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Fulltext Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro

Rahim NFC, Hussin Y, Aziz MNM, Mohamad NE, Yeap SK, Masarudin MJ, et al.

Molecules, 2021 Feb 26;26(5).
PMID: 33652694 DOI: 10.3390/molecules26051261

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012-2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC50) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.

Matched MeSH terms: Apoptosis Regulatory Proteins/genetics
Fulltext Collagen type I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media

Akhir HM, Teoh PL

Biosci Rep, 2020 12 23;40(12).
PMID: 33245097 DOI: 10.1042/BSR20201325

Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen.

Matched MeSH terms: CCAAT-Enhancer-Binding Proteins/genetics
Long-Term Health Outcome and Quality of Life Post-HSCT for IL7Rα-, Artemis-, RAG1- and RAG2-Deficient Severe Combined Immunodeficiency: a Single Center Report

Abd Hamid IJ, Slatter MA, McKendrick F, Pearce MS, Gennery AR

J Clin Immunol, 2018 08;38(6):727-732.
PMID: 30105620 DOI: 10.1007/s10875-018-0540-9

Hematopoietic stem cell transplantation (HSCT) is curative for severe combined immunodeficiency (SCID), but data on long-term impact of pre-HSCT chemotherapy, immune reconstitution and quality of life (QoL) of specific SCID genotypes are limited. We evaluated the long-term immune-reconstitution, health outcome and QoL in IL7Rα SCID, Artemis and RAG1 and 2 SCID survivors > 2 years post-HSCT in our center. Clinical data and immune reconstitution parameters were collated, and patients/families answered PedsQL generic core scale v4.0 questionnaires. Thirty-nine patients with a diagnosis of IL7Rα SCID (17 patients), Artemis SCID (8 patients) and RAG1/2 SCID (13 patients) had undergone HSCT with median age at last follow up for IL7Rα SCID, 14 years (range 4-27) and Artemis and RAG1/2 SCID, 10 years (range 2-18). Many patients have ongoing medical issues at latest follow-up [IL7Rα (73%), Artemis (85%), RAG1/2 (55%)]. Artemis SCID patients experienced more sequela than RAG1/2 SCID. Conditioned recipients with Artemis and RAG SCID had more CD4+ naïve lymphocytes compared to unconditioned recipients. All patients except those of IL7Rα SCID reported lower QoL; further subset group analysis showed parents and Artemis and RAG1/2 survivors without ongoing medical issues reported normal QoL. Conditioned recipients have superior long-term thymopoiesis, chimerism and immunoglobulin-independence. QoL was normal in those who did not have medical issues at long-term follow-up.

Matched MeSH terms: Homeodomain Proteins/genetics*
Fulltext Characterization and Transcriptome Studies of Autoinducer Synthase Gene from Multidrug Resistant Acinetobacter baumannii Strain 863

Ng CK, How KY, Tee KK, Chan KG

Genes (Basel), 2019 04 08;10(4).
PMID: 30965610 DOI: 10.3390/genes10040282

Quorum sensing (QS) is a cell-to-cell communication system that uses autoinducers as signaling molecules to enable inter-species and intra-species interactions in response to external stimuli according to the population density. QS allows bacteria such as Acinetobacter baumannii to react rapidly in response to environmental changes and hence, increase the chances of survival. A. baumannii is one of the causative agents in hospital-acquired infections and the number of cases has increased remarkably in the past decade. In this study, A. baumannii strain 863, a multidrug-resistant pathogen, was found to exhibit QS activity by producing N-acyl homoserine lactone. We identified the autoinducer synthase gene, which we named abaI, by performing whole genome sequencing analysis of A. baumannii strain 863. Using high resolution tandem triple quadrupole mass spectrometry, we reported that abaI of A. baumannii strain 863 produced 3-hydroxy-dodecanoyl-homoserine lactone. A gene deletion mutant was constructed, which confirmed the functionality of abaI. A growth defect was observed in the QS-deficient mutant strain. Transcriptome profiling was performed to determine the possible genes regulated by QS. Four groups of genes that showed differential expression were discovered, namely those involved in carbon source metabolism, energy production, stress response and the translation process.

Matched MeSH terms: Bacterial Proteins/genetics*
Fulltext Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015

Toh X, Soh ML, Ng MK, Yap SC, Harith N, Fernandez CJ, et al.

Transbound Emerg Dis, 2019 Sep;66(5):1884-1893.
PMID: 31059176 DOI: 10.1111/tbed.13218

Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub-lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.

Matched MeSH terms: Viral Proteins/genetics
Fulltext A P. falciparum NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages

Marin-Mogollon C, Salman AM, Koolen KMJ, Bolscher JM, van Pul FJA, Miyazaki S, et al.

Front Cell Infect Microbiol, 2019;9:96.
PMID: 31058097 DOI: 10.3389/fcimb.2019.00096

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line [email protected]). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The [email protected] parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.

