Publications by authors named "Duncan Smith"

263 Publications

Mesophyll photosynthetic sensitivity to leaf water potential in Eucalyptus: a new dimension of plant adaptation to native moisture supply.

New Phytol 2021 Feb 25. Epub 2021 Feb 25.

Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin, USA, 53705.

Photosynthetic sensitivity to drought is a fundamental constraint on land-plant evolution and ecosystem function. However, little is known about how the sensitivity of photosynthesis to non-stomatal limitations varies among species in the context of phylogenetic relationships. Using saplings of 10 Eucalyptus species, we measured maximum CO -saturated photosynthesis using A-c curves at several different leaf water potentials (ψ ) to quantify mesophyll photosynthetic sensitivity to ψ (MPS), a measure of how rapidly non-stomatal limitations to carbon uptake increase with declining ψ . MPS was compared to the macroclimatic moisture availability of the species' native habitats, while accounting for phylogenetic relationships. We found that species native to mesic habitats have greater MPS but higher maximum photosynthetic rates during non-water-stressed conditions, revealing a trade-off between maximum photosynthesis and drought sensitivity. Species with lower turgor loss points have less MPS, indicating coordination among photosynthetic and water-relations traits. By accounting for phylogenetic relationships among closely related species, we provide the first compelling evidence that MPS in Eucalyptus evolved in adaptive fashion with climatically determined moisture availability, opening further study of this poorly explored dimension of plant adaptation to drought.
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http://dx.doi.org/10.1111/nph.17304DOI Listing
February 2021

Mistletoes and their eucalypt hosts differ in the response of leaf functional traits to climatic moisture supply.

Oecologia 2021 Feb 17. Epub 2021 Feb 17.

Department of Botany, University of Wisconsin-Madison, Madison, WI, 53706, USA.

Trade-offs between photosynthesis and the costs of resource capture inform economic strategies of plants across environmental gradients and result in predictable variation in leaf traits. However, understudied functional groups like hemiparasites that involve dramatically different strategies for resource capture may have traits that deviate from expectations. We measured leaf traits related to gas exchange in mistletoes and their eucalypt hosts along a climatic gradient in relative moisture supply, measured as the ratio of precipitation to pan evaporation (P/E), in Victoria, Australia. We compared traits for mistletoes vs. hosts as functions of relative moisture supply and examined trait-trait correlations in both groups. Eucalypt leaf traits responded strongly to decreasing P/E, consistent with economic theory. Leaf area and specific leaf area (SLA) decreased along the P/E gradient, while C:N ratio, leaf thickness, N per area, and δC all increased. Mistletoes responded overall less strongly to P/E based on multivariate analyses; individual traits sometimes shifted in parallel with those of hosts, but SLA, leaf thickness, and N per area showed no significant change across the gradient. For mistletoes, leaf thickness was inversely related to leaf dry matter content (LDMC), with no relationship between SLA and mass-based N. In mistletoes, reduced costs of transpiration (reflecting their lack of roots) and abundant succulent leaf tissue help account for observed differences from their eucalypt hosts. Trait-based analysis of atypical functional types such as mistletoes help refine hypotheses based on plant economics and specialized adaptations to resource limitation.
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http://dx.doi.org/10.1007/s00442-021-04867-1DOI Listing
February 2021

Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2.

Front Plant Sci 2020 7;11:604663. Epub 2021 Jan 7.

Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease (COVID-19) which has recently emerged as a potential threat to global public health. SARS-CoV-2 is the third known human coronavirus that has huge impact on the human population after SARS-CoV and MERS-CoV. Although some vaccines and therapeutic drugs are currently in clinical trials, none of them are approved for commercial use yet. As with SARS-CoV, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the cell entry receptor to enter into the host cell. In this study, we have transiently produced human ACE2 fused with the Fc region of human IgG1 in and the neutralization efficacy of the plant-produced ACE2-Fc fusion protein was assessed. The recombinant ACE2-Fc fusion protein was expressed in at 100 μg/g leaf fresh weight on day 6 post-infiltration. The recombinant fusion protein showed potent binding to receptor binding domain (RBD) of SARS-CoV-2. Importantly, the plant-produced fusion protein exhibited potent anti-SARS-CoV-2 activity . Treatment with ACE2-Fc fusion protein after viral infection dramatically inhibit SARS-CoV-2 infectivity in Vero cells with an IC value of 0.84 μg/ml. Moreover, treatment with ACE2-Fc fusion protein at the pre-entry stage suppressed SARS-CoV-2 infection with an IC of 94.66 μg/ml. These findings put a spotlight on the plant-produced ACE2-Fc fusion protein as a potential therapeutic candidate against SARS-CoV-2.
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http://dx.doi.org/10.3389/fpls.2020.604663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874119PMC
January 2021

