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All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation, such as Delta (N:R203M), Iota (N:S202R), and B.1.2 (N:P199L). A new open reading frame encoding a truncated N protein called N*, which occurs in the B.1.1 lineage and subsequent lineages of the Alpha, Gamma, and Omicron variants, supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection.

All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation, such as Delta (N:R203M), Iota (N:S202R), and B.1.2 (N:P199L). A new open reading frame encoding a truncated N protein called N*, which occurs in the B.1.1 lineage and subsequent lineages of the Alpha, Gamma, and Omicron variants, supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection. Author summary: The COVID-19 pandemic was caused by SARS-CoV-2 and is characterized by waves of evolved viral variants with mutations across the viral genome. Nucleocapsid (N) is a viral protein that is critical for viral RNA packaging into particles and for viral replication in cells. Over the pandemic, N has acquired several mutations between amino acids 199 and 205 that are associated with increased infectivity. The mechanism underlying these mutations has been difficult to elucidate given the multiple roles of N protein in the viral lifecycle. Here, we used developed methods to separately determine the effect of mutations on viral particle assembly vs viral RNA replication. We found that the mutations between amino acids 199 and 205 affect N protein phosphorylation and have opposite effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation. One of the mutations found in the Alpha, Gamma, and Omicron variants of concern results in expression of a new, truncated N protein called N* that supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection. [ABSTRACT FROM AUTHOR]

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Transition metal complexes with characteristics of unique packaging in nanoparticles and remarkable cancer cell cytotoxicity have emerged as potential alternatives to platinum-based antitumor drugs. Here we report the synthesis, characterization, and antitumor activities of three new Ruthenium complexes that introduce 5-fluorouracil-derived ligands. Notably, encapsulation of one such metal complex, Ru3, within pluronic® F-127 micelles (Ru3-M) significantly enhanced Ru3 cytotoxicity toward A549 cells by a factor of four. To determine the mechanisms underlying Ru3-M cytotoxicity, additional in vitro experiments were conducted that revealed A549 cell treatment with lysosome-targeting Ru3-M triggered oxidative stress, induced mitochondrial membrane potential depolarization, and drastically reduced intracellular ATP levels. Taken together, these results demonstrated that Ru3-M killed cells mainly via a non-apoptotic pathway known as oncosis, as evidenced by observed Ru3-M-induced cellular morphological changes including cytosolic flushing, cell swelling, and cytoplasmic vacuolation. In turn, these changes together caused cytoskeletal collapse and activation of porimin and calpain1 proteins with known oncotic functions that distinguished this oncotic process from other cell death processes. In summary, Ru3-M is a potential anticancer agent that kills A549 cells via a novel mechanism involving Ru(II) complex triggering of cell death via oncosis. [ABSTRACT FROM AUTHOR]

Effective cysteine (Cys) detection is extremely important for early disease monitoring and diagnosis. In this study, a fluorescent probe (PHO) comprising 1,10-phenanthroline carboxaldehyde as the ligand and cobalt as the central ion was synthesized to detect Cys. The synthesized PHO exhibited enhanced fluorescence at 603 nm in the presence of Cys in HEPES buffer. Furthermore, the probe detected Cys concentrations as low as 0.6 μmol/L, demonstrating high sensitivity. Additionally, a strong linear relationship was established between the Cys concentration and normalized intensity of fluorescence. Importantly, the high selectivity was retained even in the presence of other interfering compounds. Consequently, this method can serve as a novel approach for detecting Cys in physiological systems. [ABSTRACT FROM AUTHOR]

With the interconnection between large power grids, the issue of security and stability has become increasingly prominent. At present, data-driven power system adaptive transient stability assessment methods have achieved excellent performances by balancing speed and accuracy, but the complicated construction and parameters are difficult to obtain. This paper proposes a stacked-GRU (Gated Recurrent Unit)-based transient stability intelligent assessment method, which builds a stacked-GRU model based on time-dependent parameter sharing and spatial stacking. By using the time series data after power system failure, the offline training is performed to obtain the optimal parameters of stacked-GRU. When the application is online, it is assessed by framework of confidence. Basing on New England power system, the performance of proposed adaptive transient stability assessment method is investigated. Simulation results show that the proposed model realizes reliable and accurate assessment of transient stability and it has the advantages of short assessment time with less complex model structure to leave time for emergency control.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts. [ABSTRACT FROM AUTHOR]

