Many individuals mount nearly identical antibody responses to SARS-CoV-2. To gain insight into how the viral spike (S) protein receptor-binding domain (RBD) might evolve in response to common ...antibody responses, we studied mutations occurring during virus evolution in a persistently infected immunocompromised individual. We use antibody Fab/RBD structures to predict, and pseudotypes to confirm, that mutations found in late-stage evolved S variants confer resistance to a common class of SARS-CoV-2 neutralizing antibodies we isolated from a healthy COVID-19 convalescent donor. Resistance extends to the polyclonal serum immunoglobulins of four out of four healthy convalescent donors we tested and to monoclonal antibodies in clinical use. We further show that affinity maturation is unimportant for wild-type virus neutralization but is critical to neutralization breadth. Because the mutations we studied foreshadowed emerging variants that are now circulating across the globe, our results have implications to the long-term efficacy of S-directed countermeasures.
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•SARS-CoV-2 spike evolves during persistent infection to resist common antibodies•Antibody affinity maturation is critical to neutralization breadth•Intra-host evolution foreshadows mutations in circulating spike variants
Structural and functional analysis of the evolution of SARS-CoV-2 in a persistently infected immunocompromised individual yields insights into mutations that foreshadow emerging viral variants worldwide.
Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults
. No vaccine or effective antiviral therapy currently exists to ...control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities
. How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å
of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers-which are largely disordered in their free state-demonstrates an example of a 'folding-upon-partner-binding' mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs.
1,5-Anhydroglucitol (1,5-AG) is sensitive to short-term glucose fluctuations and postprandial hyperglycemia, which has great potential in the clinical application of diabetes as a nontraditional ...blood glucose monitoring indicator. A large number of studies have found that 1,5-AG can be used to screen for diabetes, manage diabetes, and predict the perils of diabetes complications (diabetic nephropathy, diabetic cardiovascular disease, diabetic retinopathy, diabetic pregnancy complications, diabetic peripheral neuropathy, etc.). Additionally, 1,5-AG and β cells are also associated with each other. As a noninvasive blood glucose monitoring indicator, salivary 1,5-AG has much more benefit for clinical application; however, it cannot be ignored that its detection methods are not perfect. Thus, a considerable stack of research is still needed to establish an accurate and simple enzyme assay for the detection of salivary 1,5-AG. More clinical studies will also be required in the future to confirm the normal reference range of 1,5-AG and its role in diabetes complications to further enhance the blood glucose monitoring system for diabetes.
The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by ...the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3–0.5μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.
Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We ...show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.
► Cryo-EM images are blurred by sample motion during electron beam exposure ► The sample motion degrades signal especially at high resolution ► Aligning frames of movies recorded with a direct electron detector reduces blurring ► This procedure enhances the resolution obtained in 3D reconstructions of the sample
In cryo-EM, low-dose images are often affected by blurring caused by sample motion during beam exposure. Melody et al. show that aligning frames of movies recorded with a direct electron detector during beam exposure greatly reduces blurring, increases the efficiency of cryo-EM imaging, and enhances resolution.
Breast cancer is a common cause of cancer-related death worldwide. Chemotherapy plays an indispensable role in the conventional treatment of breast cancer, bringing some physical burdens and ...discomfort on cancer patients. Consequently, more and more patients turn to seeking the help of Complementary and Alternative Medicine (CAM), mainly traditional Chinese medicine (TCM). Xiaoyao san (XYS), a classical formula, has been shown to improve symptoms of breast cancer. An increasing number of researches suggest that compared to chemotherapy alone, Chinese herbal medicine combined with chemotherapy could increase effectiveness and reduce toxicity caused by chemotherapy. Emerging experimental research continuously demonstrated some of the components in XYS could stop breast cancer tumor cells from growing. However, the efficacy and safety of modified XYS combined with chemotherapy remain to be determined. Therefore, it is essential to evaluate the comparative effectiveness and safety of modified XYS combined with chemotherapy in-depth, thus providing clinicians and policymakers with evidence-based guidance and new treatment options.
To comprehensively evaluate the efficacy and safety of modified XYS in conjunction with chemotherapy in treating breast cancer by conducting a meta-analysis.
8 databases were systemically searched until April 3, 2022, including Web of Science PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang, Chinese Scientific Journals Database (VIP), and Chinese Biological Medical Database (CBM). Relevant randomized controlled trials (RCTs) comparing modified XYS in combination with chemotherapy versus chemotherapy alone were included. For the evaluation of methodological quality, Cochrane Collaboration was considered. Software Review Manager (version 5.4) was used for data analysis. Software STATA (version 15.0) was employed for sensitivity analysis and publication bias.
Altogether, 17 RCTs involving 1207 patients were investigated in the current review. The findings revealed that modified XYS combined with chemotherapy could lead to beneficial improvements compared to chemotherapy alone. More specifically, the combined therapy could enhance the short-term efficacy in the treatment of solid tumors (OR: 1.74; 95% CI 1.27 to 2.39; P = 0.0006; I
= 0%); improve QOL (quality of life) (OR: 3.75; 95% CI 2.58 to 5.44; P < 0.00001; I
= 0%); reduce clinical symptoms (OR: 3.69; 95% CI 1.43 to 9.49; P = 0.007; I
= 53%); ease depression (MD: -12.96; 95% CI -16.09 to -9.83; P < 0.00001; I
= 0%); increase leukocytes (OR: 0.32; 95% CI 0.20 to 0.50; P < 0.00001; I
= 0%) and platelets (OR: 0.37; 95% CI 0.20 to 0.67; P = 0.001; I
= 0%); reduce nausea and vomiting (OR: 0.26; 95% CI 0.15 to 0.44; P < 0. 00001; I
= 0%); mitigate cardiotoxicity (OR: 0.16; 95% CI 0.07 to 0.36; P<0.00001; I
= 0%); prolong survival time (OR: 2.19; 95% CI 1.03 to 4.66; P = 0.04; I
= 0%), compared to chemotherapy alone. Unfortunately, there was no statistically significant difference in damage to the liver and kidney (OR: 0.59; 95% CI 0.29 to 1.21; P = 0.15; I
= 0%).
