SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1.

Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.

Validation of evidence-based questionnaire for TCM syndrome differentiation of heart failure and evaluation of expert consensus.

BACKGROUND: Traditional Chinese Medicine (TCM) is widely used to treat heart failure (HF). Syndrome differentiation is a unique and crucial component in TCM practice for guiding disease diagnosis and treatment strategies as well as clinical research. The major bottlenecks in TCM syndrome differentiation are the diversity of the syndrome differentiation criteria and the broad spectrum of syndrome patterns, hindering evidence-based studies for clinical research. In the present study, we aim to develop an evidence-based questionnaire for the diagnosis of HF and establish a definitive set of criteria for syndrome differentiation. METHODS: We designed a TCM syndrome differentiation questionnaire for heart failure (SDQHF) based on the "TCM expert consensus for diagnosis and treatment of heart failure" (expert consensus), literature review, and various clinical guidelines. To test the reliability and efficiency of the questionnaire, we performed a large-scale multiple-center clinical trial with the recruitment of 661 HF patients. Cronbach's alpha was used to assess the internal consistency of the SDQHF. Content validity was conducted through expert review. Principal component analysis (PCA) was applied to evaluate the construct validity. We constructed a proposed model for syndrome differentiation for HF based on the PCA results. Tongue analysis was performed to verify the accuracy of syndromes derived from the proposed model and the expert consensus. An evidence-based practical questionnaire for TCM syndrome differentiation patients was developed and validated with the data from 661 HF patients. RESULTS: The syndrome differentiation criteria were constructed with five syndrome elements (qi-deficiency, yang-deficiency, yin-deficiency, blood stasis, and phlegm retention). The results revealed good convergent and discriminant validity, satisfactory internal consistency, and feasibility. The significant discoveries include: (1) A total of 91% of the derived TCM syndromes from the proposed model matched with the characterized tongue images of the syndrome patterns; (2) Qi Deficiency Syndrome is the dominant syndrome pattern for HF patients, followed by Yang-Qi Deficiency Syndrome and Qi-yin deficiency Syndrome, and finally, Yin-Yang Dual Deficiency Syndrome; (3) The majority of the HF patients had the combination of Blood Stasis and Phlegm Retention Syndromes; (4) The "Yin-Yang Dual Deficiency" Syndrome was a valid syndrome for HF, suggesting that this syndrome pattern should be included in the criteria for syndrome differentiation; and (5) Through the validation of the expert consensus, several recommendations were proposed to improve the accuracy of syndrome differentiation of HF. CONCLUSIONS: The proposed SDQHF and the criteria could be a reliable and valid tool for syndrome differentiation of heart failure with high accuracy. It is recommended to use the proposed model for evidence-based study on Chinese Medicine to diagnose and treat HF. TRIAL REGISTRATION NUMBER: The trial was registered at the Chinese Clinical Trial Registry, http://www.chictr.org.cn . (Registration No.: ChiCTR1900021929); Date: 2019-03-16.

Be Still and You Will Know: A Mixed-Method Study on Solitude and Consideration of Future Consequences Among Youth in Rehabilitation.

Although solitude is found to be undesirable to many, systematic practice of it can yield positive psychological outcomes. This mixed-method study explored the process and influence of solitude as a behavioral intervention among youths in a therapeutic community in Hong Kong. Qualitative interviews with 43 youths (67.4% male, mean age = 18.3) revealed that solitude facilitated growth in their sense of personal responsibility, increased perspective-taking, increased respect for rules, change in life attitudes, and growth in consideration of future consequences. A two-wave prospective study (n = 79, 82.3% male, mean age = 17.4) further demonstrated perceived meaningfulness in solitude predicted an increase in consideration of future consequences, but not in other types of behavioral intervention. This study preliminarily demonstrated solitude has beneficial outcomes among high-risk youths, and meaning-making can facilitate this relationship.

Mitochondrial regulation of acute extrafollicular B-cell responses to COVID-19 severity.