Matched MeSH terms: Recombinant Proteins/genetics
Fulltext Habenula kisspeptin retrieves morphine impaired fear memory in zebrafish

Sivalingam M, Ogawa S, Parhar IS

Sci Rep, 2020 11 11;10(1):19569.
PMID: 33177592 DOI: 10.1038/s41598-020-76287-9

The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10-21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.

Matched MeSH terms: Zebrafish Proteins/genetics
Quercetin interferes with the fluid volume and receptivity development of the uterus in rats during the peri-implantation period

Shahzad H, Giribabu N, Karim K, Kassim N, Muniandy S, Kumar KE, et al.

Reprod Toxicol, 2017 08;71:42-54.
PMID: 28431985 DOI: 10.1016/j.reprotox.2017.04.004

HYPOTHESIS: Quercetin could induce changes to the fluid volume and receptivity development of the uterus during peri-implantation period.
METHODS: Female rats were treated with quercetin (10, 25 and 50mg/kg/day) subcutaneously beginning from day-1 pregnancy. Uterus was harvested at day-4 (following three days quercetin treatment) for morphological, ultra-structural, protein and mRNA expressional changes and plasma sex-steroid levels analyses. In another cohort of rats, implantation rate was determined at day-6 (following five days quercetin treatment).
RESULTS: Administration of 50mg/kg/day quercetin causes increased in uterine fluid volume and CFTR expression but decreased in γ-ENaC, AQP-5, AQP-9 claudin-4, occludin, E-cadherin, integrin αnβЗ, FGF, Ihh and Msx-1expression in the uterus. Pinopodes were poorly develop, tight junctions appear less complex and implantation rate decreased. Serum estradiol levels increased but serum progesterone levels decreased.
CONCLUSIONS: Interference in the fluid volume and receptivity development of the uterus during peri-implantation period by quercetin could adversely affect embryo implantation.

Matched MeSH terms: Membrane Proteins/genetics
Fulltext New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Ang JXD, Kadir KA, Mohamad DSA, Matusop A, Divis PCS, Yaman K, et al.

Parasit Vectors, 2020 Sep 15;13(1):472.
PMID: 32933567 DOI: 10.1186/s13071-020-04345-2

BACKGROUND: Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak.
METHODS: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis.
RESULTS: Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo.
CONCLUSIONS: Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

Matched MeSH terms: Insect Proteins/genetics
Fulltext Transcription Factor ChbZIP1 from Alkaliphilic Microalgae Chlorella sp. BLD Enhancing Alkaline Tolerance in Transgenic Arabidopsis thaliana

Qu D, Show PL, Miao X

Int J Mol Sci, 2021 Feb 27;22(5).
PMID: 33673599 DOI: 10.3390/ijms22052387

Saline-alkali soil has become an important environmental problem for crop productivity. One of the most effective approaches is to cultivate new stress-tolerant plants through genetic engineering. Through RNA-seq analysis and RT-PCR validation, a novel bZIP transcription factor ChbZIP1, which is significantly upregulated at alkali conditions, was obtained from alkaliphilic microalgae Chlorella sp. BLD. Overexpression of ChbZIP1 in Saccharomyces cerevisiae and Arabidopsis increased their alkali resistance, indicating ChbZIP1 may play important roles in alkali stress response. Through subcellular localization and transcriptional activation activity analyses, we found that ChbZIP1 is a nuclear-localized bZIP TF with transactivation activity to bind with the motif of G-box 2 (TGACGT). Functional analysis found that genes such as GPX1, DOX1, CAT2, and EMB, which contained G-box 2 and were associated with oxidative stress, were significantly upregulated in Arabidopsis with ChbZIP1 overexpression. The antioxidant ability was also enhanced in transgenic Arabidopsis. These results indicate that ChbZIP1 might mediate plant adaptation to alkali stress through the active oxygen detoxification pathway. Thus, ChbZIP1 may contribute to genetically improving plants' tolerance to alkali stress.

Matched MeSH terms: Plant Proteins/genetics
Fulltext Evaluation of two transformation protocols and screening of positive plasmid introduction into Bacillus cereus EB2, a gram-positive bacterium using qualitative analyses

Sirajuddin SA, Sundram S

Braz J Microbiol, 2020 Sep;51(3):919-929.
PMID: 32078730 DOI: 10.1007/s42770-020-00241-0

Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.

Matched MeSH terms: Green Fluorescent Proteins/genetics
Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Alhaji SY, Nordin N, Ngai SC, Al Abbar A, Mei L, Abdullah S

Gene, 2020 Oct 20;758:144958.
PMID: 32683073 DOI: 10.1016/j.gene.2020.144958

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Matched MeSH terms: Green Fluorescent Proteins/genetics*
Characterization of aluminum resistant Anoxybacillus sp. SK 3-4 isolated from a hot spring

Lim JC, Goh KM, Shamsir MS, Ibrahim Z, Chong CS

J Basic Microbiol, 2015 Apr;55(4):514-9.
PMID: 25523650 DOI: 10.1002/jobm.201400621

