Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3KBTBD2.

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

Structure-based discovery of dual pathway inhibitors for SARS-CoV-2 entry.

Since 2019, SARS-CoV-2 has evolved rapidly and gained resistance to multiple therapeutics targeting the virus. Development of host-directed antivirals offers broad-spectrum intervention against different variants of concern. Host proteases, TMPRSS2 and CTSL/CTSB cleave the SARS-CoV-2 spike to play a crucial role in the two alternative pathways of viral entry and are characterized as promising pharmacological targets. Here, we identify compounds that show potent inhibition of these proteases and determine their complex structures with their respective targets. Furthermore, we show that applying inhibitors simultaneously that block both entry pathways has a synergistic antiviral effect. Notably, we devise a bispecific compound, 212-148, exhibiting the dual-inhibition ability of both TMPRSS2 and CTSL/CTSB, and demonstrate antiviral activity against various SARS-CoV-2 variants with different viral entry profiles. Our findings offer an alternative approach for the discovery of SARS-CoV-2 antivirals, as well as application for broad-spectrum treatment of viral pathogenic infections with similar entry pathways.

Asymmetric pendrin homodimer reveals its molecular mechanism as anion exchanger.

Pendrin (SLC26A4) is an anion exchanger expressed in the apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However its molecular structure has remained unknown, limiting our understanding of the structural basis of transport. Here, we determine the cryo-electron microscopy structures of mouse pendrin with symmetric and asymmetric homodimer conformations. The asymmetric homodimer consists of one inward-facing protomer and the other outward-facing protomer, representing coincident uptake and secretion- a unique state of pendrin as an electroneutral exchanger. The multiple conformations presented here provide an inverted alternate-access mechanism for anion exchange. The structural and functional data presented here disclose the properties of an anion exchange cleft and help understand the importance of disease-associated variants, which will shed light on the pendrin exchange mechanism.

Fortuitous somatic mutations during antibody evolution endow broad neutralization against SARS-CoV-2 Omicron variants.

Striking antibody evasion by emerging circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identify a clonally related antibody family from a convalescent individual. One of the members, XG005, exhibits potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members show significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface reveals how crucial somatic mutations endow XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality exhibits a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provide a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

Diagnostic value of curved planar reformation of MRI for lumbosacral nerve roots in the localization of nerve roots for adult lumbar degenerative scoliosis.

PURPOSE: To compare the diagnostic value of curved planar reformation of MRI (MRI-CPR) and 2D MRI in determining the responsible nerve in patients with adult lumbar degenerative scoliosis (ALDS). METHOD: A total of 45 patients diagnosed with ALDS were included in the study. All patients underwent MRI-CPR and 2D MRI and subsequently received surgery. These two diagnostic methods were compared with the results of surgical exploration to assess nerve root compression. RESULTS: The sensitivity and accuracy of MRI-CPR are higher than 2D MRI (93.8% vs 80.0%; 92.8% vs 77.7%, respectively). And the specificity of MRI-CPR is higher than 2D MRI (87.5% vs 68.8%). Besides, the PPV and NPV of MRI-CPR are higher than 2D MRI (96.8% vs 91.2%; 7.8.% vs 45.8%). The area (AUC) under the receiver operating characteristic curve (ROC) for MRI-CPR and 2D MRI was 0.74 and 0.91, respectively. The judgement was made by two independent radiologists, while the consistency tests for 2D MRI and MRI-CPR with Kappa values were 90.6% and 82.2%, respectively. CONCLUSIONS: The clinical diagnostic value of MRI-CPR was better than 2D MRI in the determination of the responsible nerve root. Moreover, MRI-CPR sequence images can clearly show the route of lumbosacral nerve roots and their relationship with adjacent tissues. Therefore, MRI-CPR can be an important complement to conventional 2D MRI in the diagnosis of responsible nerve roots in patients with ALDS.

Fortuitous Somatic Mutations during Antibody Evolution Endow Broad Neutralization against SARS-CoV-2 Omicron Variants.

Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

Structural Study of SARS-CoV-2 Antibodies Identifies a Broad-Spectrum Antibody That Neutralizes the Omicron Variant by Disassembling the Spike Trimer.

