The Burden of Microplastics Pollution and Contending Policies and Regulations.

The relationship between humans and plastics has become intricate due to their versatile nature and low production cost. Plastics generation has surpassed that of other manufactured products, which, coupled with the prevailing poor waste management systems, makes it a serious problem for the terrestrial and aquatic environments as its final destination. Their extensive presence has continued to pose a significant threat, not only to the aquatic ecosystem but also to the approximately 3 billion people relying on it for their livelihood. Even more disturbing were the recent findings of these plastics in food and drinking water and the evidence of human exposure, the long-term health effects of which are largely unknown. This ubiquitous phenomenon has over time put plastics under critical observation, leading to the development of many local and international policies, resolutions, and directives aimed at addressing and reversing the menace. This review provided the first snapshot of the global and local governance strategies currently aimed at mitigating plastic pollution, their limitations, and future directions. The findings of the review revealed several aspects of microplastics (MPs) pollution to be overlooked in policy formulation, a laxity in policy implementation, and an apparent lack of indices to ascertain the impact of the regulations. Furthermore, there is currently no regulation on MPs contamination of food and drinking water and an apparent lack of funding for research into the health effects of plastics and their alternatives. This, therefore, necessitates the need for a well-coordinated approach at international and national levels to scale up these policies in all countries and translate them from paper to measurable, holistic, and realizable actions that will address all forms of plastic pollution.

Polystyrene Microplastics Exposure: An Insight into Multiple Organ Histological Alterations, Oxidative Stress and Neurotoxicity in Javanese Medaka Fish (Oryzias javanicus Bleeker, 1854).

Microplastics (MPs) have become pollutants of concern due to their unknown human health effect and negative impact on terrestrial and aquatic ecosystems. There is increasing number of experimental research on MPs globally with its effects not fully understood; recent animal studies explore its effects on the intestines, yet on other vital organs. Javanese medaka fish was exposed to polystyrene microplastics (PS-MPs) beads for a period of 21 days. Histological alterations, intestinal oxidative stress, permeability and neurotoxicity were evaluated. Significant inflammatory changes and tissue damage were observed in the intestine, liver and kidney. Intestinal oxidative stress and permeability were found to be significantly increased. In the brain, neurotoxicity characterised by a significant induction of oxidative stress, lipid peroxidation and the inhibition of acetylcholinesterase enzyme were elucidated. This study provided an insight into the multiple organ effect of microplastics exposure, necessitating further exploration and identification of biomarkers to be utilised for biomonitoring population at risk in the future.

Functional prediction of de novo uni-genes from chicken transcriptomic data following infectious bursal disease virus at 3-days post-infection.

BACKGROUND: Infectious bursal disease (IBD) is an economically very important issue to the poultry industry and it is one of the major threats to the nation's food security. The pathogen, a highly pathogenic strain of a very virulent IBD virus causes high mortality and immunosuppression in chickens. The importance of understanding the underlying genes that could combat this disease is now of global interest in order to control future outbreaks. We had looked at identified novel genes that could elucidate the pathogenicity of the virus following infection and at possible disease resistance genes present in chickens. RESULTS: A set of sequences retrieved from IBD virus-infected chickens that did not map to the chicken reference genome were de novo assembled, clustered and analysed. From six inbred chicken lines, we managed to assemble 10,828 uni-transcripts and screened 618 uni-transcripts which were the most significant sequences to known genes, as determined by BLASTX searches. Based on the differentially expressed genes (DEGs) analysis, 12 commonly upregulated and 18 downregulated uni-genes present in all six inbred lines were identified with false discovery rate of q-value < 0.05. Yet, only 9 upregulated and 13 downregulated uni-genes had BLAST hits against the Non-redundant and Swiss-Prot databases. The genome ontology enrichment keywords of these DEGs were associated with immune response, cell signalling and apoptosis. Consequently, the Weighted Gene Correlation Network Analysis R tool was used to predict the functional annotation of the remaining unknown uni-genes with no significant BLAST hits. Interestingly, the functions of the three upregulated uni-genes were predicted to be related to innate immune response, while the five downregulated uni-genes were predicted to be related to cell surface functions. These results further elucidated and supported the current molecular knowledge regarding the pathophysiology of chicken's bursal infected with IBDV. CONCLUSION: Our data revealed the commonly up- and downregulated novel uni-genes identified to be immune- and extracellular binding-related, respectively. Besides, these novel findings are valuable contributions in improving the current existing integrative chicken transcriptomics annotation and may pave a path towards the control of viral particles especially towards the suppression of IBD and other infectious diseases in chickens.

Microplastics Pollution as an Invisible Potential Threat to Food Safety and Security, Policy Challenges and the Way Forward.