BACKGROUND: Patients with COVID-19 display a broad spectrum of manifestations from asymptomatic to life-threatening disease with dysregulated immune responses. Mechanisms underlying the detrimental immune responses and disease severity remain elusive. METHODS: We investigated a total of 137 APs infected with SARS-CoV-2. Patients were divided into mild and severe patient groups based on their requirement of oxygen supplementation. All blood samples from APs were collected within three weeks after symptom onset. Freshly isolated PBMCs were investigated for B cell subsets, their homing potential, activation state, mitochondrial functionality and proliferative response. Plasma samples were tested for cytokine concentration, and titer of Nabs, RBD-, S1-, SSA/Ro- and dsDNA-specific IgG. RESULTS: While critically ill patients displayed predominantly extrafollicular B cell activation with elevated inflammation, mild patients counteracted the disease through the timely induction of mitochondrial dysfunction in B cells within the first week post symptom onset. Rapidly increased mitochondrial dysfunction, which was caused by infection-induced excessive intracellular calcium accumulation, suppressed excessive extrafollicular responses, leading to increased neutralizing potency index and decreased inflammatory cytokine production. Patients who received prior COVID-19 vaccines before infection displayed significantly decreased extrafollicular B cell responses and mild disease. CONCLUSION: Our results reveal an immune mechanism that controls SARS-CoV-2-induced detrimental B cell responses and COVID-19 severity, which may have implications for viral pathogenesis, therapeutic interventions and vaccine development.

Lockd promotes myoblast proliferation and muscle regeneration via binding with DHX36 to facilitate 5' UTR rG4 unwinding and Anp32e translation.

Adult muscle stem cells, also known as satellite cells (SCs), play pivotal roles in muscle regeneration, and long non-coding RNA (lncRNA) functions in SCs remain largely unknown. Here, we identify a lncRNA, Lockd, which is induced in activated SCs upon acute muscle injury. We demonstrate that Lockd promotes SC proliferation; deletion of Lockd leads to cell-cycle arrest, and in vivo repression of Lockd in mouse muscles hinders regeneration process. Mechanistically, we show that Lockd directly interacts with RNA helicase DHX36 and the 5'end of Lockd possesses the strongest binding with DHX36. Furthermore, we demonstrate that Lockd stabilizes the interaction between DHX36 and EIF3B proteins; synergistically, this complex unwinds the RNA G-quadruplex (rG4) structure formed at Anp32e mRNA 5' UTR and promotes the translation of ANP32E protein, which is required for myoblast proliferation. Altogether, our findings identify a regulatory Lockd/DHX36/Anp32e axis that promotes myoblast proliferation and acute-injury-induced muscle regeneration.

A prebiotically plausible scenario of an RNA-peptide world.

The RNA world concept1 is one of the most fundamental pillars of the origin of life theory2-4. It predicts that life evolved from increasingly complex self-replicating RNA molecules1,2,4. The question of how this RNA world then advanced to the next stage, in which proteins became the catalysts of life and RNA reduced its function predominantly to information storage, is one of the most mysterious chicken-and-egg conundrums in evolution3-5. Here we show that non-canonical RNA bases, which are found today in transfer and ribosomal RNAs6,7, and which are considered to be relics of the RNA world8-12, are able to establish peptide synthesis directly on RNA. The discovered chemistry creates complex peptide-decorated RNA chimeric molecules, which suggests the early existence of an RNA-peptide world13 from which ribosomal peptide synthesis14 may have emerged15,16. The ability to grow peptides on RNA with the help of non-canonical vestige nucleosides offers the possibility of an early co-evolution of covalently connected RNAs and peptides13,17,18, which then could have dissociated at a higher level of sophistication to create the dualistic nucleic acid-protein world that is the hallmark of all life on Earth.

Development of RNA G-quadruplex (rG4)-targeting L-RNA aptamers by rG4-SELEX.

