Aims: Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447). Methodology and results: Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli. Conclusion, significance and impact of study GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.
Synechococcus ; Ribulose-Bisphosphate Carboxylase ; Escherichia coli ; Holoenzymes

A delayed wound healing process can lead to detrimental complications in chronic wound patients such as tissue necrosis and systemic infections. Application of immunonutrition (IN) in experimental animal models and chronic wound patients has shown promising and improved wound healing processes. IN restores the supply of essential nutrients that are critical for cell growth and tissue repair in the wounded subjects. Several commonly found nutrients in IN formulations include polyunsaturated fatty acids (PUFAs), essential amino acids, trace elements such as zinc and vitamins. Recently, some studies suggested the use of traditionally used herbs like curcumin in IN recipes due to its efficient wound healing properties. The roles and functions of IN in wound healing encompass recruitment of white blood cells, platelets and fibroblasts into the wounded area during the coagulation and inflammation phases, enhancement of fibroblast proliferation, collagen synthesis and neovascularization in the proliferation phase; and lastly, regulation of tissue re-epithelization for wound closure and recovery. In this review, the roles and functions of individual nutrients were deliberately discussed alongside their mechanisms of action in wound healing. This aims to provide a more holistic insight into the potentials of those nutrients when used as part of IN for major wound patients. Despite its remarkable effects in wound healing, several criteria should be considered in an IN formulation: the type and severity of wounds, administration timing and mode of administration, and concoction of immune-boosting nutrients in order to ensure the optimal wound healing effects.

In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.

In light of the limited protection conferred by current influenza vaccines, immunisation using universal influenza vaccines has been proposed for protection against all or most influenza sub-types. The fundamental principle of universal influenza vaccines is based on conserved antigens found in most influenza strains, such as matrix 2, nucleocapsid, matrix 1 and stem of hemagglutinin proteins. These antigens trigger cross-protective immunity against different influenza strains. Many researchers have attempted to produce the conserved epitopes of these antigens in the form of peptides in the hope of generating universal influenza vaccine candidates that can broadly induce cross-reactive protection against influenza viral infections. However, peptide vaccines are poorly immunogenic when applied individually owing to their small molecular sizes. Hence, strategies, such as combining peptides as multi-epitope vaccines or presenting peptides on vaccinia virus particles, are employed. This review discusses the clinical and laboratory findings of several multi-epitope peptide vaccine candidates and vaccinia-based peptide vaccines. The majority of these vaccine candidates have reached the clinical trial phase. The findings in this study will indeed shed light on the applicability of universal influenza vaccines to prevent seasonal and pandemic influenza outbreaks in the near future.

Each year, influenza A infections have caused tremendous death rate as high as 300,000-500,000 globally. Althoughthere are effective anti-influenza agents and vaccines, high mutational rate among influenza A viruses renders dramaticdecline in the effectiveness of anti-influenza agents or vaccines in certain individuals. The situation is further complicatedby limitations in influenza vaccine production, for instance, long production period, limited vaccine capacity and lackof cross-protection against various influenza A virus strains. To solve these issues, development of universal influenzavaccine based on conserved antigens such as non-stuctural protein 1 (NS1) has been endeavoured. NS1 protein is highlyconserved in all influenza A virus strains known by far, produced abundantly on infected cell surfaces and responsible formaintaining virulence. Furthermore, cytotoxic T-lymphocytes that are active against NS1 were also reported to be ableto avoid shedding of influenza in hosts. To better inhibit influenza infections, oral immunization has long been proposeddue to feasibility of this method to be implemented and safer for recipients while able to target influenza A viruses fromthe entry point. Lactobacillus has been vastly studied for its roles as bacterial carrier in oral vaccine development dueto its significant probiotic properties. For examples, stimulation of immune responses in oral and airway mucosal layers,high colonization in oral and airway mucosal layers and great natural adjuvant effects. In this light, influenza universaloral vaccine developed using NS1 dan Lactobacillus should be further studied in influenza oral vaccine design.

Nowadays, probiotics have been widely consumed as supplementary food for their health benefits. However, safetyevaluation for many probiotic bacteria is still lacking. Furthermore, health benefits conferred by probiotics depend onthe strains used in producing probiotic products. Therefore, it is important to examine oral toxicity of newly isolatedLactobacillus casei (Lb. casei) C1. A total of 32 Wistar (WIS) rats were divided into acute (single dose) and subacuteoral toxicity (28-days repeated dose) groups. Rats in each group were further divided into control group which receivedphosphate buffer saline (PBS) orally and treatment group that was administered orally with Lb. casei C1 (1011 CFU/ml).For acute oral toxicity, treatment was performed on day-1 and the effects were monitored subsequently for 14 days. Forsubacute oral toxicity, treatment was given daily for 28 days and the effects were observed throughout the experimentalperiod. Body weight, food and water intake of the rats were recorded. Rats in acute and subscute groups were sacrificedon day-15 and day-29, respectively. Serum was collected to determine the levels of total protein, malondialdehyde (MDA),alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and creatinine. Organs were alsoharvested for histological examination. There were no significant differences (p > 0.05) in body weight, food and waterintake between the control and treated rats in acute oral toxicity group. There were also no significant differences in theblood cell count, levels of total protein, MDA, LDH and creatinine between the control and treated rats. Similar findingswere recorded for the subacute oral toxicity group, except that the levels of ALT and AST which were significantly different(p < 0.05). When observed under a light microscope, there were no morphological changes detected in the kidney, liverand ileum of treated rats as compared to control rats in both of the experimental groups. In conclusion, Lb. casei C1exhibited no toxic effects in Wistar rats hence safe to be consumed orally.

