Potential small‐molecule drugs as available weapons to fight novel coronavirus (2019‐nCoV): A review

Since the new coronavirus known as 2019‐nCoV (severe acute respiratory syndrome coronavirus 2, SARS‐CoV‐2) has widely spread in Wuhan, China, with severe pneumonia, scientists and physicians have made remarkable efforts to use various options such as monoclonal antibodies, peptides, vaccines, small‐molecule drugs and interferon therapies to control, prevent or treatment infections of 2019‐nCoV. However, no vaccine or drug has yet been confirmed to completely treat 2019‐nCoV. In this review, we focus on the use of potential available small‐molecule drug candidates for treating infections caused by 2019‐nCoV.     

A five‐step risk management process for geriatric dental practice during SARS‐CoV‐2 pandemic

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an RNA virus that causes coronavirus infection (COVID‐19). COVID‐19 is a highly contagious disease transmitted through respiratory droplets, saliva and other contact routes. Within 10 months of its outbreak, SARS‐CoV‐2 has infected more than 23 million people around the world. Evidence suggests that older adults are the most vulnerable to infection and have an increased risk of mortality. Reduced immunity and underlying medical conditions make them risk‐prone and vulnerable to critical care. Older adults affected with the SARS‐CoV‐2 virus present with distinct clinical manifestations necessitating specific treatment needs and management protocols. While it is crucial to prevent the spread of novel coronavirus (2019‐nCoV), the role of oral healthcare workers in addressing the specific needs of ageing adult patients by adopting specific guidelines and appropriate infection control protocols is timely. This paper aims to develop specific guidelines and protocols for the dental management of geriatric patients during the COVID‐19 pandemic.     

Comparison of pulsed‐field gel electrophoresis and three rep‐PCR methods for evaluating the genetic relatedness of Stenotrophomonas maltophilia isolates

Aims: In this study, three facile repetitive‐sequence PCR (rep‐PCR) techniques have been compared with the pulsed‐field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. Methods and Results: The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR‐based methods, i.e. enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), BOX‐PCR and repetitive extragenic palindromic‐PCR (REP‐PCR). When compared with PFGE, ERIC‐PCR displayed a much lower discriminatory power, whereas BOX‐PCR and REP‐PCR had a comparable discriminatory power for close genetic‐related isolates. Conclusion: BOX‐PCR and REP‐PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. Significance and Impact of the Study: A rapid method for determining S. maltophilia’s close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia.     

Comparison of a culture‐based and a PCR‐based methods for estimating bacterial abundance on eggshells,with comments on statistical analyses

Field ornithologists have used traditional culture‐based techniques to determine the presence and abundance of microbes on surfaces such as eggshells, but culture‐independent PCR‐based methods have recently been introduced. We compared the traditional culture‐based and the real‐time PCR‐based methods for detecting and quantifying Escherichia coli on the eggshells of Eurasian Magpies (Pica pica). PCR estimates of bacterial abundance were ～10 times higher than culture‐based estimates, and the culture‐based technique failed to detect bacteria at lower densities. When both methods detected bacteria, bacterial densities determined by the two methods were positively correlated, indicating that both methods can be used to study factors affecting bacterial densities. The difference between the two methods is consistent with generally acknowledged higher sensitivity of the PCR method, but the extent of the difference in our study (10×) may have been influenced by both a PCR‐based overestimation and culture‐based underestimation of bacterial densities. Our results also illustrate that bacterial counts may sometimes produce left‐censored data (i.e., we did not detect E. coli in 62% of our samples using the culture‐based method). Specific statistical methods have been developed for analyzed left‐censored data, but, to our knowledge, have not been used by ornithologists. In future studies, investigators studying bacterial loads should provide information about the possible degree of left censoring and should justify their choice of statistical methods from the broad set of available methods, including those explicitly designed for censored data.     