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals. [ABSTRACT FROM AUTHOR]

Dynamic tracking of laser spot is a key process in the establishment of free space optical communication. In this paper, a dynamic tracking system was presented. In this system, a two-dimensional (2D) galvanometer was used to change the angle of the optical axis of the incident beam at a certain scanning frequency as optical signal jitter simulator, and another galvanometer was used to track the jitter with quadrant detector (QD) and data processing module to acquire the position information of laser spot. Results indicated that the tracking accuracy of this system mainly composed of 2D galvanometer was as high as 27.8 μrad, and its linear deviation was less than 0.013. The system could still keep the dynamic tracking of the spot stable when the jitter frequency of the optical signal was less than 1000 Hz. Those results suggested that this system could be suitable for the short distance in free space communication due to its simple structure, easy to control and low cost compared with conventional system. [ABSTRACT FROM AUTHOR]

Background: While influenza vaccines are the most effective measure for preventing influenza, uptake rates in China remain relatively low. Rapid Verbal Persuasion (RVP), with highly rapid fashion, has a strong evidence base in promoting behavior change. Despite this, it is underused or rarely evaluated in the context of vaccination. Additionally, the success of RVP implementation in vaccination clinics hinges on the motivation of vaccination staff, which remains critical even with stable contextual factors. Multifaceted incentive-based implementation strategies, which aim to enhance motivation to promote the implementation of evidence-based practices, could be advantageous. This study protocol outlines an implementation-effectiveness hybrid type 2 design to evaluate the effectiveness of both the incentive-based implementation strategies on implementation outcomes and RVP on increasing influenza vaccination rates. Method: This study will be conducted as a two-tiered cluster of randomized controlled trials over three months. Initially, 32 vaccination clinics will be randomly allocated to one of two study arms: (a) implementation of RVP or (b) no implementation. At the end of the study period, differences in influenza vaccination status between the intervention and control groups will be compared (primary outcome). Subsequently, a cluster randomized factorial trial will be conducted, involving 16 clinics implementing RVP. This trial will aim to compare the impact of various implementation strategies (different combinations of incentives) on fidelity in RVP implementation (primary outcome). Data collection for the primary outcomes will include unannounced exit interviews. Modified Poisson regression models and generalized linear mixed-effects models will be utilized to analyze the association between primary outcomes and interventions. Conclusion: The study aims to enhance the influenza vaccination rate in China by developing financial and non-financial incentives that allow vaccination staff to deliver RVP with greater motivation. Furthermore, the evidence generated from this multi-center trial will assist policymakers in improving current incentive systems within immunization services. Trial registration: Chinese Clinical Trial Registry. Trial identifier: ChiCTR2400091302 (Registration Date: October 25, 2024); ChiCTR2400091324 (Registration Date: October 25, 2024). [ABSTRACT FROM AUTHOR]

With the interconnection between large power grids, the issue of security and stability has become increasingly prominent. At present, data-driven power system adaptive transient stability assessment methods have achieved excellent performances by balancing speed and accuracy, but the complicated construction and parameters are difficult to obtain. This paper proposes a stacked-GRU (Gated Recurrent Unit)-based transient stability intelligent assessment method, which builds a stacked-GRU model based on time-dependent parameter sharing and spatial stacking. By using the time series data after power system failure, the offline training is performed to obtain the optimal parameters of stacked-GRU. When the application is online, it is assessed by framework of confidence. Basing on New England power system, the performance of proposed adaptive transient stability assessment method is investigated. Simulation results show that the proposed model realizes reliable and accurate assessment of transient stability and it has the advantages of short assessment time with less complex model structure to leave time for emergency control. [ABSTRACT FROM AUTHOR]