The continuous emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses new challenges in the fight against the coronavirus disease 2019 (COVID-19) pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of an antibody targeting a conserved and universal epitope is urgently needed. A subset of neutralizing antibodies (NAbs) against COVID-19 from convalescent patients were isolated in our previous study. In this study, we investigated the accommodation of these NAbs to SARS-CoV-2 variants of concern (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of the SARS-CoV-2 Omicron variant. In addition, we determined the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 spike (S) protein complexed with three monoclonal antibodies targeting different epitopes, including 553-49, 553-15, and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes the virus by disassembling S trimers. IgG 553-15, an antibody that neutralizes all of the VOCs except Omicron, cross-links two S trimers to form a trimer dimer, demonstrating that 553-15 neutralizes the virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 553-49 and other antibodies targeting this highly conserved epitope as promising therapeutic reagents for COVID-19. IMPORTANCE The emergence of the Omicron strain of SARS-CoV-2 caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 neutralizing antibody, 553-49, which neutralizes all variants by targeting a completely conserved novel epitope. In addition, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by cross-linking virions and that 553-60 functions by blocking receptor binding. Comparison of different receptor binding domain (RBD) epitopes revealed that the 553-49 epitope is hidden in the S trimer and keeps a high degree of conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutic reagent against the emerging Omicron and future variants of SARS-CoV-2.

An ultrapotent pan-ß-coronavirus lineage B (ß-CoV-B) neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope.

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes ß-coronavirus lineage B (ß-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-ß-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against ß-CoV-B and newly emerging SARS-CoV-2 variants of concern.

Controlling upconversion luminescence patterns in space with red emission enhancement from a single fluoride microcrystal by tuning the excitation mode.

The ability to control the upconversion (UC) luminescence patterns in space from lanthanide-doped UC luminescence materials is very important for many applications including three-dimensional color displays, optical waveguides and optical communication. In this work, the fascinating UC luminescence patterns could be adjusted from the blue or green flower-like emission pattern to the red flame-like irradiation pattern with a red luminescence enhancement from a single ß-NaYF4:Yb3+/(Tm3+ or Er3+) microcrystal (MC) by varying the excitation position. The red-to-blue (R/B) and the red-to-green (R/G) emission intensity ratios from the single MC particle that the focal point position of excitation NIR laser is on the side lace are much stronger than that in the case of the focused laser beam on the center. We think that the physical mechanism from the changes in the luminescence pattern and the emission intensity ratio is explained by the optical waveguide effect based on the total reflection effect. These results provide a new strategy for facilitating fundamental investigations of the UC micro/nano-materials, which will lead to promising applications in three-dimensional color display, optical waveguides and optical communication.

[Relationship between current smoking behaviors and household characteristics in Guangdong].

OBJECTIVE: To disclose the relationship between current smoking behaviors and household characteristics among adults in Guangdong, in order to provide scientific evidence for household tobacco control strategy. METHODS: A questionnaire survey was conducted among the adults sampled by four-stage randomized cluster sampling in ten districts of five cities in Guangdong from September to November, 2010. The questionnaire included demographic characteristics, smoking behaviors and smoking-related household characteristics. Chi-square test and unconditional logistic regression analysis were employed to explore the relationship between current smoking behaviors and household characteristics. RESULTS: This study included 2 150 respondents, with data from 2 121 eligible questionnaires used for analysis. The current smoking rates for respondents with household characteristics including per capita household income below 1 000 Yuan, three or more smokers in the family, no ban of smoking at home and treating visitors with cigarettes were 28.7%, 56.5%, 34.2% and 43.5% , respectively, and were significantly higher than those for respondents with household characteristics at other levels (P < 0.01 for all). After controlling for factors such as gender, age, personal income, education, employment status and residential location, we found that the number of family smokers, smoking restrictions at home and treating visitors with cigarettes had significant association with current smoking behaviors (P < 0.01 for all), except for per capita household income (P = 0.567). CONCLUSION: Household is one of the effective places in carrying out health education and tobacco control intervention. In order to reduce tobacco use, much effort should be made to create smoke-free homes, encourage smoking cessation and treat visitors with no cigarettes.

[Design of airborne dual channel ultraviolet-visible imaging spectrometer with large field of view, wide spectrum, and high resolution].

The ultraviolet-visible (UV-Vis 200-500 nm) imaging spectrometer is an important part of space remote sensing. Based on special requirements and practical application of the airborne UV-VIS spectrometer, a kind of scanning imaging spectrometer using area array CCD is proposed, which can meet the application requirements of large field of view, wide spectrum and high resolution. It overcomes low spatial resolution of traditional line array CCD scanning imaging spectrometer, and limited field of view of the pushbroom imaging spectrometer. In addition, dual channel was designed to reduce stray light. 400-500 nm band includes two order spectrum for 200-250 nm band, and variation of radiance from earth between the shorter wavelength (<290 nm) and the longer wavelength (>310 nm) is above three orders of magnitude. In the structure design of the system, the imaging spectrometer is composed of a two-mirror concentric telescope and two Czerny-Turner plane grating imaging spectrometers. The whole system doesn't use any additional optical elements in addition to spherical mirrors. The whole system has the advantage of simple structure, excellent performance, and very good feasibility. The modulation transfer function value of full spectrum and full field of view is above 0.6.

[Promoting of angiogenesis and osteogenesis in radial critical bone defect regions of rabbits with nano-hydroxyapatite/collagen/PLA scaffolds plus endothelial progenitor cells].