Phenanthroline impairs βAPP processing and expression, increases p53 protein levels and induces cell cycle arrest in human neuroblastoma cells.

Brain Res Bull 2021 Feb 5;170:29-38. Epub 2021 Feb 5.

Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, 73170, Thailand; Centre National de la Recherche Scientifique, 2 rue Michel Ange, Paris, 75016, France. Electronic address:

Mis-functional βAPP processing is deemed to be the major phenomenon resulting in increased neuronal cell death, impaired neurogenesis and the loss of synapses, which eventually manifest as the complex symptoms of Alzheimer's disease. Despite of several milestones having been achieved in the field of drug development, the stigma of the disorder as an incurable disease still remains. Some ADAM proteases mediate the physiological non-amyloidogenic α-secretase processing of βAPP that generates neuroprotective sAPPα production. Earlier studies have also pointed out the role of p53 in Alzheimer's disease neuropathology, although a direct link with metalloprotease activities remains to be established. In this study, we explored the consequences of α-secretase inhibition on p53 status in cultured human neuroblastoma SH-SY5Y cells by means of specific inhibitors of ADAM10 and ADAM17 and the metal chelator and general metalloprotease inhibitor phenanthroline. We establish that, beyond the ability of all inhibitors to affect sAPPα production to varying degrees, phenanthroline specifically and dose-dependently lessened βAPP expression, a phenomenon that correlated with a strong increase in p53 protein levels and a concomitant decrease of the p53-degrading calpain protease. Furthermore, treatment of cells at concentrations of phenanthroline similar to those inducing increased levels of p53 induced cell cycle arrest leading to apoptosis. Altogether, our results identify new roles of phenanthroline in perturbing βAPP, p53 and calpain biology, and suggest that the use of this compound and its derivatives as antimicrobial and anti-cancer therapies might trigger Alzheimer's disease pathogenesis.
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http://dx.doi.org/10.1016/j.brainresbull.2021.02.001DOI Listing
February 2021

Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair.

PLoS Genet 2021 Jan 25;17(1):e1009322. Epub 2021 Jan 25.

Department of Biology, New York University, New York City, New York, United State of America.

DNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability.
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http://dx.doi.org/10.1371/journal.pgen.1009322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861531PMC
January 2021

Detection of antibodies to duck tembusu virus in human population with or without the history of contact with ducks.

Transbound Emerg Dis 2021 Jan 20. Epub 2021 Jan 20.

Faculty of Veterrinary Medicine, Kasetsart University, Bangkok, Thailand.

Duck tembusu virus (DTMUV) is an emerging duck pathogen in China and other Asian countries. It is unclear whether this emerging zoonotic infection poses a threat to humans. A previous study in 2012 showed surprisingly high rates of seropositivity and positive viral detection by RT-PCR in duck farm workers in China. To understand the nature of the threat of this emerging virus, we studied the neutralizing antibody response to a local isolate of DTMUV in an at-risk population, who were workers in duck farms and residents around farming areas in Central Thailand where DTMUV had been previously detected, and in a not-at-risk population, who were people living in the same or neighbouring province, but at a distance from the farms and who had no contact with ducks. The sera from the at-risk population showed higher anti-DTMUV neutralizing antibody titres as compared with those of the not-at-risk population. However, within the at-risk population, workers with direct contact with ducks did not show higher neutralizing titres than those without direct contact. Interestingly, some people in the not-at-risk group also displayed high neutralizing antibody titres to DTMUV. These sera were tested against other endemic Flaviviruses and showed no or low cross-reactivity suggesting the specificity of the neutralizing activity against DTMUV. These data raise a possibility of DTMUV as a potential zoonotic pathogen but the mode of transmission of the virus from ducks or other possible hosts to humans should be explored further.
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http://dx.doi.org/10.1111/tbed.13998DOI Listing
January 2021

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

Nat Protoc 2021 02 13;16(2):1193-1218. Epub 2021 Jan 13.

Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.
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http://dx.doi.org/10.1038/s41596-020-00454-5DOI Listing
February 2021

A functional interaction between GRP78 and Zika virus E protein.

Sci Rep 2021 Jan 11;11(1):393. Epub 2021 Jan 11.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Nakhon Pathom, 73170, Thailand.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused significant public health concerns around the world, partly because of an association with microcephaly in babies born to mothers who were infected with ZIKV during pregnancy. As a recently emerging virus, little is known as to how the virus interacts with the host cell machinery. A yeast-2-hybrid screen for proteins capable of interacting with the ZIKV E protein domain III, the domain responsible for receptor binding, identified 21 proteins, one of which was the predominantly ER resident chaperone protein GRP78. The interaction of GRP78 and ZIKV E was confirmed by co-immunoprecipitation and reciprocal co-immunoprecipitation, and indirect immunofluorescence staining showed intracellular and extracellular co-localization between GRP78 and ZIKV E. Antibodies directed against the N-terminus of GRP78 were able to inhibit ZIKV entry to host cells, resulting in significant reductions in the levels of ZIKV infection and viral production. Consistently, these reductions were also observed after down-regulation of GRP78 by siRNA. These results indicate that GRP78 can play a role mediating ZIKV binding, internalization and replication in cells. GRP78 is a main regulator of the unfolded protein response (UPR), and the study showed that expression of GRP78 was up-regulated, and the UPR was activated. Increases in CHOP expression, and activation of caspases 7 and 9 were also shown in response to ZIKV infection. Overall these results indicate that the interaction between GRP78 and ZIKV E protein plays an important role in ZIKV infection and replication, and may be a potential therapeutic target.
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http://dx.doi.org/10.1038/s41598-020-79803-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801745PMC
January 2021

as a Transient Expression Host to Produce Auxin Analogs.

Front Plant Sci 2020 20;11:581675. Epub 2020 Nov 20.

Department of Natural Product Biosynthesis, Max Planck Institute for Chemical Ecology, Jena, Germany.

Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce "new-to-nature" derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in for industrial scale protein and product expression, this work provides insight into the capacity of to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites.
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http://dx.doi.org/10.3389/fpls.2020.581675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714751PMC
November 2020

Analysis of Tembusu virus infection of human cell lines and human induced pluripotent stem cell derived hepatocytes.

Virus Res 2021 Jan 5;292:198252. Epub 2020 Dec 5.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Molecular Medicine Graduate Program, Faculty of Science, Mahidol University, Bangkok, Thailand. Electronic address:

Tembusu virus (TMUV) causes disease in poultry, especially in ducks, resulting in abnormality in egg production and with high morbidity and mortality, resulting in great loss in duck farming industry in China and Southeast Asia. Previous studies on the pathogenesis of TMUV infection have been mostly conducted in poultry, with a few studies being undertaken in mice. While TMUV does not cause disease in humans, it has been reported that antibodies against TMUV have been found in serum samples from duck farmers, and thus data on TMUV infection in humans is limited, and the pathogenesis is unclear. In this study we investigated the cell tropism and potential susceptibility of humans to TMUV using several human cell lines. The results showed that human nerve and liver cell lines were both highly susceptible and permissive, while human kidney cells were susceptible and permissive, albeit to a lower degree. In addition, human muscle cells, lung epithelial cells, B-cells, T-cells and monocytic cells were largely refractory to TMUV infection. This data suggests that liver, neuron and kidney are potential target organs during TMUV infection in humans, consistent with what has been found in animal studies.
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http://dx.doi.org/10.1016/j.virusres.2020.198252DOI Listing
January 2021

Review a brief history of coronaviruses in Thailand.

