Portable nanopore-sequencing technology: Trends in development and applications.

Sequencing technology is the most commonly used technology in molecular biology research and an essential pillar for the development and applications of molecular biology. Since 1977, when the first generation of sequencing technology opened the door to interpreting the genetic code, sequencing technology has been developing for three generations. It has applications in all aspects of life and scientific research, such as disease diagnosis, drug target discovery, pathological research, species protection, and SARS-CoV-2 detection. However, the first- and second-generation sequencing technology relied on fluorescence detection systems and DNA polymerization enzyme systems, which increased the cost of sequencing technology and limited its scope of applications. The third-generation sequencing technology performs PCR-free and single-molecule sequencing, but it still depends on the fluorescence detection device. To break through these limitations, researchers have made arduous efforts to develop a new advanced portable sequencing technology represented by nanopore sequencing. Nanopore technology has the advantages of small size and convenient portability, independent of biochemical reagents, and direct reading using physical methods. This paper reviews the research and development process of nanopore sequencing technology (NST) from the laboratory to commercially viable tools; discusses the main types of nanopore sequencing technologies and their various applications in solving a wide range of real-world problems. In addition, the paper collates the analysis tools necessary for performing different processing tasks in nanopore sequencing. Finally, we highlight the challenges of NST and its future research and application directions.

T492I mutation alters SARS-CoV-2 properties via modulating viral non-structural proteins (preprint)

The historically dominant SARS-CoV-2 Delta variants and the currently dominant Omicron variants carry a T492I substitution within the non-structural protein 4 (NSP4). Based on a combination of in silico analyses, we predicted that the T492I mutation increases the transmissibility and adaptability of the virus. We confirmed this hypothesis by performing competition experiments in hamsters and in human airway tissue culture models. Furthermore, we show that the T492I mutation also increases the replication capacity and infectiveness of the virus, and improves its ability to evade antibody neutralization induced by previous variants. Mechanistically, the T492I mutation increases cleavage efficiency of the viral main protease NSP5 by enhancing enzyme-substrate binding, resulting in increased production of nearly all non-structural proteins processed by NSP5. Importantly, T492I mutation suppresses the viral RNA associated chemokines in monocytic macrophages, which may contribute to the attenuated pathogenicity of Omicron variants. Our results highlight the importance of the NSP4 mutation in the evolutionary dynamics of SARS-CoV-2 and identify a novel target for the development of broad-spectrum antiviral agents.

Status and Determinants of Symptoms of Anxiety and Depression among Food Delivery Drivers in Shanghai, China.

(1) Background: The psychological status of employees, especially vulnerable populations, has received considerable research attention. However, as a newly emerging and popular occupation in the gig industry, food delivery drivers have received little attention. The majority of these workers are immigrants who are already in a precarious position due to a lack of available jobs, inadequate medical care, poor diets, and communication and acculturation difficulties even before they take these jobs, which involve long working hours and exposure to the elements. (2) Methods: To examine the anxiety and depression symptoms of these workers and possible influencing factors, a cross-sectional study was conducted with a sample of food delivery drivers working for the Meituan Company (one of the largest e-platform companies in China). Anxiety and depression scales were adapted from the GAD-7, and the PHQ-9 was used to assess participants' related symptoms. Differences were compared in terms of sociodemographic, work situation, and lifestyle variables. Binary logistic regressions were conducted to analyze the effects of various factors on the two psychological dimensions. (3) Results: Among the 657 participants, the proportions of participants reporting anxiety and depression symptoms were 46.0% and 18.4%, respectively. Lack of communication with leaders (ORAN = 2.620, 95% CI: 1.528-4.493, p < 0.001; ORDE = 1.928, 95% CI: 1.039-3.577, p = 0.037) and poor sleep quality (ORAN = 2.152, 95% CI: 1.587-2.917, p < 0.001; ORDE = 2.420, 95% CI: 1.672-3.504, p < 0.001) were significant risk factors for both anxiety and depression symptoms. Women (OR = 2.679, 95% CI: 1.621-4.427, p < 0.001), those who climbed ≥31 floors per day (OR = 2.415, 95% CI: 1.189-4.905, p = 0.015), and those with a high frequency of breakfast consumption (OR = 3.821, 95% CI: 1.284-11.369, p = 0.016) were more likely to have anxiety symptoms. Participants who earned less than 5000 RMB (OR = 0.438, 95% CI: 0.204-0.940, p = 0.034), were unwilling to seek medical help (OR = 3.549, 95% CI: 1.846-6.821, p < 0.001), or had a high frequency of smoking (OR = 5.107, 95% CI: 1.187-21.981, p = 0.029) were more likely to be depressive. (4) Conclusion: The existence of communication channels with leaders and good sleep quality are protective factors for anxiety and depression symptoms. Participants who were female, climbed ≥31floors per day, and had a high frequency of eating breakfast were more likely to have anxiety symptoms, while earning less, unwillingness to seek medical help, and a high frequency of smoking were risk factors for depression symptoms.

Paving the way for precise diagnostics of antimicrobial resistant bacteria.

