Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.
Two female and one male adult hookworms were recovered from a female patient in Thailand. Based on gross and microscopic morphology, the three hookworms are members of Necator americanus. Phylogenetic reconstruction based on partial NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequences shows that these hookworms belong to the same genetic lineage as N. americanus adult worm from Zhejiang, China. The male and female hookworms were genetically distinct, belonging to two different nad1-haplotypes. This is the first report targeting the nad1 gene on the identification and genetic characterization of the human hookworms originated from infected patient. The nad1 gene marker is useful for species and higher taxa differentiation of hookworms.
The focus of the current study was to disrupt the Toxo 5699 gene via CRISPR/Cas9 to evaluate the effects of gene disruption on the parasite lytic cycle. In the present work, a single plasmid expressing both the guide RNA and Cas9 nuclease together with a selectable marker of human dihydrofolate reductase (DHFR) was introduced into Toxoplasma gondii. Targeted disruption of the Toxo 5699 gene was carried out via the CRISPR/Cas9 system and confirmed by PCR, sequencing, and immunofluorescence microscopy. Disrupted and nondisrupted control parasites were allowed to invade HS27 cell monolayers and plaques were counted. The average number of plaques from three replicates per group was obtained between the disrupted and non-disrupted T. gondii RH strain and was compared using a onetailed t-test. It was observed that there was a significant decrease in number and size of plaque formation in the Toxo 5699 gene disrupted parasite line. This is an indication that the Toxo 5699 gene may play a role in the lytic cycle of the parasite, particularly during the replication phase and thus would be a novel target for disruption or silencing. The Toxo 5699 gene presented in the current work is an important part of the T. gondii lytic cycle, therefore meriting further inquiry into its potential as a target for further genetic-silencing or disruption studies.
This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the fimbrial protein of Pasteurella multocida B:2 following intranasal vaccination against hemorrhagic septicemia in goats. Goats were vaccinated intranasal with 10(6) CFU/mL of the recombinant vaccine (vaccinated group) and 10(6) CFU/mL of pET32/LIC vector without fimbrial protein (control group). All three groups were kept separated before all goats in the three groups were challenged with 10(9) CFU/mL of live pathogenic P. multocida B:2. During the course of study, both serum and lung lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay. It was found that goats immunized with the inactivated recombinant vaccine developed a strong and significantly (p
Duck Tembusu virus (DTMUV), a newly emerging virus in ducks, was first reported in China in 2010. However, an unknown severe contagious disease associated with severe neurological signs and egg production losses in ducks, resembling to DTMUV infection, was observed in Thailand since 2007. To determine the presence of DTMUV in 2007, the clinical samples from affected ducks collected in 2007 were tested for DTMUV using pathological and virological analyses. Gross and histopathological lesions of affected ducks were mostly restricted to the ovary, brain and spinal cord, and correlated with the presence of flavivirus antigen in the brain and spinal cord samples. Subsequently, DTMUV was identified by RT-PCR and nucleotide sequencing of the polyprotein gene. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was a unique virus, belonged within DTMUV cluster 1, but distinctively separated from the Malaysian DTMUV, which was the most closely related DTMUV. It is interesting to note that the 2007 Thai DTMUV was genetically different from the currently circulating Thai and Chinese DTMUVs, which belonged to cluster 2. Our findings indicated that the 2007 Thai DTMUV emerged earlier from a common ancestor with the recently reported DTMUVs; however, it was genetically distinctive to any of the currently circulating DTMUVs. In conclusion, our data demonstrated the presence of DTMUV in the Thai ducks since 2007, prior to the first report of DTMUV in China in 2010. This study indicates that DTMUV may have circulated in the region long before 2010 and highlights high genetic diversity of DTMUVs in Asia.
cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.
A novel human leukocyte antigen-B allele officially named B*3589, was found in an indigenous individual of Jehai ethnicity when sequencing was performed to investigate human genome variation in a research project. B*3589 differs form B*3505 in a point mutation at codon 169 (CGC to TGC) resulting in an amino acid change from Arg to Cys.
Strategies to resolve B*18 alleles which carry a deletion in intron 1 close to the 5' end of exon 2 relative to other HLA-B alleles or a null allele mutation in exon 1 and to resolve ambiguities among allele combinations including B*18 are described. B*18 allele frequencies from volunteer donors recruited for two hematopoietic stem cell registries show the presence of two alleles, B*180101 and B*1802, in a population from Singapore and only B*180101 in African-Americans.
Ankylosing spondylitis (AS) is a chronic inflammatory disorder with predilection for the axial skeleton, leading to progressive restricted mobility and deformity of the spine. The fundamental mechanism involves autoimmunity orchestrated by T cells. Similar to other rheumatic diseases, the complex interplay of cytokines such as tumour necrosis factor alpha, interleukin-6 (IL 6) and interleukin-10 (IL 10) has been implicated in the pathogenesis of the disease. Despite extensive research over the past decades, the treatment options for AS, are limited. Non steroidal antiinflammatory drugs are the first line of therapy, whereas anti TNF drugs are administered for refractory cases which fail to respond to the treatment. There have been conflicting views on the correlation of IL 6 with disease activity in AS. As such, the debate on the role of anti IL6 in AS is still ongoing. Anti IL 6 such as tocilizumab and siltuximab have proven efficacy based on the large randomized controlled trials. The Food and Drug Administration (FDA) has approved these drugs for treating rheumatoid arthritis and systemic juvenile idiopathic arthritis. Researchers have adventurously experimented anti IL 6 therapy in AS but the conclusions made were not consolidated into international guidelines or consensus statement for clinical practice. In the present review, we explore the role of anti IL6 in the treatment of AS based on the cumulative evidence over recent years.