Authors:
Duncan R Smith

J Virol Methods 2021 03 4;289:114034. Epub 2020 Dec 4.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phutthamonthon Sai 4 Road, Salaya, Nakhon Pathom, 73170, Thailand. Electronic address:

As with many countries around the world, Thailand is currently experiencing restrictions to daily life as a consequence of the worldwide transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is the third respiratory syndrome coronavirus to be introduced into Thailand, following previous importation of cases of the severe acute respiratory syndrome coronavirus (SARS) and the Middle East respiratory syndrome coronavirus (MERS). Unlike SARS and MERS, SARS-CoV-2 was able to establish local transmission in Thailand. In addition to the imported coronaviruses, Thailand has a number of endemic coronaviruses that can affect livestock and pet species, can be found in bats, as well as four human coronaviruses that are mostly associated with the common cold. This article seeks to review what is known on both the endemic and imported coronaviruses in Thailand.
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http://dx.doi.org/10.1016/j.jviromet.2020.114034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831773PMC
March 2021

Andrographolide and Its 14-Aryloxy Analogues Inhibit Zika and Dengue Virus Infection.

Molecules 2020 Oct 30;25(21). Epub 2020 Oct 30.

School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, China.

Andrographolide is a labdene diterpenoid with potential applications against a number of viruses, including the mosquito-transmitted dengue virus (DENV). In this study, we evaluated the anti-viral activity of three 14-aryloxy analogues (ZAD-1 to ZAD-3) of andrographolide against Zika virus (ZIKV) and DENV. Interestingly, one analogue, ZAD-1, showed better activity against both ZIKV and DENV than the parental andrographolide. A two-dimension (2D) proteomic analysis of human A549 cells treated with ZAD-1 compared to cells treated with andrographolide identified four differentially expressed proteins (heat shock 70 kDa protein 1 (HSPA1A), phosphoglycerate kinase 1 (PGK1), transketolase (TKT) and GTP-binding nuclear protein Ran (Ran)). Western blot analysis confirmed that ZAD-1 treatment downregulated expression of HSPA1A and upregulated expression of PGK1 as compared to andrographolide treatment. These results suggest that 14-aryloxy analogues of andrographolide have the potential for further development as anti-DENV and anti-ZIKV agents.
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http://dx.doi.org/10.3390/molecules25215037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662321PMC
October 2020

Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana.

Sci Rep 2020 10 19;10(1):17698. Epub 2020 Oct 19.

Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.
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http://dx.doi.org/10.1038/s41598-020-74904-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573609PMC
October 2020

Even when the seasons change our allometry stays the same. A Commentary on: 'Corner's rules pass the test of time: little effect of phenology on leaf-shoot and other scaling relationships'.

Authors:
Duncan D Smith

Ann Bot 2020 11;126(7):iii-iv

University of Wisconsin-Madison, Department of Botany, Madison, WI, USA.

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http://dx.doi.org/10.1093/aob/mcaa166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684695PMC
November 2020

Analysis of the virus propagation profile of 14 dengue virus isolates in Aedes albopictus C6/36 cells.

BMC Res Notes 2020 Oct 12;13(1):481. Epub 2020 Oct 12.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthon Sai 4, Salaya, Nakhon Pathom, 73170, Thailand.

Objective: The mosquito transmitted RNA virus dengue virus (DENV) shows significant variation as a consequence of the lack of proofreading activity of the RNA-dependent RNA polymerase that synthesizes new virus genomes. How this variation affects DENV replication, and how this in turn impacts drug development remains largely unknown. Given the technical limitations in working with large numbers of isolates few studies have sought to investigate this area. This study used a panel of 14 DENV isolates of different serotypes and origins to determine how much virus replication in Aedes albopictus C6/36 cells was affected by DENV variability.