Breast cancer is one of the commonest cancers among women. Conventional therapies cause adverse side effects inpatients. Cytokine immunotherapy such as interleukin-27 (IL-27) has been sought as an alternative cancer treatment inrecent years. IL-27 has been shown to improve anticancer immunity and anti-angiogenesis in cancers, however, its effecton apoptotic and anti-apoptotic gene expression especially in breast cancers is yet to be explored. Cytotoxicity of IL-27in non-cancerous (184b5) and cancerous (MCF-7 and MDA-MB-231) breast cell lines was first determined for 24-72h in this study. The results indicated that IL-27 treatment did not retard 184b5 cell growth, however, did inhibit MCF-7(48 h) and MDA-MB-231 (72 h) cell growth with IC50 at 442 and 457 ng/ml, respectively. Apoptotic (TRAIL, FADD, FAS,caspase-3 and caspase-8) and anti-apoptotic (BCL-2, AKT, and COX-2) genes were then amplified from untreated (control)and treated breast cancer cells and studied. TRAIL, caspase-3, caspase-8 gene expression was significantly (p < 0.05)upregulated in treated MCF-7 (442 ng/ml) and MDA-MB-231 (457 ng/ml) cells. Expression of FADD and FAS genes wasnot detected in both control and treated MCF-7 and MDA-MB-231 cells. COX-2 gene was also not expressed by MCF-7cells, but reduced significantly (p < 0.05) in treated MDA-MB-231 cells. In MDA-MB-231 cells, IL-27 treatment seemedto slightly enhance the expression of AKT and BCL-2 genes which, on the other hand, was downregulated in treatedMCF-7 cells. Conclusively, IL-27 is able to inhibit breast cancer cell growth and regulate apoptotic and anti-apoptoticgene expression in breast cancer cells.

Mutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the localfermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study alsoinvolved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence andabsence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on bothS. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenicactivity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as comparedto the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence ofmetabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagensand suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food developmentas a supplement product to prevent development of colon cancer.

Aims: Endophytic fungi are microorganisms that live asymptomatically within plant tissues, producing a wide range of metabolites, including compounds potentially useful for drug development. We investigated endophytic fungi from Maliau Basin, Sabah to identify strains producing bioactive compounds, notably with antimicrobial activity. Methodology and results: A total of 23 plants were sampled yielding 345 endophytic fungal isolates. Of these, 44 isolates were screened for antimicrobial activity against nine species of bacteria and fungi, revealing 14 endophytes producing bioactive metabolites. Crude fungal extracts were obtained from broth cultures of endophytic isolates with promising activity while the fungal strains were identified using molecular methods. The crude extract of endophyte MB4 WA10, isolated from Callophyllum sp. (bintangor) showed IC50 of 2.6 mg/mL against S. aureus and 0.6 mg/mL against B. subtilis while the extract of MB22 WA16, an isolate identified as Valsaceae sp., was also active against S. aureus with an IC50 of 1.37 mg/mL. Another isolate, namely MB5 L4 (WA), was identified as a Phomopsis sp. and its extract was the most active against S. aureus with an IC50 of 1 mg/mL. The HPLC fraction of this fungal extract with the highest inhibition (92.37%) of S. aureus was purified for compound isolation and identification. A polyketide compound, 2,3-dihydro-2- hydroxy-2,4-dimethyl-5-trans-propenylfuran-3-one (C9H12O3), with molecular weight of 168.192 was identified based on mass spectral and NMR data analysis. This previously identified compound is known to have other antimicrobial properties. Conclusion, significance and impact of study Rainforests in Malaysia, especially Maliau Basin, harbour many species of fungal endophytes, producing useful bioactive compounds that may be explored for further potential uses, including antimicrobial activity.

The occasional influenza pandemics and the seasonal influenza epidemics have destroyed millions of lives since the last century. It is therefore necessary to understand the virus replication patterns as this provides essential information on the virus infectivity, pathogenicity and spread patterns. This study aimed to investigate the replication of avian influenza A virus H5N1 (A/Chicken/Malaysia/5858/2004) in MDCK cells. In this study, the TCID50 (50% tissue culture infectious dose) of AIV H5N1 was first determined. The MDCK cells were then infected with AIV H5N1 at TCID50 for 0-48 h. The CPE (cytopathic effect) was observed and cell death was determined hourly. The virus-infected cells and media were subsequently collected for gene analysis. The results showed that the TCID50 of AIV H5N1 was 10-9 dilution. The CPE percentage showed a strong and positive correlation with the infection period (r = 1.0, n = 9, p < 0.01). The amount of a highly conserved influenza viral gene, M2 gene amplified from infected media (r = 0.471, n = 9, p= > 0.05) and infected cell (r = 0.73, n = 9, p < 0.05) were also positively correlated with the infection period. In conclusion, although CPE started to be observed in the early time points of infection, however, the M2 gene was only amplified from the infected media and cells after 48 h and 24 h, respectively. This signifies that AIV H5N1 used in this study is pathogenic and it is able to cause severe cytopathology to host cells even at low virus load.
Influenza, Human ; Influenza A Virus, H5N1 Subtype