The existing evidence suggests modified XYS combined with chemotherapy leads to beneficial improvements in the management of breast cancer, which may serve as a promising therapy for breast cancer in clinical practice. Given the limited number of high quality RCTs, more rigorous, scientific, double-blinded, large-scale, multi-center clinical trials are warranted further.
https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022357860.
False negative interferon-γ release assay (IGRA) results constitute the major dilemma for the diagnosis of tuberculosis (TB) infections. Herein, we conducted a cohort study to compare the host ...immunological response to TB-specific antigens between active TB patients with positive and negative IGRA results and control groups. A total of 274 laboratory-confirmed TB patients were included in our analysis, consisting of 221 were IGRA positive and 53 were IGRA negative. Patients with the elderly were identified as an independent risk factor for negative IGRA results. In addition, the elevated level of IL-4 and the decreased levels of IFN-γ, IL-2, IL-6, IL-1β, and IL-12 in IGRA negative TB relative to IGRA positive TB group, demonstrating a significant difference in Th1/Th2 paradigm between two groups. The IFN-γ&IL-2 based assay could correctly identify 247 out of 307 MTB-infected individuals 271 TB patients and 36 individuals with latent TB infection (LTBI), demonstrating a sensitivity of 80.5%. Then the IFN-γ and IL-4 were applied to distinguish healthy control and IGRA-negative group. When using the stepwise algorithm, the sensitivity for detecting
Mycobacterium tuberculosis
(MTB) infections was significantly increased from 80.5% to 89.6%. Additionally, patients with negative IGRA results had a conversion to culture-negative status longer than those with positive IGRA results. In conclusion, a stepwise algorithm outperforms IGRA assays to accurately identify MTB infections by the combination IFN-γ, IL-2, and IL-4. Further study is needed to evaluate the accuracy of our diagnostic algorithm in the LTBI population.
Abstract
Background
Metagenomic next-generation sequencing (mNGS) has become a powerful tool for pathogen detection, but the value of human sequencing reads generated from it is underestimated.
...Methods
A total of 138 patients with pleural effusion (PE) were diagnosed with tuberculous pleurisy (TBP, N = 82), malignant pleural effusion (MPE, N = 35), or non-TB infection (N = 21), whose PE samples all underwent mNGS analysis. Clinical TB tests including culture, Acid-Fast Bacillus (AFB) test, Xpert, and T-SPOT, were performed. To utilize mNGS for MPE identification, 25 non-MPE samples (20 TBP and 5 non-TB infection) were randomly selected to set human chromosome copy number baseline and generalized linear modeling was performed using copy number variant (CNV) features of the rest 113 samples (35 MPE and 78 non-MPE).
Results
The performance of TB detection was compared among five methods. T-SPOT demonstrated the highest sensitivity (61% vs. culture 32%, AFB 12%, Xpert 35%, and mNGS 49%) but with the highest false-positive rate (10%) as well. In contrast, mNGS was able to detect TB-genome in nearly half (40/82) of the PE samples from TBP subgroup, with 100% specificity. To evaluate the performance of using CNV features of the human genome for MPE prediction, we performed the leave-one-out cross-validation (LOOCV) in the subcohort excluding the 25 non-MPE samples for setting copy number standards, which demonstrated 54.1% sensitivity, 80.8% specificity, 71.7% accuracy, and an AUC of 0.851.
Conclusion
In summary, we exploited the value of human and non-human sequencing reads generated from mNGS, which showed promising ability in simultaneously detecting TBP and MPE.
Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm ...region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.
Tuberculosis (TB) is caused by
(
) and remains a major health threat worldwide. However, a detailed understanding of the immune cells and inflammatory mediators in
-infected tissues is still lacking. ...Tuberculous pleural effusion (TPE), which is characterized by an influx of immune cells to the pleural space, is thus a suitable platform for dissecting complex tissue responses to
infection.
We employed singe-cell RNA sequencing to 10 pleural fluid (PF) samples from 6 patients with TPE and 4 non-TPEs including 2 samples from patients with TSPE (transudative pleural effusion) and 2 samples with MPE (malignant pleural effusion).
Compared to TSPE and MPE, TPE displayed obvious difference in the abundance of major cell types (e.g., NK, CD4+T, Macrophages), which showed notable associations with disease type. Further analyses revealed that the CD4 lymphocyte population in TPE favored a Th1 and Th17 response. Tumor necrosis factors (TNF)-, and XIAP related factor 1 (XAF1)-pathways induced T cell apoptosis in patients with TPE. Immune exhaustion in NK cells was an important feature in TPE. Myeloid cells in TPE displayed stronger functional capacity for phagocytosis, antigen presentation and IFN-γ response, than TSPE and MPE. Systemic elevation of inflammatory response genes and pro-inflammatory cytokines were mainly driven by macrophages in patients with TPE.
We provide a tissue immune landscape of PF immune cells, and revealed a distinct local immune response in TPE and non-TPE (TSPE and MPE). These findings will improve our understanding of local TB immunopathogenesis and provide potential targets for TB therapy.
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