Results: The results showed that there was considerable variation, with peak titers ranging from 6Log10 to 8Log10, and maximum titer being reached from day 3 to day 9 post infection. While strains from DENV 1 and 4 serotypes showed considerable uniformity, DENV 2 and 3 strains showed much greater variation. Overall, these results show that serotype specific strain variation can have a significant impact on DENV replication, suggesting that studies either investigating DENV pathogenesis or developing drug therapeutics should consider the contribution of DENV variability.
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http://dx.doi.org/10.1186/s13104-020-05325-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552352PMC
October 2020

Hold on Tight: Lagging-Strand DNA Polymerases Synthesize Multiple Okazaki Fragments without Letting Go.

Mol Cell 2020 10;80(1):6-8

Department of Biology, New York University, New York, NY, USA. Electronic address:

Kapadia et al. (2020) use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.
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http://dx.doi.org/10.1016/j.molcel.2020.09.010DOI Listing
October 2020

Patterns of behaviour in nursing staff actioning the afferent limb of the rapid response system (RRS): A focused ethnography.

J Adv Nurs 2020 Dec 30;76(12):3548-3562. Epub 2020 Sep 30.

School of Health Sciences, City University of London, London, UK.

Aim: To improve understanding of afferent limb behaviour in acute hospital ward settings, to define and specify who needs to do what differently and to report what afferent limb behaviours should be targeted in a subsequent multi-phase, theory-based, intervention development process.

Design: Focused ethnography was used including direct observation of nursing staff enacting afferent limb behaviours and review of vital signs charts.

Methods: An observation guide focused observation on "key moments" of the afferent limb. Descriptions of observations from between 7 January 2019-18 December 2019 were recorded in a field journal alongside reflexive notes. Vital signs and early warning scores from charts were reviewed and recorded. Field notes were analysed using structured content analysis. Observed behaviour was compared with expected (policy-specified) behaviour.

Results: Observation was conducted for 300 hr. Four hundred and ninety-nine items of data (e.g., an episode of observation or a set of vital signs) were collected. Two hundred and eighty-nine (58%) items of data were associated with expected (i.e. policy-specified) afferent limb behaviour; 210 (42%) items of data were associated with unexpected afferent limb behaviour (i.e. alternative behaviour or no behaviour). Ten specific behaviours were identified where the behaviour observed deviated (negatively) from policy or where no action was taken when it should have been. One further behaviour was seen to expedite the assessment of a deteriorating patient by an appropriate responder and was therefore considered a positive deviance.

Conclusion: Afferent limb failure has been described as a problem of inconsistent staff behaviour. Eleven potential target behaviours for change are reported and specified using a published framework.

Impact: Clear specification of target behaviour will allow further enquiry into the determinants of these behaviours and the development of a theory-based intervention that is more likely to result in behaviour change and can be tested empirically in future research.
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http://dx.doi.org/10.1111/jan.14551DOI Listing
December 2020

Production of Zika Virus Virus-Like Particles.

Methods Mol Biol 2021 ;2183:183-203

Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus and thus present a similar antigenic profile. These properties make them good candidates for vaccine development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation of the VLP is achieved through western blotting and electron microscopy.
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http://dx.doi.org/10.1007/978-1-0716-0795-4_10DOI Listing
January 2021

Estimating tissue expander volume and skin availability using VECTRA 3D imaging software.

J Plast Reconstr Aesthet Surg 2021 03 25;74(3):644-710. Epub 2020 Aug 25.

State Burn Service, Fiona Stanley Hospital, 11 Robin Warren Drive, Murdoch, WA 6150, Australia.

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http://dx.doi.org/10.1016/j.bjps.2020.08.070DOI Listing
March 2021

Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein.

Sci Rep 2020 09 2;10(1):14493. Epub 2020 Sep 2.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Rd, Salaya, 73170, Nakorn Pathom, Thailand.

Dengue virus (DENV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADE) of infection. Phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DENV infection. This study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection.
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http://dx.doi.org/10.1038/s41598-020-71407-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467932PMC
September 2020

Oxyresveratrol Inhibits IL-1β-Induced Inflammation via Suppressing AKT and ERK1/2 Activation in Human Microglia, HMC3.

Int J Mol Sci 2020 Aug 22;21(17). Epub 2020 Aug 22.

Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.

