Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.Activation-induced cytidine deaminase (AID), which is expressed in antigen-stimulated mature B cells, is essential for Ig somatic hypermutation (SHM) and class-switch recombination (CSR) (1, 2). AID induces DNA breaks at the variable (V) and switch (S) regions during SHM and CSR, respectively (3, 4). Although both processes are initiated by AID-induced DNA cleavage, point mutations at the V region are executed mostly by error-prone DNA repair whereas CSR is accomplished by recombination of cleaved ends at donor and acceptor S regions (5, 6). However, the detailed mechanisms by which AID carries out the two mechanistically distinct functions for SHM and CSR have yet to be uncovered (7). Studies on AID mutants revealed that AID’s N- and C-terminal domains are distinctly required for its DNA-cleavage and recombination functions, respectively (8–10). Mutations at the N terminus of AID impair SHM as well as CSR whereas those at the C terminus abrogate CSR only and show increased SHM activity. Recent studies demonstrated that the CSR process after DNA cleavage, including the synapsis formation between cleaved ends, is impaired with the C-terminally defective AID, indicating that AID’s C terminus confers a CSR-specific recombination function, independent of AID’s DNA cleavage function, by an unknown mechanism (11, 12).AID belongs to the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide) family of cytidine deaminases (CDDs) and shows high sequence homology with APOBEC1 (A1) (1, 13, 14), which edits apolipoprotein B (APOB) mRNA. The APOB mRNA editing ability of A1 is highly dependent on its cofactors, A1CF/ACF (15, 16) and RBM47 (17), both of which belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Recently, two A1CF-like hnRNPs, hnRNP K and hnRNP L, were identified as the cofactors of AID and found to be involved in the cleavage and recombination of DNA, respectively (18). Because the N and C termini of AID differentially regulate two functions of AID—cleavage and recombination, respectively—we speculated that the AID termini would be critical for function-coupled cofactor association. For instance, the N or C terminus of AID may function as a molecular switch that induces an AID–AID interaction, enabling AID to exert distinct physiological functions through its association with cofactors. Regrettably, however, there is little structural information available that can explain any of AID’s regulatory modes of action, including its cofactor association mechanisms, in the context of its physiological functions.Although a significant amount of structural information is available for a number of APOBEC family members, the 3D structures of A1 and AID are yet to be resolved (19, 20). The CDD family of enzymes exists in nature in a variety of structural forms, including monomeric, dimeric, and tetrameric forms, and comparative structural modeling using the yeast CDD structure predicts a dimeric structure for both A1 and AID (21, 22). On the other hand, homology modeling with the APOBEC2 (A2) crystal structure, which seems to be a tetramer composed of two head-to-head interacting dimers, predicts that AID forms a tetramer (23). Notably, A2 was later reported to exist as a monomer in solution (24). Similarly, an atomic force microscopic (AFM) study found that AID exists in the cell predominantly as a monomer associated with a single-strand DNA substrate (25). However, the same AFM dataset was interpreted differently by another group of investigators, who concluded that AID probably forms an A2-like tetramer in solution (26). The modeling of AID’s catalytic pocket in reference to eight APOBEC family members suggested that most of the AID–DNA complex remains in an inactive state due to occlusion by the substrate DNA, which may explain its weak catalytic activity for cleaving DNA in vitro (27).One of the limitations of the computational modeling of AID’s structure is that AID’s N-and C-terminal sequences are substantially different from those of other APOBEC members and thus reside outside the modeling template. Although the structural outcome of a protein can differ by a variety of reasons, including the methods applied (28), none of the AID studies mentioned above explain why the C-terminal deletion of AID leads to the loss of CSR function only. Therefore, model-based computational simulation may not explain the physiological structure–function relationship of AID in B cells.Here, we explored AID’s structure–function relationship using a bimolecular fluorescence complementation (BiFC) assay, which detects homo- or heteromeric protein–protein interactions in live cells (29, 30). For the homomeric interaction assay, the target protein is fused to two nonfluorescent halves of a green or red fluorescent protein. An interaction between two of the target proteins brings the two nonfluorescent halves of the fluorescent protein into close proximity, reconstituting the fluorescence. The BiFC assay thus allows a rapid analysis of the dimerization of a protein of interest in live cells.By combining this assay with other biochemical approaches, such as coimmunoprecipitation (co-IP) and glycerol gradient sedimentation, we revealed the presence of both monomeric and dimeric forms of AID in analyzed cells. Intriguingly, C-terminal AID mutants that lost CSR function showed a severe dimerization defect, suggesting that AID’s C terminus is required to stabilize the dimeric structure that is required for CSR. We also showed that the AID monomer and dimer associate with different RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. Based on these findings, we propose that the monomeric AID–RNP complex includes hnRNP K (18) and contributes to the DNA cleavage function of AID whereas the dimeric AID–RNP complexes include hnRNP L (18), hnRNP U (31), or Serpine mRNA-binding protein 1 (SERBP1) (32) and contribute to the recombination step of CSR.     