RNA G-quadruplex (rG4)-SELEX is a method that generates L-RNA aptamers to target an rG4 structure of interest, which can be applied to inhibit G-quadruplex-mediated interactions that have important roles in gene regulation and function. Here we present a Protocol Extension substantially modifying an existing SELEX protocol to describe in detail the procedures involved in performing rG4-SELEX to identify rG4-specific binders that can effectively suppress rG4-peptide and rG4-protein associations. This Protocol Extension improves the speed of aptamer discovery and identification, offering a suite of techniques to characterize the aptamer secondary structure and monitor binding affinity and specificity, and demonstrating the utility of the L-RNA aptamer. The previous protocol mainly describes the identification of RNA aptamers against proteins of interest, whereas in this Protocol Extension we present the development of an unnatural RNA aptamer against an RNA structure of interest, with the potential to be applicable to other nucleic acid motifs or biomolecules. rG4-SELEX starts with a random D-RNA library incubated with the L-rG4 target of interest, followed by binding, washing and elution of the library. Enriched D-aptamer candidates are sequenced and structurally characterized. Then, the L-aptamer is synthesized and used for different applications. rG4-SELEX can be carried out by an experienced molecular biologist with a basic understanding of nucleic acids. The development of rG4-targeting L-RNA aptamers expands the current rG4 toolkit to explore innovative rG4-related applications, and opens new doors to discovering novel rG4 biology in the near future. The duration of each selection cycle as outlined in the protocol is ~2 d.

HLA-B*58:01 screening to prevent allopurinol-induced severe cutaneous adverse reactions in Chinese patients with chronic kidney disease.

Human leukocyte antigen (HLA)-B*58:01 allele is a significant risk factor for allopurinol-induced severe cutaneous adverse reactions (SCARs) which is potentially fatal. In some studies, chronic kidney disease (CKD) was also implicated to compound the risk of SCARs. We aim to investigate if pre-treatment HLA-B*58:01 screening can prevent allopurinol-induced SCARs in Chinese patients with CKD and its cost-effectiveness. We prospectively recruited Chinese CKD patients who required allopurinol during 2011-2015 and performed pre-treatment HLA testing (HLA screening group). Patients tested positive for HLA-B*58:01 were refrained from allopurinol while those tested negative were prescribed allopurinol. The incidence of SCARs in the HLA screening group was compared with the historical control in previous 5 years and the cost-effectiveness of HLA testing was analyzed. In the historical control (2006-2010), 3605 patients on allopurinol were screened, 22 out of 1027 (2.14%) CKD Chinese patients newly started on allopurinol developed SCARs, including 6 SJS/TEN. In the HLA screening group, 28 out of 192 patients (14.6%) tested HLA-B*58:01 positive were advised to avoid allopurinol; 156 out of 164 HLA-B*58:01-negative patients received allopurinol and none developed SCARs. The incidence rate of SCARs was significantly lower in the HLA screening group compared with controls (0% vs 2.14% respectively, p = 0.037*). The targeted HLA screening approach was associated with lower healthcare costs compared with no HLA screening (US$ 92,430 vs US$ 281,226). Pre-treatment HLA-B*58:01 screening is cost-effective to target on patients with CKD in Chinese to prevent allopurinol-induced SCARs.

Nasal prevention of SARS-CoV-2 infection by intranasal influenza-based boost vaccination in mouse models.

BACKGROUND: Vaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal. METHODS: Since mucosal immunity is critical for nasal prevention, we investigated the efficacy of an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2. FINDINGS: Substantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity. INTERPRETATION: Our results demonstrated that intranasal influenza-based boost vaccination induces mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems. FUNDING: This study was supported by the Research Grants Council Collaborative Research Fund, General Research Fund and Health and Medical Research Fund in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program and matching fund from Shenzhen Immuno Cure BioTech Limited; the Health@InnoHK, Innovation and Technology Commission of Hong Kong; National Program on Key Research Project of China; donations from the Friends of Hope Education Fund; the Theme-Based Research Scheme.

Tandem bispecific antibody prevents pathogenic SHIVSF162P3CN infection and disease progression.