Flexible Nano‐felts of Carbide‐Derived Carbon with Ultra‐high Power Handling Capability

Nano‐fibrous felts (nano‐felts) of carbide‐derived carbon (CDC) have been developed from the precursor of electrospun titanium carbide (TiC) nano‐felts. Conformal transformation of TiC into CDC conserves main features of the precursor including the high interconnectivity and structural integrity; the developed TiC‐CDC nano‐felts are mechanically flexible/resilient, and can be used as electrode material for supercapacitor application without the addition of any binder. After synthesis through chlorination of the precursor at 600 °C, the TiC‐CDC nano‐fibers show an average pore size of ～1nm, a high specific surface area of 1390 m²/g; and the nano‐fibers have graphitic carbon ribbons embedded in a highly disordered carbon matrix. Graphitic carbon is preserved from the precursor nano‐fibers where a few graphene layers surround TiC nanocrystallites. Electrochemical measurements show a high gravimetric capacitance of 110 F/g in aqueous electrolyte (1 M H₂SO₄) and 65 F/g in organic electrolyte (1.5 M TEA‐BF₄ in acetonitrile). Because of the unique microstructure of TiC‐CDC nano‐felts, a fade of the capacitance of merely 50% at a high scan rate of 5 V/s is observed. A fade of just 15% is observed for nano‐felt film electrodes tested in 1 M H₂ SO₄ at 1 V/s, resulting in a high gravimetric capacitance of 94 F/g. Such a high rate performance is only known for graphene or carbon‐onion based supercapacitors, whereas binders have to be used for the fabrication of those supercapacitors.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Current trends in large‐scale viral surveillance methods in mosquitoes

Vector‐borne and zoonotic infectious diseases are serious public health concerns that affect approximately half of the world's population. In particular, arthropod‐borne viruses (arboviruses) have contributed to more mortality and morbidity worldwide with the emergence of dengue, chikungunya, yellow fever, and Zika virus diseases. The infections have scaled up due to urbanization, globalization, and international mobility. Traditionally, the spread of mosquito‐borne viral diseases to humans was considered a low health priority concern. However, their categorization as emerging infectious diseases and public health emergencies of international concern has heightened the attention given by the government, academia, research, and industry for the development of timely, cost‐efficient, and sustainable solutions. The urgency has increased in the wake of global climate change. The focus on effective interventions includes epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants. In this review, we discuss the etiology and predisposition of mosquito‐borne viruses that are detrimental to public health and economically damaging when disseminated as epidemics. We focus on the large‐scale virus surveillance methods with special reference to innovations and interventions in molecular detection science and technologies that include viral nucleic acid isolation, polymerase chain reaction (PCR)‐based diagnostics, and high‐throughput sequencing technologies. In addition, we discuss the development of a viral RNA extraction and PCR‐based diagnostic kit (Invirustech) that can extract viral RNA from mosquitoes with verified applications in PCR‐based molecular diagnostics of Pan‐flavivirus.     

Molecular Targets for the Testing of COVID‐19

The pandemic outbreaks of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), spread all over the world in a short period of time. Efficient identification of the infection by SARS‐CoV‐2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID‐19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid‐February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID‐19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS‐CoV‐2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID‐19 virus.     

Genome‐wide identification of urinary cell‐free microRNAs for non‐invasive detection of bladder cancer

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non‐invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT‐PCR. Whole‐genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR‐31‐5p, miR‐93‐5p and miR‐191‐5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2‐miRNA‐based urinary DxScore (miR‐93‐5p, miR‐31‐5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post‐operatively. In conclusion, we identified and independently validated cell‐free urinary miRNAs as promising biomarkers enabling non‐invasive detection of BCA.     

Colistin and tigecycline resistance in carbapenemase‐producing Gram‐negative bacteria: emerging resistance mechanisms and detection methods

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase‐producing Gram‐negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans‐complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT‐PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC‐FGH‐IJK, mexAB‐XY‐oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr‐1 gene conferring resistance to colistin was identified via WGS, trans‐complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI‐TOF MS, micro‐array and real‐time multiplex PCR hold much promise for the future as new detection tools.     

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T₀ transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T₀ transgenic plants, which will be widely used in the area of plant gene editing.     