Oxyresveratrol (OXY), a major phytochemical component derived from several plants, has been proved to have several pharmacological properties. However, the role of OXY in regulating neuroinflammation is still unclear. Here, we focused mainly on the anti-neuroinflammatory effects at the cellular level of OXY in the interleukin-1 beta (IL-1β)-stimulated HMC3 human microglial cell line. We demonstrated that OXY strongly decreased the release of IL-6 and MCP-1 from HMC3 cells stimulated with IL-1β. Nevertheless, IL-1β could not induce the secretion of TNF-α and CXCL10 in this specific cell line, and that OXY did not have any effects on reducing the basal level of these cytokines in the sample culture supernatants. The densitometry analysis of immunoreactive bands from Western blot clearly indicated that IL-1β does not trigger the nuclear factor-kappa B (NF-κB) signaling. We discovered that OXY exerted its anti-inflammatory role in IL-1β-induced HMC3 cells by suppressing IL-1β-induced activation of the PI3K/AKT/p70S6K pathway. Explicitly, the presence of OXY for only 4 h could strongly inhibit AKT phosphorylation. In addition, OXY had moderate effects on inhibiting the activation of ERK1/2. Results from immunofluorescence study further confirmed that OXY inhibited the phosphorylation of AKT and ERK1/2 MAPK upon IL-1β stimulation in individual cells. These findings suggest that the possible anti-inflammatory mechanisms of OXY in IL-1β-induced HMC3 cells are mainly through its ability to suppress the PI3K/AKT/p70S6K and ERK1/2 MAPK signal transduction cascades. In conclusion, our study provided accumulated data that OXY is able to suppress IL-1β stimulation signaling in human microglial cells, and we believe that OXY could be a probable pharmacologic agent for altering microglial function in the treatment of neuroinflammation.
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http://dx.doi.org/10.3390/ijms21176054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504001PMC
August 2020

Limited physiological acclimation to recurrent heatwaves in two boreal tree species.

Tree Physiol 2020 12;40(12):1680-1696

Department of Botany, University of Wisconsin, 322 Birge Hall, Madison, WI 53706, USA.

The intensity of extreme heat and drought events has drastically risen in recent decades and will likely continue throughout the century. Northern forests have already seen increases in tree mortality and a lack of new recruitment, which is partially attributed to these extreme events. Boreal species, such as paper birch (Betula papyrifera) and white spruce (Picea glauca), appear to be more sensitive to these changes than lower-latitude species. Our objectives were to investigate the effects of repeated heatwaves and drought on young paper birch and white spruce trees by examining (i) responses in leaf gas exchange and plant growth and (ii) thermal acclimation of photosynthetic and respiratory traits to compare ecophysiological responses of two co-occurring, yet functionally dissimilar species. To address these objectives, we subjected greenhouse-grown seedlings to two consecutive summers of three 8-day long, +10 °C heatwaves in elevated atmospheric CO2 conditions with and without water restriction. The data show that heatwave stress reduced net photosynthesis, stomatal conductance and growth-more severely so when combined with drought. Acclimation of both photosynthesis and respiration did not occur in either species. The combination of heat and drought stress had a similar total effect on both species, but each species adjusted traits differently to the combined stress. Birch experienced greater declines in gas exchange across both years and showed moderate respiratory but not photosynthetic acclimation to heatwaves. In spruce, heatwave stress reduced the increase in basal area in both experimental years and had a minor effect on photosynthetic acclimation. The data suggest these species lack the ability to physiologically adjust to extreme heat events, which may limit their future distributions, thereby altering the composition of boreal forests.
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http://dx.doi.org/10.1093/treephys/tpaa102DOI Listing
December 2020

Flavaglines as natural products targeting eIF4A and prohibitins: From traditional Chinese medicine to antiviral activity against coronaviruses.

Eur J Med Chem 2020 Oct 15;203:112653. Epub 2020 Jul 15.