Technological advances, coupled with increasing demands by consumers, have led to a drastic increase in plastic production. After serving their purposes, these plastics reach our water bodies as their destination and become ingested by aquatic organisms. This ubiquitous phenomenon has exposed humans to microplastics mostly through the consumption of sea food. This has led the World Health Organization (WHO) to make an urgent call for the assessment of environmental pollution due to microplastics and its effect on human health. This review summarizes studies between 1999 and 2020 in relation to microplastics in aquatic ecosystems and human food products, their potential toxic effects as elicited in animal studies, and policies on their use and disposal. There is a paucity of information on the toxicity mechanisms of microplastics in animal studies, and despite their documented presence in food products, no policy has been in place so far, to monitor and regulates microplastics in commercial foods meant for human consumption. Although there are policies and regulations with respect to plastics, these are only in a few countries and in most instances are not fully implemented due to socioeconomic reasons, so they do not address the problem across the entire life cycle of plastics from production to disposal. More animal research to elucidate pathways and early biomarkers of microplastic toxicity that can easily be detected in humans is needed. This is to create awareness and influence policies that will address this neglected threat to food safety and security.

Phytochemical and anti-inflammatory properties of Scurrula ferruginea (Jack) Danser parasitising on three different host plants elucidated by NMR-based metabolomics.

INTRODUCTION: Scurrula ferruginea (Jack) Danser is a hemi-parasitic shrub that is widely used as a traditional herbal medicine. OBJECTIVES: To elucidate the anti-inflammatory activity of S. ferruginea parasitising on three different hosts of Vitex negundo L., Micromelum minutum (G. Forst.) Wight & Arn. and Tecoma stans (L.) Juss ex HBK., as well as, to determine the metabolite differences related to their anti-inflammatory properties. MATERIALS AND METHODS: Two plant parts of S. ferruginea, stems and leaves, were extracted in water. The freeze-dried stem of S. ferruginea grown on T. stans was liquid-liquid partitioned into several solvents. Their potential anti-inflammatory activity was assessed via inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced RAW 264.7 macrophage cells. The metabolite variation was examined using proton nuclear magnetic resonance (1 H-NMR) combined with multivariate data analysis (MVDA). RESULTS: Scurrula ferruginea stems parasitising on T. stans and V. negundo which were freeze dried exhibited higher anti-inflammatory activity with IC50 values of 114.47 ± 2.96 and 118.87 ± 2.31 µg/mL, respectively. The mid-polar ethyl acetate fraction of S. ferruginea hosted on T. stans displayed the highest NO inhibition with 84.80 ± 0.45% at 200 µg/mL. Principal component analysis (PCA) indicated notable and clear discriminations among the different plant parts and host plants based on the identified metabolites. Furthermore, partial least squares (PLS) regression model suggested the anti-inflammatory bioactivity might be associated with the presence of choline, isoleucine, catechin, leucine and chlorogenic acid. CONCLUSION: This study suggests S. ferruginea could serve as a potential anti-inflammatory agent, highlighting the importance of T. stans as the host plant.

Differential expression of immune-related genes in the bursa of Fabricius of two inbred chicken lines following infection with very virulent infectious bursal disease virus.

Among different inbred chickens' lines, we previously showed that lines P and N of Institute for Animal Health, Compton, UK are the most susceptible and the least affected lines, respectively, following infection with very virulent infectious bursal disease virus (vvIBDV). In this study, the differential expressions of 29 different immune-related genes were characterized. Although, birds from both lines succumbed to infection, line P showed greater bursal lesion scores and higher viral copy numbers compared to line N. Interestingly, line N showed greater down-regulation of B cell related genes (BLNK, TNFSF13B and CD72) compared to line P. While up-regulation of T-cell related genes (CD86 and CTLA4) and Th1 associated cytokines (IFNG, IL2, IL12A and IL15) were documented in both lines, the expression levels of these genes were different in the two lines. Meanwhile, the expression of IFN-related genes IFNB, STAT1, and IRF10, but not IRF5, were up-regulated in both lines. The expression of pro-inflammatory cytokines (IL1B, IL6, IL18, and IL17) and chemokines (CXCLi2, CCL4, CCL5 and CCR5) were up-regulated in both lines with greater increase documented in line P compared to line N. Strikingly, the expression of IL12B was detected only in line P whilst the expression of IL15RA was detected only in line N. In conclusion, the bursal immunopathology of IBDV correlates more with expression of proinflammatory response related genes and does not related to expression of B-cell related genes.

Complete genome sequence of fowl adenovirus-8b UPM04217 isolate associated with the inclusion body hepatitis disease in commercial broiler chickens in Malaysia reveals intermediate evolution.