The antimicrobial resistance (AMR) crisis from bacterial pathogens is frequently emerging and rapidly disseminated during the sustained antimicrobial exposure in human-dominated communities, posing a compelling threat as one of the biggest challenges in humans. The frequent incidences of some common but untreatable infections unfold the public health catastrophe that antimicrobial-resistant pathogens have outpaced the available countermeasures, now explicitly amplified during the COVID-19 pandemic. Nowadays, biotechnology and machine learning advancements help create more fundamental knowledge of distinct spatiotemporal dynamics in AMR bacterial adaptation and evolutionary processes. Integrated with reliable diagnostic tools and powerful analytic approaches, a collaborative and systematic surveillance platform with high accuracy and predictability should be established and implemented, which is not just for an effective controlling strategy on AMR but also for protecting the longevity of valuable antimicrobials currently and in the future.

Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients.

The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (-2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70-80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.

Inhibition of SARS-CoV-2 pseudovirus invasion by ACE2 protecting and Spike neutralizing peptides: An alternative approach to COVID19 prevention and therapy.

SARS-CoV-2 invades host cells mainly through the interaction of its spike-protein with host cell membrane ACE2. Various antibodies targeting S-protein have been developed to combat COVID-19 pandemic; however, the potential risk of antibody-dependent enhancement and novel spike mutants-induced neutralization loss or antibody resistance still remain. Alternative preventative agents or therapeutics are still urgently needed. In this study, we designed series of peptides with either ACE2 protecting or Spike-protein neutralizing activities. Molecular docking predicted that, among these peptides, ACE2 protecting peptide AYp28 and Spike-protein neutralizing peptide AYn1 showed strongest intermolecular interaction to ACE2 and Spike-protein, respectively, which were further confirmed by both cell- and non-cell-based in vitro assays. In addition, both peptides inhibited the invasion of pseudotype SARS-CoV-2 into HEK293T/hACE2 cells, either alone or in combination. Moreover, the intranasal administration of AYp28 could partially block pseudovirus invasion in hACE2 transgenic mice. Much more importantly, no significant toxicity was observed in peptides-treated cells. AYp28 showed no impacts on ACE2 function. Taken together, the data from our present study predicted promising preventative and therapeutic values of peptides against COVID-19, and may prove the concept that cocktail containing ACE2 protecting peptides and spike neutralizing peptides could serve as a safe and effective approach for SARS-CoV-2 prevention and therapy.

Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus.

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/µl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Large-scale single-cell analysis reveals critical immune characteristics of COVID-19 patients (preprint)

Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 205 COVID-19 patients and controls to create a comprehensive immune landscape. Lymphopenia and active T and B cell responses were found to coexist and associated with age, sex and their interactions with COVID-19. Diverse epithelial and immune cell types were observed to be virus-positive and showed dramatic transcriptomic changes. Elevation of ANXA1 and S100A9 in virus-positive squamous epithelial cells may enable the initiation of neutrophil and macrophage responses via the ANXA1-FPR1 and S100A8/9-TLR4 axes. Systemic up-regulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and designing effective therapeutic strategies for COVID-19.

Modeling the Opening SARS-CoV-2 Spike: an Investigation of its Dynamic Electro-Geometric Properties (preprint)

The recent COVID-19 pandemic has brought about a surge of crowd-sourced initiatives aimed at simulating the proteins of the SARS-CoV-2 virus. A bottleneck currently exists in translating these simulations into tangible predictions that can be leveraged for pharmacological studies. Here we report on extensive electrostatic calculations done on an exascale simulation of the opening of the SARS-CoV-2 spike protein, performed by the Folding@home initiative. We compute the electric potential as the solution of the non-linear Poisson-Boltzmann equation using a parallel sharp numerical solver. The inherent multiple length scales present in the geometry and solution are reproduced using highly adaptive Octree grids. We analyze our results focusing on the electro-geometric properties of the receptor-binding domain and its vicinity. This work paves the way for a new class of hybrid computational and data-enabled approaches, where molecular dynamics simulations are combined with continuum modeling to produce high-fidelity computational measurements serving as a basis for protein bio-mechanism investigations.

Tocilizumab treatment in severe COVID-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis (preprint)

ABSTRACTDespite the current devastation of the COVID-19 pandemic, several recent studies have suggested that the immunosuppressive drug Tocilizumab can powerfully treating inflammatory responses that occur in this disease. Here, by employing single-cell analysis of the immune cell composition of severe-stage COVID-19 patients and these same patients in post Tocilizumab-treatment remission, we have identified a monocyte subpopulation specific to severe disease that contributes to inflammatory storms in COVID-19 patients. Although Tocilizumab treatment attenuated the strong inflammatory immune response, we found that immune cells including plasma B cells and CD8+ T cells still exhibited an intense humoral and cell-mediated anti-virus immune response in COVID-19 patients after Tocilizumab treatment. Thus, in addition to providing a rich, very high-resolution data resource about the immune cell distribution at multiple stages of the COVID-19 disease, our work both helps explain Tocilizumab’s powerful therapeutic effects and defines a large number of potential new drug targets related to inflammatory storms.Competing Interest StatementJingwen Fang is the executive officer of HanGen BiotechView Full Text