INSERM U 1260, Regenerative Nanomedicine (RNM), FMTS, 11 Rue Humann, 67000, Strasbourg, France. Electronic address:

Flavaglines are cyclopenta[b]benzofurans found in plants of the genus Aglaia, several species of which are used in traditional Chinese medicine. These compounds target the initiation factor of translation eIF4A and the scaffold proteins prohibitins-1 and 2 (PHB1/2) to exert various pharmacological activities, including antiviral effects against several types of viruses, including coronaviruses. This review is focused on the antiviral effects of flavaglines and their therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
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http://dx.doi.org/10.1016/j.ejmech.2020.112653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362831PMC
October 2020

Characterization of extended-spectrum-β-lactamase producing Klebsiella pneumoniae phage KP1801 and evaluation of therapeutic efficacy in vitro and in vivo.

Sci Rep 2020 07 16;10(1):11803. Epub 2020 Jul 16.

Institute of Molecular Biosciences, Mahidol University, Bangkok, 73170, Thailand.

Extended spectrum β lactamase-producing Klebsiella pneumoniae (ESBL-KP) is being reported with high morbidity and mortality rates and is considered as the highest priority for new antimicrobial strategies. To develop an alternative antimicrobial agent, phage KP1801 with broad lytic activity was isolated. The genome of phage KP1801 was double stranded DNA of 49,835 base pairs, with a GC content of 50.26%. There were 75 putative open reading frames. Phage KP1801 was classified as being in the order Caudovirales, belonging to the Siphoviridae family. About 323 proteins were detected by shotgun proteome analysis. The phage inhibited biofilm formation and reduced pre-formed biofilm in a dose dependent manner. Scanning electron microscopic studies demonstrated a membrane damage of bacterial cells treated with phage, resulting in cell death. Prophylactic and therapeutic efficacies of the phage were evaluated in Galleria mellonella. Administration of ESBL-KP infection with phage significantly improved the survival of G. mellonella. The number of intracellular bacteria in larvae showed a significant decrease compared with untreated control while the number of phage increased. These studies suggested that phage KP1801 has the potential for development as an alternative for antibiotics and biocontrol agents against ESBL-KP infection.
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http://dx.doi.org/10.1038/s41598-020-68702-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367294PMC
July 2020

Activity of vitamin D receptor agonists against dengue virus.

Sci Rep 2020 07 2;10(1):10835. Epub 2020 Jul 2.

Institute of Molecular Biosciences, Mahidol University, Salaya, 73170, Thailand.

Infections with the mosquito-transmitted dengue virus (DENV) are a pressing public health problem in many parts of the world. The recently released commercial vaccine for DENV has encountered some problems, and there is still no effective drug to treat infections. Vitamin D has a well characterized role in calcium and phosphorus homeostasis, but additionally has a role in the immune response to bacterial and viral pathogens. In this study a number of fused bicyclic derivatives of 1H-pyrrolo[1,2]imidazol-1-one with vitamin D receptor (VDR) agonist activity were evaluated for possible anti-DENV activity. The results showed that five of the compounds were able to significantly inhibit DENV infection. The most effective compound, ZD-3, had an EC value of 7.47 μM and a selective index of 52.75. The compounds were only effective when used as a post-infection treatment and treatment significantly reduced levels of infection, virus output, DENV protein expression and genome copy number. These results suggest that these VDR agonists have the potential for future development as effective anti-DENV agents.
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http://dx.doi.org/10.1038/s41598-020-67783-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331731PMC
July 2020

Comparative analysis of a Thai congenital-Zika-syndrome-associated virus with a Thai Zika-fever-associated virus.

Arch Virol 2020 Aug 30;165(8):1791-1801. Epub 2020 May 30.

Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom, Thailand.

In this study, we compared the characteristics of two strains of Zika virus (ZIKV) isolated in Thailand, one isolated from a febrile patient and one isolated from tissues of a fetus medically terminated due to congenital Zika syndrome (CZS). Replication profiles showed that the isolate from the fetal tissues replicated significantly more slowly than the fever-associated isolate in human lung A549 cells during the first 24 hours postinfection but showed a similar growth profile over longer-term infection. A much smaller difference was observed in Aedes albopictus C6/36 cells. In a quasispecies analysis, a high proportion (approximately 20%) of nonfunctional genomes was identified, caused by an adenine insertion in the prM gene. This insertion was found to be present in two Thai fever strains and as such may represent a common feature of Thai endemic ZIKV. Comparison between viral RNA copy number and viral titer showed that the isolate from fetal tissues was produced more efficiently than the fever-associated isolate. Together, these results suggest that different ZIKV isolates differ in their replication capacity, and this might contribute to the fetotropic potential of a particular strain.
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http://dx.doi.org/10.1007/s00705-020-04667-7DOI Listing
August 2020

Effective production of recombinant Δ60VP1 chicken anemia virus protein in Escherichia coli and its application to a serodiagnostic indirect ELISA.

