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.

METHODS: The BDNF target sequence was detected on a capture probe attached on aluminum microcomb electrodes on the silicon wafer surface. A capture-target-reporter sandwich-type assay was performed to enhance the detection of the BDNF target.

RESULTS: The limit of detection was noticed to be 100 aM. Input of a reporter sequence at concentrations >10 aM improved the detection of the target sequence by enhancing changes in the generated currents. Control experiments with noncomplementary and single- and triple-mismatches of target and reporter sequences did not elicit changes in current levels, indicating the selective detection of the BDNF gene sequence.

METHODS: Polymerase chain reaction (PCR) was used to detect the presence of sasX, qacA/B and mupA genes from 47 paired MRSA isolates. A paired isolate was defined as one nasal swab (colonising) isolate and clinical isolate that caused infection in the same patient. 22 selected paired isolates were subjected to multilocus sequence typing (MLST). The genetic relatedness among the isolates and association between the putative genes with epidemic sequence types (STs) were investigated.

RESULTS: 7 (14.9%, n = 14) paired isolates were positive for the sasX gene. qacA/B genes were positive in 7.4% (n = 7) of the isolates, from three paired isolates and one clinical isolate whose paired colonising isolate was negative. The paired sample of three patients were positive for both genes. The mupA gene was not detected in all the isolates. MLST revealed two epidemic STs, ST22 and ST239, and a novel ST4649. sasX and qacA/B genes were found in ST239 in 29.5% (n = 13) and 13.6% (n = 6) of cases, respectively. Gene co-existence occurred in 13.6% (n = 6) of MRSA ST239 and 2.3% (n = 1) of MRSA ST4649.

AREAS COVERED: Literature was searched in different resources for eligible studies. The pooled risk ratio was measured using RevMan software, with p<0.05 (two-sided) set as statistically significant.

METHODS: Microbiological samples from 71 subjects with infected root canals were aseptically collected, and cultured on Sabouraud dextrose agar, and C. albicans was identified using multiplex polymerase chain reaction, and the isolates were further subtyped using ABC genotyping system. Their relative virulence was compared using further four archival samples of endodontic origin from another geographical region, and four more salivary isolates, as controls. The virulence attributes compared were biofilm formation, and production of phospholipase and haemolysin, and the susceptibility to nystatin, amphotericin B, ketoconazole, and fluoconazole was also tested.

METHOD: Information on demographic characteristics, dietary vitamin D intake from supplement and food, time spent outdoors, skin type and clothing were collected using a questionnaire. Plasma total 25-hydroxyvitamin D (25OHD) levels were measured using an Ultra-High-Performance Liquid Chromatography (UHPLC). Maternal GC single nucleotide polymorphisms (SNPs) (rs4588 and rs7041) were determined using restriction fragment length polymorphism (RFLP) technique.

Materials and Methods: Patients with opioid dependence (n = 148) were recruited from MMT clinics. Pain sensitivity, severity of the opiate withdrawal syndrome, and sleep quality were assessed using cold pressor test (CPT), Subjective Opiate Withdrawal Scale (SOWS-M), and Pittsburgh Sleep Quality Index (PSQI)-Malay, respectively. Deoxyribonucleic acid (DNA) was extracted from whole blood, and then was used for genotyping of Val96Ala, Leu141Leu, Val154Ile, Pro310Ser, Ser311Cys, TaqI A, -141C Ins/Del, and A-241G polymorphisms.

Results: Among 148 patients, 8.1% (n = 12), 60.8% (n = 90), 27.7% (n = 41), and 29.1% (n = 43) had at least one risk allele for Ser311Cys, TaqI A, -141C Ins/Del, and A-241G polymorphisms, respectively. There were no significant differences in pain responses (pain threshold, tolerance, and intensity), SOWS, and PSQI scores between DRD2 polymorphisms.