Although progress has been made on constructing potent bi-specific broadly neutralizing antibody (bi-bNAb), few bi-bNAbs have been evaluated against HIV-1/AIDS in non-human primates (NHPs). Here, we report the efficacy of a tandem bi-bNAb, namely BiIA-SG, in Chinese-origin rhesus macaques (CRM) against the CRM-adapted R5-tropic pathogenic SHIVSF162P3CN challenge. Pre-exposure BiIA-SG injection prevents productive viral infection in 6 of 6 CRMs with unmeasurable proviral load, T cell responses, and seroconversion. Single BiIA-SG injection, at day 1 or 3 post viral challenge, significantly reduces peak viremia, achieves undetectable setpoint viremia in 8 of 13 CRMs, and delays disease progression for years in treated CRMs. In contrast, 6 of 8 untreated CRMs develop simian AIDS within 2 years. BiIA-SG-induced long-term protection is associated with CD8+ T cells as determined by anti-CD8ß antibody depletion experiments. Our findings provide a proof-of-concept that bi-bNAb treatment elicits T cell immunity in NHPs, which warrant the clinical development of BiIA-SG for HIV-1 prevention and immunotherapy.

Proteomic and Transcriptome Profiling of G-Quadruplex Aptamers Developed for Cell Internalization.

Nucleic acid medicine is expected to be among the most promising next-generation therapies. Applications of nucleic acid in vivo are still challenging as a result of the difficulties in direct cell penetration without external assistance. To facilitate the cellular delivery of therapeutic nucleic acid, we developed cell-penetrating aptamers using cell-internalization Systematic Evolution of Ligands by EXponential enrichment (SELEX). Moreover, C20-4 min, a G-quadruplex-forming DNA aptamer, was discovered, showing a higher cell-penetrating capacity compared with other candidates, including AS1411. To verify the formation and understand the G-quadruplex folding topologies of enriched aptamer motifs, characteristic circular dichroism (CD) spectral features are analyzed. The CD spectra of C20-4 min strongly support the formation of parallel G-quadruplexes. Systematic analyses of the G-quadruplex regulation pathway have been performed by combining aptamer pull-down with mass spectrometry. We profiled G-quadruplex aptamers interacting with cellular proteins during internalization and identified helicases and GTPase proteins as cellular interacting partners. In addition, whole transcriptome analysis was performed to study the effects of G-quadruplex aptamers, revealing differentially expressed genes involved in the regulation of GTPase functions. Integrative analyses of transcriptome and proteomic have aided in understanding the functional hierarchy of molecular players in G-quadruplex nucleic acid mechanisms of internalization, which might facilitate developing a novel delivery system.

Robust SARS-CoV-2 infection in nasal turbinates after treatment with systemic neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a burst in the upper respiratory portal for high transmissibility. To determine human neutralizing antibodies (HuNAbs) for entry protection, we tested three potent HuNAbs (IC50 range, 0.0007-0.35 µg/mL) against live SARS-CoV-2 infection in the golden Syrian hamster model. These HuNAbs inhibit SARS-CoV-2 infection by competing with human angiotensin converting enzyme-2 for binding to the viral receptor binding domain (RBD). Prophylactic intraperitoneal or intranasal injection of individual HuNAb or DNA vaccination significantly reduces infection in the lungs but not in the nasal turbinates of hamsters intranasally challenged with SARS-CoV-2. Although postchallenge HuNAb therapy suppresses viral loads and lung damage, robust infection is observed in nasal turbinates treated within 1-3 days. Our findings demonstrate that systemic HuNAb suppresses SARS-CoV-2 replication and injury in lungs; however, robust viral infection in nasal turbinate may outcompete the antibody with significant implications to subprotection, reinfection, and vaccine.

Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA-Peptide World.

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.

Specific Binding of a d-RNA G-Quadruplex Structure with an l-RNA Aptamer.

G-quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4-binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l-RNA aptamer that targets a d-RNA G-quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l-RNA aptamer, Ap3-7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA-rG4-RHAU53 binding. Our approach and findings open a new door in further developing G4-specific tools for diverse applications.

G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications.