OBJECTIVE: To explore the roles of nano-hydroxyapatite/collagen/PLA (nHAC/PLA) plus endothelial progenitor cells (EPCs) in repairing segmental bone defects of rabbit radius and enhancing angiogenesis and new bone formation. METHODS: EPCs isolated from New Zealand white rabbit bone marrow were cultured, identified and seeded into nHAC/PLA scaffolds. And the growth of EPCs in scaffolds was observed under scanning electron microscopy (SEM). Thirty-six were randomly divided into 3 groups to establish segmental bone defect models in radii. Two groups were implanted with EPCs/scaffolds constructs (group A, n = 16) and scaffolds alone (group B, n = 16) respectively. The remaining four rabbits were used as negative control (group C) and nothing was implanted. Animals were sacrificed at different timepoints and radii harvested to undergo radiological examination, histological examination and microvessle density test. RESULTS: These cells isolated from bone marrow were confirmed as EPCs. SEM showed that EPCs attached to the nHAC/PLA scaffolds, grew and proliferated well. Animal experiments revealed that radiological scores (5w: 2.25 ± 0.50 vs 1.00 ± 0.00; 10w: 2.75 ± 0.50 vs 1.75 ± 0.50; 15w: 4.25 ± 0.50 vs 3.0 ± 0.0; each P < 0.05), percentage of new bone formation area in bone defect regions (5w: 29.0% ± 3.5% vs 8.1% ± 0.8%; 10w: 63.4% ± 5.5% vs 16.6% ± 1.3%; 15w: 96.0% ± 4.3% vs 34.0% ± 6.6%; each P < 0.05) and microvessel density (2w: 13.5 ± 0.9 vs 4.3 ± 1.0; 5w:9.8 ± 0.7 vs 4.8 ± 0.3; 10w: 7.0 ± 0.4 vs 4.5 ± 0.4; each P < 0.05) in group A were significantly higher than those in group B. No new bone formation occurred in group C. CONCLUSION: The composite structure of EPCs-nHAC/PLA can enhance angiogenesis and new bone formation in segmental bone defects in rabbit radii. It may become a potential candidate of promoting revascularization of tissue engineering bone and repairing large bone defects.

Epidemiological investigation of an outbreak of pandemic influenza A (H1N1) 2009 in a boarding school: serological analysis of 1570 cases.

BACKGROUND: A large number of 2009 pandemic influenza A (H1N1) infections were localized in school populations. OBJECTIVES: To describe the epidemiology, clinical features and risk factors associated with an outbreak that occurred at a vocational boarding school in Guangzhou, P.R. China. STUDY DESIGN: Data were collected prospectively and retrospectively through the use of on-site doctors and a post-outbreak survey and blood collection. The survey was used to confirm symptoms, and to investigate a series of flu-related factors such as dormitory conditions, health habits, vaccine history and population contact history. Blood samples were taken for serological analysis. Pandemic H1N1 infection was initially confirmed by a real-time RT-PCR assay. Following the identification of the outbreak by the Guangzhou CDC on September 4, cases were diagnosed symptomatically and retrospectively by serological analysis using the hemagglutination inhibition assay and a neutralization assay. RESULTS: The infection rate was 32% (505/1570) and the attack rate was 22.2% (349/1570). The asymptomatic infection rate was 9.9% (156/1570). Sharing a classroom (OR=2.17, 95% CI: 1.62-2.91) and dormitory space (OR=2.32, 95% CI: 1.84-2.93) was associated with higher rates of infection. Opening windows for ventilation was the only control measure that significantly protected against infection. CONCLUSION: Social isolation and quarantine should be used to prevent the spread of infection. Ventilation and a control of air flow between classrooms and dorms should be implemented as possible. School closures may be effective if implemented early.

[Risk assessment of H5N1 human infection after an outbreak of avian influenza in water fowl].

OBJECTIVE: To evaluate the risk of human infection after the outbreak of avian influenza H5N1 in animals, and probe the possibility for virus transmission. METHODS: By means of field epidemiological study, molecular epidemiology, serology and emergency surveillance, persons who had ever closely contacted with sick or dead poultry were observed. While, the RT-PCR and gene sequencing method were used to detect H5 nucleic acid from environmental swabs from 4 epidemic spots, and hemagglutination inhibition assay was also used to detect H5 antibody. RESULTS: Of 22 environmental swabs detected from 4 epidemic spots, one was positive for H5 nucleic acid, and the homogeneity was 95.9% as compared with H5N1 virus A/China/GD01/2006 (H5N1) found in Guangzhou in 2006 by gene sequence analysis. 62 environmental swabs from live poultry stalls of food markets near epidemic spot were detected negative. Six of 68 blood samples of contacts were positive for H9 antibody, and all were negative for H5 antibody. 68 throat swabs of contacts were detected negative for H5 nucleic acid. No close contact was found abnormal after 7 days medical observation. 337 influenza-like cases were reported in emergency surveillance, and no suspicious case was found. CONCLUSION: The current outbreak of H5N1 avian influenza in water fowls has not yet caused further transmission, and human avian influenza case has not been observed. It indicates that the ability of H5N1 virus to transmit to human is not strong yet, and the risk of human infection for H5N1 is still low.