The Anoxybacillus sp. SK 3-4, previously isolated from a hot spring, was screened for its heavy metals resistance (Al(3+), Mn(2+), Cu(2+), Co(2+), Zn(2+), and Ni(2+)) and the strain was found to be most resistant to aluminum. Significant growth of the strain was observed when it was grown in medium containing aluminum (200 mg L(-1)-800 mg L(-1)) with relative growth rates ranging between 77% and 100%. A gene encoding the aluminum resistance protein (accession number: WP_021095658.1) was found in genome of strain SK 3-4, which revealed high sequence identity (>95%) to its homologues from Anoxybacillus species. Sequence comparisons with two functionally characterized aluminum resistance proteins, namely G2alt and ALU1-P, showed 97% and 81% of sequence identity, respectively. Four putative metal binding sites were detected in SK 3-4 aluminum resistance protein and G2alt at same amino acid residue positions of 186, 195, 198, and 201. Strain SK 3-4 was found to be able to remove aluminum from aqueous solution. This study demonstrated that Anoxybacillus sp. SK 3-4 could be applied in the treatment of aluminum contaminated wastewater.

Matched MeSH terms: Bacterial Proteins/genetics
Molecular investigation of a gene encoding organic solvent-tolerant alkaline protease from Pseudomonas aeruginosa strain K

Rahman RN, Geok LP, Wong CF, Basri M, Salleh AB

J Basic Microbiol, 2010 Apr;50(2):143-9.
PMID: 20082370 DOI: 10.1002/jobm.200900133

A gene encoding an organic solvent-stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX-4T-1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 degrees C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli.

Matched MeSH terms: Bacterial Proteins/genetics*
Cloning and characterization of poly(3-hydroxybutyrate) biosynthesis genes from Pseudomonas sp. USM 4-55

Tan Y, Neo PC, Najimudin N, Sudesh K, Muhammad TS, Othman AS, et al.

J Basic Microbiol, 2010 Apr;50(2):179-89.
PMID: 20082371 DOI: 10.1002/jobm.200900138

Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).

Matched MeSH terms: Bacterial Proteins/genetics
Degradation of MicroRNA miR-466d-3p by Japanese Encephalitis Virus NS3 Facilitates Viral Replication and Interleukin-1β Expression

Jiang H, Bai L, Ji L, Bai Z, Su J, Qin T, et al.

J Virol, 2020 07 16;94(15).
PMID: 32461319 DOI: 10.1128/JVI.00294-20

Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1β (IL-1β) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.

Matched MeSH terms: Viral Nonstructural Proteins/genetics
Fulltext Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

Nyon MP, Du L, Tseng CK, Seid CA, Pollet J, Naceanceno KS, et al.

Vaccine, 2018 03 27;36(14):1853-1862.
PMID: 29496347 DOI: 10.1016/j.vaccine.2018.02.065

Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377-588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.

Matched MeSH terms: Recombinant Proteins/genetics
High level expression of thermostable lipase from Geobacillus sp. strain T1

Leow TC, Rahman RN, Basri M, Salleh AB

Biosci Biotechnol Biochem, 2004 Jan;68(1):96-103.
PMID: 14745170

A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.

Matched MeSH terms: Recombinant Fusion Proteins/genetics
Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment

Abd Wahid MA, Megat Mohd Noor MJ, Goto M, Sugiura N, Othman N, Zakaria Z, et al.

Biosci Biotechnol Biochem, 2017 Aug;81(8):1642-1649.
PMID: 28585494 DOI: 10.1080/09168451.2017.1329617

The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

Matched MeSH terms: Recombinant Proteins/genetics
GSK-3beta phosphorylation and alteration of beta-catenin in hepatocellular carcinoma

Ban KC, Singh H, Krishnan R, Seow HF

Cancer Lett, 2003 Sep 25;199(2):201-8.
PMID: 12969793

The aim of this study is to investigate the potential correlation between the expression of phosphorylated glycogen synthase kinase-3beta (phospho-GSK-3beta) and beta-catenin, and the mutations of beta-catenin gene at the consensus GSK-3beta phosphorylation site. The reason for this approach is to gain a better understanding of the molecular mechanisms of hepatocarcinogenesis in Malaysia. The expression of phospho-GSK-3beta and beta-catenin by immunohistochemistry and the mutations of beta-catenin were studied in 23 hepatocellular carcinoma (HCC) and surrounding tissues. Overexpression of phospho-GSK-3beta and beta-catenin was found in 12/23 (52.2%) and 13/23 (56.5%) in HCC tissues, 6/23 (26.1%) and 9/23 (39.1%) in surrounding tissues, respectively. Overexpression of beta-catenin in HCC tissues compared to the surrounding liver tissue was found to be higher in HCC tissues (p=0.015). In addition, we found that the expression of phospho-GSK-3beta was related with the accumulation of beta-catenin in surrounding tissues (p<0.05). The expression of phospho-GSK-3beta and its association with the development of HCC is reported for the first time. In addition, this is the first report from Malaysia which shows that there are no mutations at the GSK-3beta consensus phosphorylation sites on beta-catenin gene in all 23 paired HCC and surrounding tissues. This result differed from HCC in geographical areas with high aflatoxin exposure.

Matched MeSH terms: Cytoskeletal Proteins/genetics

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