN infection in the tumour and relative FN abundance with available clinical data were conducted.

RESULTS: FN was more abundant in the cancerous tissue compared to non-cancerous tissue (p = 0.0025). FN infection in the tumour was significantly associated with lymph node metastasis (p = 0.047) and cancer staging (p = 0.032), but not with other clinicopathologic variables. In double-positive patients where FN was detected in both cancerous and non-cancerous tissue, the expression fold-change of FN, calculated using 2-ΔΔCT formula, was significantly higher in patients with tumour size equal to or greater than 5 cm (p = 0.033) and in KRAS-mutated patients (p = 0.046).

METHODS AND RESULTS: To explore role of neddylation, HBV-replicating HepG2.2.15.7 cells and HBV-infected HepG2-hNTCP-30 cells were treated with siNEDD8 and MLN4924, a potent and selective NEDD8-activating enzyme inhibitor. Cell viability, intracellular and extracellular HBV DNA, covalently closed circular DNA (cccDNA), HBsAg, HBeAg, and HBcrAg were measured to assess the consequences of the various treatments on viral replication. Our data showed that HBV infection increased NEDD8 expression in human liver cell lines. Symmetrically, NEDD8 knockdown by siRNA or MLN4924 treatments decreased HBV replication in HepG2.2.15.7 and HepG2-hNTCP-30 cells. Notably, HBsAg, and HBeAg secretions were strongly suppressed in the culture supernatants, but not the HBcrAg. These results indicate that the suppression of NEDD8 decreases HBV replication. However, cccDNA steady level confirms once again its persistence and longevity in chronic infection.