Guanine (G)-quadruplexes (G4s) are unique nucleic acid structures that are formed by stacked G-tetrads in G-rich DNA or RNA sequences. G4s have been reported to play significant roles in various cellular events in both macro- and micro-organisms. The identification and characterization of G4s can help to understand their different biological roles and potential applications in diagnosis and therapy. In addition to biophysical and biochemical methods to interrogate G4 formation, G4 fluorescent turn-on ligands can be used to target and visualize G4 formation both in vitro and in cells. Here, we review several representative classes of G4 fluorescent turn-on ligands in terms of their interaction mechanism and application perspectives. Interestingly, G4 structures are commonly identified in DNA and RNA aptamers against targets that include proteins and small molecules, which can be utilized as G4 tools for diverse applications. We therefore also summarize the recent development of G4-containing aptamers and highlight their applications in biosensing, bioimaging, and therapy. Moreover, we discuss the current challenges and future perspectives of G4 fluorescent turn-on ligands and G4-containing aptamers.

Spectroscopic analysis reveals the effect of a single nucleotide bulge on G-quadruplex structures.

Here we investigate and reveal the effect of bulge position and bulge identity on G-quadruplexes using label-free spectroscopic techniques. Notably, we report significant differences in the spectroscopic features of bulged DNA and RNA G-quadruplexes, and demonstrate that intrinsic fluorescence can be generally used to detect the formation of canonical and non-canonical G-quadruplexes.

Structural analysis and cellular visualization of APP RNA G-quadruplex.

RNA G-quadruplexes (rG4s) are emerging structural motifs that are of pivotal importance in chemistry and biology; however, the current structural information of rG4s is limited, with their folding status and functions in cells remaining elusive. Here, we develop and employ a multi-disciplinary approach to characterize the structure, formation and function of an individual rG4 of interest in vitro and in cells. We apply this strategy to a biologically important rG4 in amyloid precursor protein (APP) transcript and reveal distinct structural features of APP rG4. Notably, we visualize the formation of APP rG4 in cells using an APP-specific G-quadruplex-triggered fluorogenic hybridization (GTFH) probe and report that the regulatory role of APP rG4 in translation is dependent on rG4 thermostability, providing evidence to the existence and significance of the stable rG4 structure in gene regulation.

Structural analysis reveals the formation and role of RNA G-quadruplex structures in human mature microRNAs.

Here we identify hundreds of RNA G-quadruplex (rG4) candidates in microRNAs (miRNAs), characterize the miRNA structure and miRNA-mRNA interactions on several mammalian-conserved miRNAs, and reveal the formation of rG4s in miRNAs. Notably, we study the effect of these rG4s in cells and uncover the role of rG4s in miRNA-mediated post-transcriptional regulation.

A Cancer Cell-Selective and Low-Toxic Bifunctional Heterodinuclear Pt(IV)-Ru(II) Anticancer Prodrug.

Although different types of metal-based anticancer complexes have been synthesized, novel complexes to reduce the serious side effect of cisplatin and conquer cancer metastasis are still highly desired. Here, we report the synthesis, characterization, and biological activity of a novel heterodinuclear Pt(IV)-Ru(II) anticancer prodrug. The Pt(IV)-Ru(II) complex exhibits good stability in both water and PBS solution. Biological evaluation revealed that this bifunctional Pt(IV)-Ru(II) complex utilizes the advantages of two metal centers to have both cytotoxicity and antimetastatic property as designed. Although the complex has comparable cytotoxicities to cisplatin in tested cancer cell lines, this prodrug selectively kills cancer but not normal cells, and the IC50 values of the Pt(IV)-Ru(II) complex are 7-10 times higher than those of cisplatin toward normal cells. The cancer cell selectivity is further demonstrated by a cancer-normal cell coculture system. In addition, the antimetastatic properties of the heterodinuclear complex are assessed by using highly metastatic human breast cancer cells, and the results show that the migration and invasion of cancer cells are effectively restrained after the treatment. Moreover, the Pt(IV)-Ru(II) complex displays lower toxicity than cisplatin in developing zebrafish embryos. We, therefore, report an example of heterodinuclear Pt(IV)-Ru(II) complex not only to defeat both drug resistance and cancer metastasis but also having significantly improved cancer cell selectivity and reduced in vivo toxicity than cisplatin.