Single-cell profiling reveals Müller glia coordinate retinal intercellular communication during light/dark adaptation via thyroid hormone signaling

Light adaptation enables the vertebrate visual system to operate over a wide range of ambient illumination. Regulation of phototransduction in photoreceptors is considered a major mechanism underlying light adaptation. However, various types of neurons and glial cells exist in the retina, and whether and how all retinal cells interact to adapt to light/dark conditions at the cellular and molecular levels requires systematic investigation. Therefore, we utilized single-cell RNA sequencing to dissect retinal cell-type-specific transcriptomes during light/dark adaptation in mice. The results demonstrated that, in addition to photoreceptors, other retinal cell types also showed dynamic molecular changes and specifically enriched signaling pathways under light/dark adaptation. Importantly, Müller glial cells (MGs) were identified as hub cells for intercellular interactions, displaying complex cell‒cell communication with other retinal cells. Furthermore, light increased the transcription of the deiodinase Dio2 in MGs, which converted thyroxine (T4) to active triiodothyronine (T3). Subsequently, light increased T3 levels and regulated mitochondrial respiration in retinal cells in response to light conditions. As cones specifically express the thyroid hormone receptor Thrb, they responded to the increase in T3 by adjusting light responsiveness. Loss of the expression of Dio2 specifically in MGs decreased the light responsive ability of cones. These results suggest that retinal cells display global transcriptional changes under light/dark adaptation and that MGs coordinate intercellular communication during light/dark adaptation via thyroid hormone signaling.

Structures and function of locked conformations of SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

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

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 upregulation 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. HIGHLIGHTSO_LILarge-scale scRNA-seq analysis depicts the immune landscape of COVID-19 C_LIO_LILymphopenia and active T and B cell responses coexist and are shaped by age and sex C_LIO_LISARS-CoV-2 infects diverse epithelial and immune cells, inducing distinct responses C_LIO_LICytokine storms with systemic S100A8/A9 are associated with COVID-19 severity C_LI

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

A thermostable, closed, SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

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

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

Observation on clinical effect of Shufeng Jiedu Capsule combined with Arbidol Hydrochloride Capsule in treatment of COVID-19 / 中草药

Objective: To observe the clinical effect of Shufeng Jiedu Capsule combined with Arbidol Hydrochloride Capsule in the treatment of COVID-19. Methods: From January 31, 2020 to February 11, 2020, 70 patients with COVID-19 diagnosed and treated in Bozhou people's hospital were selected. According to the different treatments, they were divided into two groups: 40 patients in combination group and 30 patients in Arbidol Hydrochloride Capsule group. Patients in both groups were given routine Arbidol Hydrochloride Capsule orally. On this basis, the combination group was given oral Shufeng Jiedu Capsule for 10 d. The antipyretic time and the disappearance time of dry cough, nasal congestion, runny nose, sore throat, fatigue, diarrhea and other symptoms of patients in two groups were compared. The negative conversion ratio and negative conversion time of novel coronavirus (SARS-CoV-2) were also compared between two groups of patients. Results: There were significant differences in antipyretic time, the disappearance time of dry cough, nasal congestion, runny nose, pharyngeal pain, fatigue, diarrhea, and novel coronavirus negative conversion time in the combination treatment group compared with the control group (P < 0.05). The negative conversion time of combination treatment group was significantly shorter than Arbidol Hydrochloride Capsule group (P < 0.05). Conclusion: The combination of Shufeng Jiedu Capsule and Arbidol Hydrochloride Capsule was better than Arbidol Hydrochloride Capsule alone in the treatment of COVID-19, which could significantly shorten the symptoms improvement time and negative conversion time of the clinical patients.

Rapid Identification of Alkaloids in Evodia rutaecarpa by HPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To obtain the information of alkaloids in Evodia rutaecarpa by HPLC-Q-TOF-MS/MS. Method:Inter Sustain-C18 column (4.6 mm×250 mm,5 μm) was used with 0.2% formic acid water-acetonitrile as the mobile phase for gradient elution. The column temperature was 25℃,the volume flow rate was 1.0 mL·min-1,and the sample volume is 5 μL. The detection wavelength was 245 nm,and the chromatographic effluent was detected and analyzed by using both positive and negative ions. Result:According to molecular ion peaks and secondary mass spectrometry characteristic fragment ions,as well as the mass spectrometry information of reference substances and relevant literature reports,more than 40 major peaks were analyzed,and 21 alkaloids were identified from the methanol extract of E. rutaecarpa, including 10 kinds of indole alkaloids,10 kinds of quinolone alkaloids,and 1 kind of ephedrine. Main types of alkaloids in E. rutaecarpa were basically clarified. And the research found that the alkaloids have a good response mainly in the positive mode. Conclusion:Based on HPLC-Q-TOF-MS/MS technology, high-performance liquid chromatography (HPLC) separation,mass spectrometry determination of molecular mass,pyrolysis data,literature analysis and retrieval were performed to quickly,accurately and comprehensively identify alkaloids in E. rutaecarpa, so as to provide a scientific basis for the further extraction and separation of the chemical constituents of E. rutaecarpa.

Veratrum nigrum inhibits the estrogenic activity of salvia miltiorrhiza bunge in vivo and in vitro.

BACKGROUND: As recorded in the 18 incompatible medicaments of Traditional Chinese Medicine theory, the combined use of Salvia miltiorrhiza bunge (SM) and Veratrum nigrum (VN) could induce toxicity and has been prohibited for thousands of years in China. However, the theory has been validated due to lack of evidence. Previous studies have focused on the chemical constituents that are responsible for the toxicity of the two agents. PURPOSE: This study offers preliminary insight into the pharmacodynamics and mechanism of estrogenic activity responsible for their incompatibility. STUDY DESIGN: We undertook a characterization of the interaction between estrogenic activities of SM and VN using in vivo models of immature and ovariectomized (OVX) mice, and in vitro studies focused on the estrogen receptor (ER) pathway for further mechanism. METHODS: Immature and OVX mice were treated intragastrically with SM at doses of 1.6, 3.2 g/kg, or combine with 0.045 g/kg VN and 0.005 g/kg the ER antagonist ICI182, 780 for elucidating the effects on estrogenic activity in reproductive tissues, E2 secretion, and the ER mechanism. ERα/ß binding experiments and ERα/ß transcriptional activity were performed in order to evaluate the biological action exerted through ERs. RESULTS: VN decreased the estrogenic efficacy of SM in promoting the development of the uterus and vagina in immature mice, and reversing the atrophy of reproductive tissues in OVX mice. VN interfered with the estrogenic efficacy of SM by decreasing the serum estradiol and the upregulation of ERα and ERß expressions in reproductive tissues by treatment with SM. VN antagonized the estrogenic efficacy of SM in promoting the viability of MCF-7 cells and stimulating the binding ability with ERα and ERß, and increasing ERα/ß-estrogen response element (ERE) luciferase activity. CONCLUSIONS: This study provided evidence that the combined use of SM and VN could induce unfavorable effects. VN decreased the estrogenic activity of SM, which might be related to the regulation of estrogen secretion and ERs through the ER-ERE pathway.