Assessment of reproducibility in depletion and enrichment workflows for plasma proteomics using label‐free quantitative data‐independent LC‐MS

Quantitation in plasma‐based proteomics necessitates the reproducible removal of highly abundant proteins to enable the less abundant proteins to be visible to the mass spectrometer. We have evaluated immunodepletion (proteoprep20) and enrichment (Bio‐Rad beads), as the current predominant approaches. Label‐free analysis offers an opportunity to estimate the effectiveness of this approach without incorporating chemical labels. Human plasma samples were used to quantitatively assess the reproducibility of these two methods using nano‐LC‐data‐independent acquisition MS. We have selected 18 candidate proteins and a comparison of both methodologies showed that both of the methods were reproducible and fell below 20% residual SD. With the same candidate proteins, individual inter‐day variability for the samples was also processed, allowing us to monitor instrument reproducibility. Overall, a total of 131 proteins were identified by both methods with 272 proteins identified by enrichment and 200 identified by immunodepletion. Reproducibility of measurements of the amount of protein in the processed sample for individual proteins is within analytically acceptable standards for both methodologies. This enables both methods to be used for biomarker studies. However, when sample is limited, enrichment is not suitable as larger volumes (>1.0 mL) are required. In experiments where sample is not limited then a greater number of proteins can be reliably identified using enrichment.     

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

SARS‐CoV‐2 lgM/lgG antibody detection confirms the infection after three negative nucleic acid detection

An ongoing outbreak of viral pneumonia was caused by a novel coronavirus in China in 2019. By March 19, over 200 thousand confirmed cases of SARS‐CoV‐2 infection and over 9000 deaths have been reported throughout the world. For this infectious disease, nucleic acid detection is still the gold standard for pathogenic detection. However, nucleic acid detection takes a long time and has relatively high "false negative"; therefore, we need urgently a convenient and accurate detection method to make up for this deficiency. In this article, we will show such technical characteristics of lgM/lgG serum antibody detection, compared with nucleic acid detection.     

Insertion site‐based polymorphism markers open new perspectives for genome saturation and marker‐assisted selection in wheat

In wheat, the deployment of marker‐assisted selection has long been hampered by the lack of markers compatible with high‐throughput cost‐effective genotyping techniques. Recently, insertion site‐based polymorphism (ISBP) markers have appeared as very powerful new tools for genomics and genetic studies in hexaploid wheat. To demonstrate their possible use in wheat breeding programmes, we assessed their potential to meet the five main requirements for utilization in MAS: flexible and high‐throughput detection methods, low quantity and quality of DNA required, low cost per assay, tight link to target loci and high level of polymorphism in breeding material. Toward this aim, we developed a programme, IsbpFinder, for the automated design of ISBP markers and adapted three detection methods (melting curve analysis, SNaPshot^® Multiplex System and Illumina BeadArray technology) for high throughput and flexible detection of ISBP or ISBP‐derived SNP markers. We demonstrate that the high level of polymorphism of the ISBPs combined with cost‐effective genotyping methods can be used to efficiently saturate genetic maps, discriminate between elite cultivars, and design tightly linked diagnostic markers for virtually all target loci in the wheat genome. All together, our results suggest that ISBP markers have the potential to lead to a breakthrough in wheat marker‐assisted selection.     

Effects of inactivation methods on the analysis of Bacillus atrophaeus endospores using real‐time PCR and MALDI‐TOF‐MS

A wide range of analytical methods are available for the detection and identification of biological warfare agents. These technologies are often hampered in their performance when the inactivated samples are analyzed. To work with pathogens outside of biosafety level 3 laboratories, a complete inactivation is mandatory when appropriate protection equipment is unavailable. When methods of inactivation are used, the detection of bacteria becomes more difficult. In contrast to measuring viable organisms, inactivation steps can have a massive impact on the intrinsic cellular information. This study examined the effects of autoclaving and chemical inactivation methods on Bacillus spores using biological warfare detection setups like real‐time PCR and MALDI‐TOF‐MS. Here, the inactivation of Bacillus atrophaeus spores with formaldehyde, which is a suggested model for biological warfare spore agents, was compared with other inactivation reagents like Wofasteril^®E400, a commercially available decontaminant based on peroxyacetic acid. With Wofasteril^®E400 the critical factor of inactivation time was reduced to about 15 min and a limit of detection of 8500 spores by PCR was still measurable using five‐times‐washed spores. It has also been shown that MALDI‐TOF‐MS peak information can be hampered by inactivation methods.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

Loop‐mediated isothermal amplification of single pollen grains

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field‐enabled, high‐throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop‐mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field‐based, high‐throughput analysis.