The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component ...have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.
Abstract
Hospital outbreaks of COVID19 result in considerable mortality and disruption to healthcare services and yet little is known about transmission within this setting. We characterise within ...hospital transmission by combining viral genomic and epidemiological data using Bayesian modelling amongst 2181 patients and healthcare workers from a large UK NHS Trust. Transmission events were compared between Wave 1 (1st March to 25th July 2020) and Wave 2 (30th November 2020 to 24th January 2021). We show that staff-to-staff transmissions reduced from 31.6% to 12.9% of all infections. Patient-to-patient transmissions increased from 27.1% to 52.1%. 40%-50% of hospital-onset patient cases resulted in onward transmission compared to 4% of community-acquired cases. Control measures introduced during the pandemic likely reduced transmissions between healthcare workers but were insufficient to prevent increasing numbers of patient-to-patient transmissions. As hospital-acquired cases drive most onward transmission, earlier identification of nosocomial cases will be required to break hospital transmission chains.
Cloning is indispensable in molecular biology. Here we developed an in vivo homologous recombination-based cloning procedure and determined the optimal conditions. This procedure required two PCR ...products to be amplified from a gene of interest and desired plasmid vector. The 5’ ends of both primers that amplified one product had nucleotide sequences complementary to that used to amplify the other product. Once the mixture of those PCR products was introduced into Escherichia coli DH5α competent cells, transformants carried plasmids in which the gene of interest had been properly cloned. Optimizing the cloning conditions, at least 12-nucleotides overlaps between the terminal ends of two fragments were required to generate desired plasmids. This value was much shorter than the length of overlaps required for the same procedure employed in the yeast system. Therefore, this procedure is expected to be an attractive alternative for cloning in the E. coli system.
Biochemical analyses of protein translocation intermediates formed during early stages of protein import into chloroplasts under restricted energy conditions identified many components involved in ...protein translocation. Whereas limited information is available at the latter stages of import, as it is difficult to pause the movement of precursor once it was released from the early intermediates. To address this problem, we have attempted to obtain post-early intermediates to plug translocation channel by precursor proteins carrying a tightly-folded structure at their C-termini. To this end, we prepared the recombinant precursor protein whose chloroplastic targeting signal was fused to dihydrofolate reductase from Escherichia coli, known to fold tightly in the presence of its substrate analogue methotrexate. If the precursor was treated with methotrexate prior to the import reaction, the amount of processed precursor was reduced. However, the processed precursor was recovered in the soluble fraction after fractionation, indicating that methotrexate was released from the precursor, which suggested the presence of strong unfolding activity within chloroplasts.
B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous ...transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.
Hospital outbreaks of COVID19 result in considerable mortality and disruption to healthcare services and yet little is known about transmission within this setting. We characterise within hospital ...transmission by combining viral genomic and epidemiological data using Bayesian modelling amongst 2181 patients and healthcare workers from a large UK NHS Trust. Transmission events were compared between Wave 1 (1st March to 25th J'uly 2020) and Wave 2 (30th November 2020 to 24th January 2021). We show that staff-to-staff transmissions reduced from 31.6% to 12.9% of all infections. Patient-to-patient transmissions increased from 27.1% to 52.1%. 40%-50% of hospital-onset patient cases resulted in onward transmission compared to 4% of community-acquired cases. Control measures introduced during the pandemic likely reduced transmissions between healthcare workers but were insufficient to prevent increasing numbers of patient-to-patient transmissions. As hospital-acquired cases drive most onward transmission, earlier identification of nosocomial cases will be required to break hospital transmission chains.
Innovative plant breeding technology is an absolute necessity to enhance agriculture production in order to have an ambition of feeding nutritious food to the ever-increasing population. Current ...advances in CRISPR/Cas genome editing technology have led to effective targeted changes in most plants that promise to accelerate crop improvement. Here we discussed the discovery of CRISPR/Cas technology, associated manipulations for plant genome editing and its potential applications in the plant breeding. We emphasized mainly on the most essential applications of CRISPR/Cas genome editing in crop improvement, such as crop trait improvement (yield and biotic/abiotic stress tolerance), developments in optimizing gene regulation, strategies for generating virus resistance in plants, and the use of high throughput mutant libraries. Finally, the challenges and opportunities for plant breeding in precision agriculture and its bright future discussed.
Wheat is one of the most important staple grains in the world and the leading source of calories, production is limited by biotic stress. There is a number of pathogen attacks on wheat crops, ...depending on environmental conditions. In some cases, more than one crop pathogen attack leads to higher damage or decrease susceptibility. There are very few studies in the field of multiple pathogen interactions; in this study, we analyzed the co-infectionof wheat with fungal and bacterial pathogens. Field isolated Xanthomonas translucens and Xanthomonas compestris bacteria have been used against GM-322 and PDKV varieties co-infected with Fusarium fungus spp. In our experiment, we used Fusarium oxysporum, Fusarium graminearum, Fusarium equitus. Compared to the combined effect of the fungus and bacteria, we measured the length and width of the infected leaf part. We have observed that there is more susceptibility to X. compestris and F. graminearium in the GM-322 wheat variety. The second susceptible cvs was PDKV when we co-infected F. oxysporum and then X. translucens fungal and bacterial infected symptom analysis showed yellow stripes on the leaf surface of the wheat crop. We observed head blight in wheat when it was infected with F. graminearum and X. compestris. As a result, we concluded that varietal susceptibility also depends on co-infection pathogen attacks and their synergetic interaction.
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