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1.
Viruses ; 14(3)2022 03 06.
Article in English | MEDLINE | ID: covidwho-1765949

ABSTRACT

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.


Subject(s)
Adenoviruses, Human , Adenoviruses, Simian , Adenoviridae/genetics , Adenoviruses, Human/genetics , Adenoviruses, Simian/genetics , Animals , DNA , Genetic Vectors/genetics , Humans , Mammals
2.
Industrial Management & Data Systems ; 122(3):592-621, 2022.
Article in English | ProQuest Central | ID: covidwho-1752273

ABSTRACT

Purpose>With the rapid development of cloud computing, most software firms face the significant choice of whether they should change the versioning strategy of enterprise software from releasing the on-premise version to the software-as-a-service (SaaS) version. Data being generated and hosted on SaaS vendors' servers brings multiple effects. It enables customers to enjoy the flexibility of accessing data and using the software remotely, named the “portability” effect. However, on the other hand, the cumulative data resources on the cloud also provide a clear target for external attacks, leading to the concern of information security. Considering these, the authors hope to offer insights for software firms by exploring the strategy selection problem.Design/methodology/approach>Taking the portability effect and security risks of the SaaS licensing model into account, the authors develop a two-period model to figure out the market segmentation and identify the feasible conditions for employing three alternative strategies. Comparative statics analyses are conducted to explore the influencing mechanism of exogenous factors on strategy selection. The authors also discuss the strategy selection in the presence of the network effect and the security loss faced by users of on-premise software.Findings>One significant finding is that the on-premise strategy can be excluded when the potential loss from security risks is small. Under this circumstance, the dual version strategy is optimal provided that the increase of customer valuation caused by portability effect is below a threshold. Otherwise, the SaaS strategy generates the highest profit. When the potential loss from security risks turns large, the on-premise strategy, the dual version strategy and the SaaS strategy are the optimal options in order as the portability effect on customer valuation gets stronger.Originality/value>Previous literature has insufficiently addressed the versioning issue of enterprise software. In this paper, the distinctive features of the SaaS model are considered, and differentiated results compared with previous work are obtained. The research results provide guidelines for software firms in deciding their product releases in the future.

3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-325253

ABSTRACT

Coronavirus Disease 2019 (COVID-19) has caused great casualties and becomes almost the most urgent public health events worldwide. Computed tomography (CT) is a significant screening tool for COVID-19 infection, and automated segmentation of lung infection in COVID-19 CT images will greatly assist diagnosis and health care of patients. However, accurate and automatic segmentation of COVID-19 lung infections remains to be challenging. In this paper we propose a dilated dual attention U-Net (D2A U-Net) for COVID-19 lesion segmentation in CT slices based on dilated convolution and a novel dual attention mechanism to address the issues above. We introduce a dilated convolution module in model decoder to achieve large receptive field, which refines decoding process and contributes to segmentation accuracy. Also, we present a dual attention mechanism composed of two attention modules which are inserted to skip connection and model decoder respectively. The dual attention mechanism is utilized to refine feature maps and reduce semantic gap between different levels of the model. The proposed method has been evaluated on open-source dataset and outperforms cutting edges methods in semantic segmentation. Our proposed D2A U-Net with pretrained encoder achieves a Dice score of 0.7298 and recall score of 0.7071. Besides, we also build a simplified D2A U-Net without pretrained encoder to provide a fair comparison with other models trained from scratch, which still outperforms popular U-Net family models with a Dice score of 0.7047 and recall score of 0.6626. Our experiment results have shown that by introducing dilated convolution and dual attention mechanism, the number of false positives is significantly reduced, which improves sensitivity to COVID-19 lesions and subsequently brings significant increase to Dice score.

4.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-322274

ABSTRACT

Background: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods: : We tried to develop a o ne-tube detection platform based on R T- R PA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and D NA E ndonuclease- T arg e ted C RISPR T rans R eporter ( DETECTR ) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19. Results: : The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input. Conclusions: : The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

5.
Carbohydr Polym ; 269: 118345, 2021 Oct 01.
Article in English | MEDLINE | ID: covidwho-1271581

ABSTRACT

This work reports novel chitosan functionalized graphene oxide (GO) nanocomposites combined fluorescence imaging and therapeutic functions in one agent, which can serve as a promising alternative to alleviate related diseases caused hyperinflammation. Briefly, GO was designed to be conjugated with chitosan, fluorescein-labeled peptide, toll-like receptor 4 antibody and hydroxycamptothecin/aloe emodin. We have demonstrated that such nanocomposites could effectively achieve active targeted delivery of pro-apoptotic and anti-inflammatory drugs into inflammatory cells and cause cells apoptosis by acid-responsive drug release. Moreover, confocal fluorescence imaging confirms that the drug-induced inflammatory cells apoptosis could be visualized the light-up fluorescence of fluorescein activated by caspase-3. Meanwhile, inflammatory-related biomarkers have down-regulated after the nanocomposites' treatment in both vitro and vivo experiments consistent with the results in histological sections. In summary, the bifunctional nanocomposites that possess anti-inflammation and fluorescence imaging could serve as a promising therapeutic agent for reducing hyperinflammation caused by numerous diseases.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Apoptosis/physiology , Drug Carriers/chemistry , Inflammation/drug therapy , Nanocomposites/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Antibodies/immunology , Camptothecin/analogs & derivatives , Camptothecin/chemistry , Camptothecin/therapeutic use , Cattle , Cell Line , Chitosan/chemistry , Drug Liberation , Emodin/chemistry , Emodin/therapeutic use , Fluorescent Dyes/chemistry , Graphite/chemistry , Humans , Lipopolysaccharides , Mammary Glands, Human/drug effects , Mammary Glands, Human/pathology , Mastitis/chemically induced , Mastitis/drug therapy , Mastitis/pathology , Mice , Toll-Like Receptor 4/immunology
6.
Comput Biol Med ; 135: 104526, 2021 08.
Article in English | MEDLINE | ID: covidwho-1252628