[Needs on information related to influenza pandemic by the public].

OBJECTIVE: To understand the information needs on human infection with avian influenza (H5N1), A (H1N1) and influenza pandemic in different time periods and create well-targeted messages by the public, so as to develop communication with the public effectively when human infection with avian influenza (H5N1), A (H1N1) and influenza pandemic occur. METHODS: Data were collected through questionnaire over telephone calls. The questionnaire was self-designed, revised after pilot testing. RESULTS: The effective response rate in this telephone survey was 54.33%, higher than those in Beijing and Shanghai. The respondents felt that government's propaganda during the influenza virus-active period was more helpful and instructive than those in the period when influenza was relatively inactive (chi(2) = 17.41, P = 0.000). Trust to the government by the public was higher in the influenza virus-active than in the relatively inactive period (chi(2) = 8.15, P = 0.004). As to the information needs, the respondents reflected that during the time period of relative influenza inactivity, they viewed the basic knowledge of human infection with avian influenza (H5N1) as their top priority, while in the influenza virus-active period, the feasible preventive measures was their top priority. The pandemic-related information needs in the influenza virus-active period and in the time period of relative influenza inactivity were similar, demonstrating that the respondents had no perceptual knowledge of influenza pandemic and had poor awareness on the pandemic. The respondents were not used to wear a mask to cover their mouths and noses when sneezing, but were quite knowledgeable about the following preventive measures as: avoid going out and at the crowded areas, wash hands frequently etc. Around 2% to 6% of the respondents did not have any information needs. CONCLUSION: The response rate in this telephone survey was fairly high, indicating that telephone survey was well accepted by Guangzhou residents. The public was satisfied with the government's propaganda activities and trusting the government. Public health professionals should develop communication messages closely related to the epidemic situation to target the information needs from the public.

[Epidemiological study on injuries among children in Beijing].

OBJECTIVE: To explore the morbidity, burdens caused and risk factors of injuries among children and to provide information for policy making on prevention. METHODS: By PPS sampling method, we selected 28 000 households in Beijing and using a questionnaire developed by UNICEF, TASC and China CDC. RESULTS: The morbidity of child injury in Beijing was 2.25%, higher than that of adults (1.56%) with 6-9 year group the highest (2.83% ). The morbidity of boys was 3.04% higher than that of girls (1.37%). Among the 7 death cases, 3 were caused by injury(2 RTA, 1 drowning). The three leading causes of injuries were fall, animal bite and road traffic accident in children. Among children aged 0-5 years, fall, animal and burn/machine injuries were the leading causes. Among children aged 0-5 years, the leading causes were hurt by animals, fall and RTA with the leading causes as fall, sprain and RTA. The average medical cost of injury cases was 1617.57 Yuan. There were 3500 children (injured orphan) whose parents were injured leading to death or disability in Beijing in 2003. CONCLUSION: Injury was the leading cause of child deaths in Beijing with children of 6-9 years old having the highest morbidity with RTA the most severe type of injuries in Beijing which affects both children and their parents.

Germline splicing mutations of CDKN2A predispose to melanoma.

Coding mutations of the CDKN2A gene on chromosome 9p21 cosegregate with 25-60% of familial melanoma cases, but there remains a number of 9p21-linked kindreds that lack germline coding mutations of CDKN2A. We sequenced CDKN2A exons 1alpha, 2, 3, and the adjacent intronic regions in 167 melanoma-prone families (at least two affected first-degree relatives), and detected four splice site variations, three of which cosegregate with the disease. RT-PCR experiments verified that these three variants, including an AGgt to ATgt mutation that demonstrates a founder effect, do affect splicing. While an exon 1alpha splice donor site mutation incompletely abolishes splicing, the correctly spliced mRNA yields a protein (Q50P) that cannot effectively interact with CDK4. We also performed RT-PCR on mRNA from 16 melanoma-prone kindreds to search for cryptic splice sites deep within introns, but identified no splice variants. Meanwhile, we screened 139 affected families using allele-specific PCR for the recently discovered IVS2-105A>G mutation, but found only one family that possesses this alteration. We conclude that splice site mutations do predispose to disease in a subset of melanoma-prone kindreds. Characterization of additional splice site variants and other noncoding alterations of CDKN2A should allow us to detect a wider range of mutations in at-risk patients.