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SummaryMonitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. The availability of patient hospital records is crucial for linking the genomic sequence information to virus function during the course of infections. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. From the assembled sequences, we estimate the SARS-CoV-2 effective population size and infection rate and outline the epidemiological dynamics of import and transmission events during this period in Saudi Arabia. We show that two consecutive mutations (R203K/G204R) in the SARS-CoV-2 nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein by mass-spectrometry analysis. Furthermore, analysis of the host cell transcriptome suggests that the mutant N protein results in dysregulated interferon response genes. We provide crucial information in linking the R203K/G204R mutations in the N protein as a major modulator of host-virus interactions and increased viral load and underline the potential of the nucleocapsid protein as a drug target during infection.

Molecular diagnosis and surveillance of pathogens such as SARS‐CoV‐2 depend on nucleic acid isolation. Pandemics at the scale of COVID‐19 can cause a global shortage of proprietary commercial reagents and BSL‐2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open‐source method, magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real‐world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol‐chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID‐19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS‐CoV‐2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field‐deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens., One important bottleneck in the diagnosis and surveillance of COVID‐19 is the shortage of kits for RNA extraction. Magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS) is an open‐source, safe, fast, and scalable method for RNA extraction. MAVRICS rivals commercial kits but requires minimal materials, and thus could become an enabling technology for widespread community testing of diverse pathogens.

Highlights • RT-LAMP coupled with CRISPR-Cas12 provides a sensitive and specific virus detection platform. • iSCAN sensitivity and specificity are comparable with RT-qPCR. • iSCAN is a 1 h detection module that can help in testing in low resource areas. • iSCAN can be developed as a one-pot assay. • iSCAN reagents can be produced locally and deployed for SARS-CoV2 detection., The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript™ III Platinum™ One-step RT-qPCR and TaqPath™ 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility.

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method calledMagnetic-nanoparticle-AidedViralRNAIsolation ofContagiousSamples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. MAVRICS is scalable and thus could become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics.

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

This paper is to design a Smart Forklift model which reduces accident rates. Forklift is a powered industrial truck used to lift and move material over short distance. It is one of the most used machines employed for several types of tasks. Forklift is used on small as well as large scale on multiple locations. Various forklifts are available according to load capacity. Forklifts for general load purpose have capacity from 1 to 5 tons, and that of for heavier loads it has capacity of 50 tons. It has major drawback of severe accidents because of less safety features. To avoid these unwanted accidents, Smart Forklift is introduced, with arduino, gyro sensor, which decreases the rate of accidents and increases the safety of driver as well as vehicle. This design includes a small model which will detect problem whenever Forklift is in danger mode and a security threat indication is sent through a buzzer and light indication to driver.

This paper describes design of Biometric Voting Machine Using Fingerprint Scanner and Arduino for voting in institutes and organizations. Indian constitution empowers its citizen to exercise right to vote. Election decides the future of country, so that the system used for voting should be trustworthy. The conventional system for voting is ballot paper and Electronic voting machine too, has many flaws and trust issues. To eradicate malpractice and defrauding of the above methods of voting, we have designed an advanced system by using arduino and Fingerprint module. In this system, a person has to register a fingerprint ID with the system which will be centrally stored in arduino. In organizations, educational institutes, a co-operative bank, maximum number of votes elect head of organization that holds the office of public interest. For confirmation of voter, the name of the candidate will be displayed on LCD for whom the voter has cast a vote. It has simple hardware design and it is easily accessible. In case user wants to remove any of stored ID then the user need to press DEL key, after pressing DEL key, LED will ask to select ID that is to be deleted. After pressing OK key, the selected ID will be deleted and LCD will display that which ID has been deleted successfully. This system is flexible to use.

A critical bottleneck in COVID‐19 diagnosis and surveillance is the shortage of kits for RNA extraction. In article number 2000068, Mo Li and co‐workers develop a DIY, open‐source, safe, and fast method for magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS). MAVRICS rivals commercial kits in performance and could become an enabling technology for community testing and wastewater monitoring in the current and future pandemics.

The huge popularity of voice and data services in mobile communication facilitates ubiquitous infrastructure that has been mainly driven by the scalability of their architectures and the flexibility of their search facilities. Such systems are usually designed as unstructured point-to-point networks and support highly versatile search algorithms. Developing search algorithms is difficult in unstructured peer-to-peer networks. Flooding and Random Walk are two typical search algorithms. Flooding covers most of the nodes by searching randomly. In the unstructured network peers are interconnected randomly, they rely on Flooding query messages to discover objects of interest. It generates a large amount of query messages which produces network traffic. Random walk search algorithms are used in mobile networks because of their dynamic nature. But it only generates a fixed amount of query messages at each peer but would take longer search time. Thus we propose development of unstructured architecture for voice and data services in mobile communication. In this architecture we are focusing on interconnection of various mobile devices. One way is to improve the algorithm for selecting path, second way to modify the routing algorithm by considering new characteristics for less signaling overhead.

The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In T. gondii and other apicomplexan parasites, many subunit components necessary for proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species., Author summary The mitochondrial F-type ATP synthase, which is a major source of ATP in eukaryotic cells, is a unique nanomotor. The enzyme consists of two subcomplexes called the F1 and FO sectors. The F1 sector is the site of ATP synthesis, while the FO sector couples proton translocation to the rotary motion of the enzyme. FO sector subunits also form the peripheral stalk (stator), which holds the nonrotating parts of the enzyme together. F1 sector subunits are highly conserved among eukaryotes, while most FO sector subunits appear to be diverse and remain unknown in many species, including in the important human pathogen Toxoplasma gondii. In this study, we have partially purified the T. gondii F-type ATP synthase and analyzed its proteome, using mass spectrometry. This resulted in the identification of 20 novel subunits of the enzyme, including extremely diversified key FO sector subunits. Many of these novel T. gondii proteins are conserved in related apicomplexan parasites, such as the malaria parasite, and thus might be good drug targets. Conservation of many of these proteins in related free-living alveolate species provides insights on their evolutionary history and can potentially facilitate further biochemical and structural studies on this unusual enzyme.

In view of rapidly growing trend of mobile communication, there is tremendous demand for wireless technologies. Mobile communication networks are getting more and more complex with variety of services they offer, variety of devices connected to the network, variety of possible interconnections they have to make and network topology they use. As the number of mobile customers are increased, network has to provide services to all mobile customers. However, as the mobile devices moves between networks signaling overhead causes real time data traffic. Developing routing algorithms for mobile communication is a key challenge. In this paper we present a detail review of hybrid routing protocols for mobile communication. We consider both protocols (a)Proactive routing protocol which is based on periodic exchanges that updates the routing tables to all possible destinations, even if no traffic (b) Reactive routing protocol which is based on on-demand route discoveries that updates routing tables only for the destination that has traffic going through. There are two ways to enhance the performance of unstructured architecture for voice and data services in mobile communication. One way is to improve the algorithm for selecting path, second way to modify the routing algorithm by considering new characteristics for less signaling overhead.