Southern Thai Muslims (STM)--from Nakon Si Thammarat, whose ancestors come mainly from Malaysia--constitute more than half of all Thai Muslims which, in total, represent approximately 10% of the country's population. The most common A2 subtypes in STM were A*0203 (n=15), A*0201 (n=8) and A*0207 (n=7). In this study, samples with unexpected amplification patterns were sequenced. Three individuals were indicative of a novel A2 allele, now known as A*02012.
beta-D-N-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of β-hexosaminidases to analyze the evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history of evolution of plant β-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato β-hexosaminidase (β-Hex-Sl) was predicted by homology modeling using 1now as a template. Structural conformity studies of the best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl hydrolase20 superfamily domain-II including the (β/α)8 barrel in the central part. The α and β contents of the modeled structure were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330) and Glu(331) could play important roles in enzyme-catalyzed reactions. The predicted model provides a structural framework that can act as a guide to develop a hypothesis for β-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in plant kingdom.
This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80 ± 0.27 mg of oFGH compared to 1.75 ± 0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P < 0.05) and fourth weeks (P < 0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production.
N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.
Conservation is imperative for the Asian snakeheads Channa striata, as the species has been overfished due to its high market demand. Using maternal markers (mitochondrial cytochrome c oxidase subunit 1 gene (COI)), we discovered that evolutionary forces that drove population divergence did not show any match between the genetic and morphological divergence pattern. However, there is evidence of incomplete divergence patterns between the Borneo population and the populations from Peninsular Malaysia. This supports the claim of historical coalescence of C. striata during Pleistocene glaciations. Ecological heterogeneity caused high phenotypic variance and was not correlated with genetic variance among the populations. Spatial conservation assessments are required to manage different stock units. Results on DNA barcoding show no evidence of cryptic species in C. striata in Malaysia. The newly obtained sequences add to the database of freshwater fish DNA barcodes and in future will provide information relevant to identification of species.
The cyclic AMP- (cAMP-) dependent protein kinase A signaling pathway is one of the major signaling pathways responsible for regulation of the morphogenesis and pathogenesis of several pathogenic fungi. To evaluate the role of this pathway in the plant pathogenic fungus, Colletotrichum gloeosporioides, the gene encoding the catalytic subunit of cAMP-dependent protein kinase A, CgPKAC, was cloned, inactivated, and the mutant was analyzed. Analysis of the Cgpkac mutant generated via gene replacement showed that the mutants were able to form appressoria; however, their formation was delayed compared to the wild type. In addition, the mutant conidia underwent bipolar germination after appressoria formation, but no appressoria were generated from the second germ tube. The mutants also showed reduced ability to adhere to a hydrophobic surface and to degrade lipids localized in the appressoria. Based on the number of lesions produced during a pathogenicity test, the mutant's ability to cause disease in healthy mango fruits was reduced, which may be due to failure to penetrate into the fruit. These findings indicate that cAMP-dependent protein kinase A has an important role in regulating morphogenesis and is required for pathogenicity of C. gloeosporioides.
Unspecialized cells that can renew themselves and give rise to multiple differentiated cell types are termed stem cells. The objective of this study was to characterize and investigate, through molecular and biochemical analyses, the stemness of cells derived from isolated mononucleated cells that originated from peripheral blood. The isolated mononucleated cells were separated according to their physical characteristics (adherent and suspension), after 4 to 7 days into a 14-day culturing period in complete medium. Our results revealed that adherent and suspension cells were positive for mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC) markers, respectively. Differentiation of adherent cells into osteoblasts was associated with expression of the OPN gene and increasing ALP enzyme activity, while differentiation of suspension cells into osteoclasts was associated with expression of the TRAP gene and increasing TRAP enzyme activity. In conclusion, molecular and biochemical analyses showed that mononucleated cells consist of MSC (adherent) and HSC (suspension), and both cell types are able to differentiate into specialized cells from their respective lineage: osteoblast (MSC) and osteoclast (HSC).
Basal stem rot (BSR) disease caused by the fungus Ganoderma boninense is the most serious disease affecting the oil palm; this is because the disease escapes the early disease detection. The biocontrol agent Trichoderma harzianum can protect the disease only at the early stage of the disease. In the present study, the expression levels of three oil palm (Elaeis guineensis Jacq.) chitinases encoding EgCHI1, EgCHI2, and EgCHI3 at 2, 5, and 8 weeks inoculation were measured in oil palm leaves from plants treated with G. boninense or T. harzianum alone or both.