The main aim of our study was to explore the genome sequence of the inclusion body hepatitis associated Fowl adenovirus serotype 8b (FAdV-8b) UPM04217 and to study its genomic organisation. The nucleotide sequence of the whole genome of FAdV-8b UPM04217 was determined by using the 454 Pyrosequencing platform and the Sanger sequencing method. The complete genome was found to be 44,059 bp long with 57.9% G + C content and shared 97.5% genome identity with the reference FAdV-E genome (HG isolate). Interestingly, the genome analysis using ORF Finder, Glimmer3 and FGENESV predicted a total of 39 open reading frames (ORFs) compared to the FAdV-E HG that possessed 46 ORFs. Fourteen ORFs located within the central genomic region and 16 ORFs located within the left and right ends of the genome were assigned as being the high protein-coding regions. The fusion of the small ORFs at the right end terminal specifically in ORF22 and ORF33 could be the result of gene truncation in the FAdV-E HG. The frame shift mutation in ORF25 and other mutations in ORF13 and ORF17 might have lead to the emergence of genes that could have different functions. Besides, one of the minor capsid components, pVI, in FAdV-8b UPM04217 shared the highest similarity of 93% with that of FAdV-D, while only 47% similarity was found with FAdV-E. From the gene arrangement layout of the FAdV genome, FAdV-8b UPM04217 showed intermediate evolution between the FAdV-E HG and the FAdV-D although it was apparently more similar to the FAdV-E HG.

Genome sequencing and analysis of Salmonella enterica subsp. enterica serovar Stanley UPM 517: Insights on its virulence-associated elements and their potentials as vaccine candidates.

Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.

Molecular characterization of fowl adenovirus isolate of Malaysia attenuated in chicken embryo liver cells and its pathogenicity and immunogenicity in chickens.

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens.

RNA sequencing of kidney and liver transcriptome obtained from wild cynomolgus macaque (Macaca fascicularis) originating from Peninsular Malaysia.

OBJECTIVE: Using high-throughput RNA sequencing technology, this study aimed to sequence the transcriptome of kidney and liver tissues harvested from Peninsular Malaysia cynomolgus macaque (Macaca fascicularis). M. fascicularis are significant nonhuman primate models in the biomedical field, owing to the macaque's biological similarities with humans. The additional transcriptomic dataset will supplement the previously described Peninsular Malaysia M. fascicularis transcriptomes obtained in a past endeavour. RESULTS: A total of 75,350,240 sequence reads were obtained via Hi-seq 2500 sequencing technology. A total of 5473 significant differentially expressed genes were called. Gene ontology functional categorisation showed that cellular process, catalytic activity, and cell part categories had the highest number of expressed genes, while the metabolic pathways category possessed the highest number of expressed genes in the KEGG pathway analysis. The additional sequence dataset will further enrich existing M. fascicularis transcriptome assemblies, and provide a dataset for further downstream studies.

Bursal transcriptome profiling of different inbred chicken lines reveals key differentially expressed genes at 3 days post-infection with very virulent infectious bursal disease virus.

Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.

Whole-Genome Sequence of Salmonella enterica subsp. enterica Serovar Typhimurium Strain UPM 260, Isolated from a Broiler Chicken in Perak, Malaysia.

Salmonella enterica subsp. enterica serovar Typhimurium is one of several well-categorized Salmonella serotypes recognized globally. Here, we report the whole-genome sequence of S Typhimurium strain UPM 260, isolated from a broiler chicken.

Complete Genome Sequence of Pasteurella multocida Serotype A Strain PMTB2.1 Isolated from Buffaloes That Died of Septicemia in Malaysia.

Pasteurella multocida causes pneumonic pasteurellosis and hemorrhagic septicemia (HS) in large ruminants. In this study, we determined the complete genome sequence of P. multocida strain PMTB2.1 capsular serotype A isolated from buffaloes that died of septicemia.

Cell Wall-Treated Lactococcus lactis Increases the Plasmid Transfer Efficiency of Internal Ribosome Entry Site-Incorporated Lactococcal Bicistronic Vector into DF1 Cells.

This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 µg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.

RNA sequencing (RNA-Seq) of lymph node, spleen, and thymus transcriptome from wild Peninsular Malaysian cynomolgus macaque (Macaca fascicularis).

The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53-63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development.

IRES-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system.

Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis) has been gaining more interests for its positive prospects in genetic engineering-related applications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be able to express multiple genes in the eukaryotic system. Therefore, a cassette containing an internal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infectious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene, green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048 harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicistronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation system followed by SDS-PAGE and Western blot analysis to rapidly verify its functionality. Immunoblotting profiles showed the presence of 49 and 29kDa bands that corresponds to the sizes of the VP2 and GFP proteins respectively. This preliminary result shows that the newly constructed lactococcal bicistronic vector can co-express multiple genes in a eukaryotic system via the IRES element thus suggesting its feasibility to be used for transfection of in vitro cell cultures and vaccine delivery.