J Virol Methods 2020 May 21;282:113887. Epub 2020 May 21.

Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address:

Chicken anemia virus (CAV) causes severe anemia and immunosuppression in chickens. VP1 is the main capsid protein, and is suitable for diagnostic kit development, however, it has 24 arginine residues in the first forty N-terminal amino acids of the protein causing toxicity to bacteria leading to reduced prokaryotic expression. In this study, a 60 amino acid N-terminally truncated VP1 (Δ60VP1) which removes the toxic region was expressed in Escherichia coli and the resultant insoluble recombinant protein was purified by Ni-NTA affinity chromatography with anionic denaturing detergents. The high amounts of purified Δ60VP1 produced (150 mg/L) retained appropriate antigenicity and the antigen was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of CAV. One hundred fifty-two chicken serum samples (n = 152) were evaluated using the newly developed Δ60VP1 indirect ELISA (cutoff value = 7.58 % S/P). The sensitivity and specificity of the Δ60VP1 indirect ELISA were 87.50 % and 95.31 %, respectively, while the agreement between the Δ60VP1 indirect ELISA and the commercial IDEXX CAV ELISA was 90.79 % (kappa = 0.814). In this study, we have developed an alternative VP1 production platform in E. coli by truncating the N-terminal 60 amino acids (Δ60VP1) and using anionic denaturing detergents during the purification to successfully solubilize the insoluble Δ60VP1. The antigen was purified with high yield and good immunoreactivity, and an indirect ELISA was developed. The assay could potentially be applied to large-scale CAV serosurveillance.
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http://dx.doi.org/10.1016/j.jviromet.2020.113887DOI Listing
May 2020

Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation.

PLoS Genet 2020 05 7;16(5):e1008755. Epub 2020 May 7.

Department of Biology, New York University, New York, NY, United States of America.

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.
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http://dx.doi.org/10.1371/journal.pgen.1008755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237047PMC
May 2020

Proteomic analysis of CHIKV-infected human fibroblast-like synoviocytes: Identification of host factors potentially associated with CHIKV replication and cellular pathogenesis.

Microbiol Immunol 2020 Jun 12;64(6):445-457. Epub 2020 May 12.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes arthralgic fever. Fibroblast-like synoviocytes play a key role in joint damage in inflammatory arthritides and can additionally serve as target cells for CHIKV infection. To gain a better understanding of CHIKV-induced arthralgia, the interaction between CHIKV and synoviocytes was investigated at the protein level. A gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS/MS) approach was used to examine protein expression from primary human fibroblast-like synoviocytes (HFLS) infected with clinical isolates of CHIKV at 12 and 24 hr post infection. Our analysis identified 259 and 241 proteins of known function that were differentially expressed (>1.5 or <-1.5 fold change) following CHIKV infection at 12 and 24 hpi, respectively. These proteins are involved in cellular homeostasis, including cellular trafficking, cytoskeletal organization, immune response, metabolic process, and protein modification. Some of these proteins have previously been reported to participate in arthralgia/arthritis and the death of infected cells. Our results provide information on the CHIKV-induced modulation of cellular proteins of HFLS at an early stage of infection, as well as highlighting biological processes associated with CHIKV infection in the main target cells of the joint.
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http://dx.doi.org/10.1111/1348-0421.12793DOI Listing
June 2020

Discordant Activity of Kaempferol Towards Dengue Virus and Japanese Encephalitis Virus.

Molecules 2020 Mar 10;25(5). Epub 2020 Mar 10.

Institute of Molecular Biosciences, Mahidol University, Salaya 73170, Thailand.

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.
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http://dx.doi.org/10.3390/molecules25051246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179415PMC
March 2020