ABSTRACT

Coronavirus Disease 2019 (COVID-19) has become one of the most urgent public health events worldwide due to its high infectivity and mortality. Computed tomography (CT) is a significant screening tool for COVID-19 infection, and automatic segmentation of lung infection in COVID-19 CT images can assist diagnosis and health care of patients. However, accurate and automatic segmentation of COVID-19 lung infections is faced with a few challenges, including blurred edges of infection and relatively low sensitivity. To address the issues above, a novel dilated dual attention U-Net based on the dual attention strategy and hybrid dilated convolutions, namely D2A U-Net, is proposed for COVID-19 lesion segmentation in CT slices. In our D2A U-Net, the dual attention strategy composed of two attention modules is utilized to refine feature maps and reduce the semantic gap between different levels of feature maps. Moreover, the hybrid dilated convolutions are introduced to the model decoder to achieve larger receptive fields, which refines the decoding process. The proposed method is evaluated on an open-source dataset and achieves a Dice score of 0.7298 and recall score of 0.7071, which outperforms the popular cutting-edge methods in the semantic segmentation. The proposed network is expected to be a potential AI-based approach used for the diagnosis and prognosis of COVID-19 patients.


Subject(s)
COVID-19 , Image Processing, Computer-Assisted , Tomography, X-Ray Computed , COVID-19/diagnostic imaging , Humans
7.
Aging (Albany NY) ; 13(9): 12301-12307, 2021 05 06.
Article in English | MEDLINE | ID: covidwho-1220256

ABSTRACT

Patients with pre-existing chronic diseases are more susceptible to coronavirus disease 2019 (COVID-19), yet the underlying causes of increased risk are of infection remain unclear. Angiotensin-converting- enzyme 2 (ACE2), the cell surface receptor that recognizes the coronavirus spike protein has protective effects against inflammation and chronic hyperglycemia in animal models. The roles of ACE2 in severe SARS-CoV-2 infections remains ambiguous due to contradictory findings. In this study, we aimed to investigate the relationship between human plasma ACE2 levels in diabetics and the high risk of severe SARS-CoV-2 infection. First, the medical records of 245 patients with SARS-CoV-2-positive who have chronic diseases were analyzed. We also recruited 404 elderly subjects with comorbid chronic diseases such as diabetes mellitus, coronary heart disease, cerebrovascular disease, hypertension and obesity, and investigated the ACE2 plasma levels. Plasma concentrations of ACE2 were much lower (2973.83±2196.79 pg/mL) in diabetics with chronic disease than in healthy controls (4308.21±2352.42 pg/ml), and the use of hypoglycemia drugs was associated with lower circulating concentrations of ACE2 (P=1.49E-08). Diabetics with lower plasma levels of ACE2 may be susceptible to severe COVID-19. Our findings suggest that the poor prognosis in patients with diabetes infected with SARS-CoV-2 may be due to low circulating ACE2 levels.


Subject(s)
Angiotensin-Converting Enzyme 2/blood , COVID-19/blood , Diabetes Mellitus/blood , Aged , COVID-19/enzymology , Diabetes Mellitus/enzymology , Female , Humans , Male , Middle Aged , SARS-CoV-2
8.
J Transl Med ; 19(1): 74, 2021 02 16.
Article in English | MEDLINE | ID: covidwho-1088600

ABSTRACT

BACKGROUND: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. METHODS: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19. RESULTS: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input. CONCLUSIONS: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , CRISPR-Cas Systems/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/genetics , SARS-CoV-2/isolation & purification , Base Sequence , Humans , Limit of Detection , RNA, Viral/genetics , Reference Standards , SARS-CoV-2/genetics
9.
Environ Int ; 147: 106361, 2021 02.
Article in English | MEDLINE | ID: covidwho-987643

ABSTRACT

Corona virus disease 2019 has spread worldwide, and appropriate drug design and screening activities are required to overcome the associated pandemic. Using computational simulation, blockade mechanism of SARS-CoV-2 spike receptor binding domain (S RBD) and human angiotensin converting enzyme 2 (hACE2) was clarified based on interactions between RBD and hesperidin. Interactions between anti-SARS-CoV-2 drugs and therapy were investigated based on the binding energy and druggability of the compounds, and they exhibited negative correlations; the compounds were classified into eight common types of structures with highest activity. An anti-SARS-CoV-2 drug screening strategy based on blocking S RBD/hACE2 binding was established according to the first key change (interactions between hesperidin and S RBD/hACE2) vs the second key change (interactions between anti-SARS-CoV-2 drugs and RBD/hACE2) trends. Our findings provide valuable information on the mechanism of RBD/hACE2 binding and on the associated screening strategies for anti-SARS-CoV-2 drugs based on blocking mechanisms of pockets.


Subject(s)
COVID-19 , Pharmaceutical Preparations , Humans , Peptidyl-Dipeptidase A , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
10.
EBioMedicine ; 61: 103036, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-844322

ABSTRACT

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Subject(s)
Bacterial Proteins/metabolism , Betacoronavirus/genetics , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endodeoxyribonucleases/metabolism , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nasal Cavity/virology , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reference Standards , SARS-CoV-2 , Viral Proteins/genetics , Viral Proteins/metabolism
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