Polygenic risk scores for prediction of breast cancer risk in Asian populations

PurposeNon-European populations are under-represented in genetics studies, hindering clinical implementation of breast cancer polygenic risk scores (PRSs). We aimed to develop PRSs using the largest available studies of Asian ancestry and to assess the transferability of PRS across ethnic subgroups.MethodsThe development data set comprised 138,309 women from 17 case-control studies. PRSs were generated using a clumping and thresholding method, lasso penalized regression, an Empirical Bayes approach, a Bayesian polygenic prediction approach, or linear combinations of multiple PRSs. These PRSs were evaluated in 89,898 women from 3 prospective studies (1592 incident cases).ResultsThe best performing PRS (genome-wide set of single-nucleotide variations [formerly single-nucleotide polymorphism]) had a hazard ratio per unit SD of 1.62 (95% CI = 1.46-1.80) and an area under the receiver operating curve of 0.635 (95% CI = 0.622-0.649). Combined Asian and European PRSs (333 single-nucleotide variations) had a hazard ratio per SD of 1.53 (95% CI = 1.37-1.71) and an area under the receiver operating curve of 0.621 (95% CI = 0.608-0.635). The distribution of the latter PRS was different across ethnic subgroups, confirming the importance of population-specific calibration for valid estimation of breast cancer risk.ConclusionPRSs developed in this study, from association data from multiple ancestries, can enhance risk stratification for women of Asian ancestry.     

Interstitial Lung Disease in a Patient with Chronic Granulomatous Disease

Background

Chronic granulomatous disease (CGD) is an inherited phagocytes defect, characterized by defects of NADPH-oxidase and inability of bacterial killing, which leads to recurrent life-threatening infections. Respiratory problems, which are the major cause of morbidity in CGD, usually result from recurrent severe infections; however, vigorous inflammatory response could also cause respiratory diseases.

Case Presentation

Herein, an 11 year-old patient with CGD is presented who suffered from chronic cough and dyspnea for 7 years. Considering the results of chest X-ray, high-resolution computed tomography, and pulmonary function test, the diagnosis of interstitial lung disease was made.

Conclusion

Early recognition of manifestations associated with CGD and appropriate treatment could prevent further complications and reduce morbidity and mortality in this group of patients.     

Blood flow quantification of the human retina with MRI

The purpose of this study was to investigate the feasibility of measuring blood flow to the retina using arterial spin labeling MRI, a quantitative, noninvasive tomographic technique. Blood flow imaging was performed in a single axial slice through both eyes of five healthy volunteers with no history of retinal diseases. The imaging was optimized to minimize the errors from motion and nonuniform magnetic fields caused by proximity to the sinuses. Key hemodynamic factors for flow quantification, including arterial transit delay and the apparent decay time of the signal, were estimated by repeated measurements with different arterial spin labeling timing. A clearly elevated signal, consistent with the anatomical location of the retina, was observed in all subjects. The measured blood flow to a 1 cm × 1.47 cm section of the retina, centered on the fovea, was 1.75 ± 0.54 µL/mm²/min (total blood flow of 261 ± 87 µL/min). The arterial transit delay from a labeling plane 5 cm below the slice was 1137 ± 288 ms. These results establish the feasibility of measuring blood flow to the retina with MRI, and support the future characterization of the healthy and diseased ocular circulation with this method. Copyright © 2010 John Wiley & Sons, Ltd.     

Whole lung lavage with intermittent double lung ventilation. A modified technique for managing pulmonary alveolar proteinosis

Whole lung lavage is still the most effective treatment for pulmonary alveolar proteinosis. We report a 21-year-old male diagnosed with pulmonary alveolar proteinosis by open lung biopsy and who underwent whole lung lavage with a modified technique. He showed significant improvement in clinical and functional parameters. The technique of intermittent double lung ventilation during lavage procedure keeps the oxygen saturation in acceptable limits in patients at risk for severe hypoxemia and allows the procedure to be completed in a single setting.     

Transfer of blastocysts derived from frozen-thawed cleavage stage embryos improved ongoing pregnancy

Purpose

The aim of our study was to compare the transfer of embryos that are cryopreserved in cleavage stage after thawing with the transfer of embryos after thawing and culture in sequential media until blastocyst formation.

Methods

In this prospective clinical study, we have evaluated 134 cycles of ART treatment for infertility. Frozen embryos were thawed and then cultured in sequential media until blastocyst stage in blastocyst group and were compared with thawed embryos in cleavage stage group.

Results

Implantation rate was significantly higher in blastocyst group (30?%) compared to cleavage group (17?%). No statistical differences were reported in chemical and clinical pregnancy rates between groups. Ongoing pregnancy rate was significantly higher in blastocyst group compared to cleavage group (42.9 vs. 24.6?%).

Conclusions

Our results indicated that blastocyst formation after thawing of cleavage stage embryos is a good predictor for embryo viability and pregnancy outcome.     

Giant cell myocarditis following COVID‐19 successfully treated by immunosuppressive therapy

It has been shown that severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), by coronavirus disease 2019 (COVID‐19), can lead to multi‐organ impairment including cardiac involvement and immunological problems. Acute myocarditis is one of serious and fatal complications of COVID‐19. In this case report, we present a 46‐year‐old lady with a history of lichen planus dermatitis who has developed a rapidly progressive heart failure after an episode of COVID‐19. The pathologic examination of her endomyocardial biopsy specimens was compatible with GCM, and she was successfully treated with a combined immunosuppressive therapy regimen.     

Serious events following COVID‐19 vaccination with ChAdOx1 nCoV‐19 vaccine (Vaxzevria): A short case series from Iran

In 2020, the SARS‐COV‐2 disease (COVID‐19) imposed huge challenges on the health, economic, and political systems, and by the end of the year, hope had been born with the release of COVID‐19 vaccines aimed at bringing the pandemic to an end. However, the COVID‐19 vaccination programs have sparked several concerns and ongoing debates over safety issues. Here, we presented three cases of patients with serious adverse events, encephalopathy, vaccine‐induced thrombotic thrombocytopenia, and leukocytoclastic vasculitis, after receiving the ChAdOx1 nCoV‐19 vaccine. Therefore, it is critical to investigate and report the occurrence of adverse reactions following vaccination, particularly serious ones, as it contributes to the growing body of research and assists clinicians in better diagnosing and managing them.