Stepwise generation of AID knock-in and conditional knockout mice from a single gene-targeting event.

Activation-induced cytidine deaminase (AID) encoded by the Aicda gene initiates class-switch recombination and somatic hypermutation of immunoglobulin genes. In addition to this function, AID is also implicated in the epigenetic regulation in pluripotent stem cells and in the oncogenesis of lymphoid and non-lymphoid origins. To examine AID's role in specific cell types, we developed mouse strains of conditional knockout (Aicda-FL) and knock-in with a red fluorescent protein gene (RFP) inserted into the Aicda locus (Aicda-RFP). These two strains were obtained from a single targeting event in embryonic stem cells by a three-loxP or tri-lox strategy. Partial and complete recombination among the three loxP sites in the Aicda-RFP locus gave rise to Aicda-FL and AID-deficient loci (Aicda-KO), respectively, after mating Aicda-RFP mice with Cre-expressing mice driven by tissue-non-specific alkaline phosphate promoter. We confirmed RFP expression in B cells of germinal centers of intestine-associated lymphoid tissue. Mice homozygous for each allele were obtained and were checked for AID activity by class-switch and hypermutation assays. AID activity was normal for Aicda-FL but partially and completely absent for Aicda-RFP and Aicda-KO, respectively. Aicda-FL and Aicda-RFP mice would be useful for studying AID function in subpopulations of B cells and in non-lymphoid cells.

Claw Toe: Anatomic Guide for Injection of Botulinum Toxin into Foot Muscles.

BACKGROUND: Poor response to injection of botulinum toxin (BoNT) into the flexor digitorum longus (FDL) muscle has been reported especially in patients with claw foot deformity. We previously advocated BoNT injection into the flexor hallucis longus (FHL) muscle in such patients. Here, we determined the functional and anatomical relationships between FHL and FDL. METHODS: Toe flexion pattern was observed during electrical stimulation of FHL and FDL muscles in 31 post-stroke patients with claw-foot deformity treated with BoNT. The FHL and FDL tendon arrangement was also studied in five limbs of three cadavers. RESULTS: Electrical stimulation of the FHL muscle elicited big toe flexion in all 28 cases examined and second toe in 25, but the response was limited to the big toe in 3. FDL muscle stimulation in 29 patients elicited weak big toe flexion in 1 and flexion of four toes (2nd to 5th) in 16 patients. Cadaver studies showed division of the FHL tendon with branches fusing with the FDL tendon in all five limbs examined; none of the tendons was inserted only in the first toe. No branches of the FDL tendon merged with the FHL tendon. CONCLUSION: Our results showed coupling of FHL and FDL tendons in most subjects. Movements of the second and third toes are controlled by both the FDL and FHL muscles. The findings highlight the need for BoNT injection in both the FDL and FHL muscles for the treatment of claw-toe deformity.

Vascular endothelial growth factor-A is an Immunohistochemical biomarker for the efficacy of bevacizumab-containing chemotherapy for duodenal and jejunal adenocarcinoma.

BACKGROUND: The efficacy and safety of bevacizumab-containing chemotherapy for patients with metastatic duodenal and jejunal adenocarcinoma (mDJA) are unclear. The present study aimed to evaluate the efficacy of bevacizumab and to explore immunohistochemical markers that can predict the efficacy of bevacizumab for patients with mDJA. METHODS: This multicentre study included patients with histologically confirmed small bowel adenocarcinoma who received palliative chemotherapy from 2008 to 2017 at 15 hospitals. Immunostaining was performed for vascular endothelial growth factor-A (VEGF-A), TP53, Ki67, ß-catenin, CD10, MUC2, MUC5AC, MUC6, and mismatch repair proteins. RESULTS: A total of 74 patients were enrolled, including 65 patients with mDJA and 9 with metastatic ileal adenocarcinoma. Patients with mDJA who received platinum-based chemotherapy with bevacizumab as first-line treatment tended to have a longer progression-free survival and overall survival than those treated without bevacizumab (P = 0.075 and 0.077, respectively). Multivariate analysis extracted high VEGF-A expression as a factor prolonging progression-free survival (hazard ratio: 0.52, 95% confidence interval: 0.30-0.91). In mDJA patients with high VEGF-A expression, those who received platinum-based chemotherapy with bevacizumab as a first-line treatment had significantly longer progression-free survival and tended to have longer overall survival than those treated without bevacizumab (P = 0.025 and P = 0.056, respectively), whereas no differences were observed in mDJA patients with low VEGF-A expression. CONCLUSION: Immunohistochemical expression of VEGF-A is a potentially useful biomarker for predicting the efficacy of bevacizumab-containing chemotherapy for patients with mDJA.

Prophylactic clipping not effective in preventing post-polypectomy bleeding for < 20-mm colon polyps: A multicenter, open-label, randomized controlled trial.

BACKGROUND AND AIM: Prophylactic clipping (PC) after polypectomy has the potential to prevent post-polypectomy bleeding (PPB). We aimed to evaluate the effectiveness of PC in preventing PPB for < 20-mm polyps. METHODS: This multicenter, open-label, randomized controlled trial conducted from December 2013 to June 2017 at 10 institutions randomly assigned 1080 patients with < 20-mm colon polyps to the non-PC and PC groups. Allocation factors were institution, antiplatelet drug use, and polyp number. The primary endpoint was differences in PPB rates between the groups. The severity of PPB and post-procedural abdominal symptoms were also investigated. These endpoints in intention-to-treat and per-protocol (PP) analyses were evaluated. RESULTS: We investigated 1039 patients with 2960 lesions. There was no significant difference between the groups in characteristics including age, sex, hypertension, diabetes, hyperlipidemia, antiplatelet drug use, and lesion characteristics such as type and size. Excluding the clip used in the non-PC group, intraoperative bleeding, and deviation of protocol, 903 patients were investigated in PP analysis. There was no significant difference in the PPB rate between the non-PC and PC groups (2.7% vs 2.3%, P = 0.6973 [intention-to-treat analysis]; 3.0 vs 2.4%, P = 0.7353 [PP analysis]). Severe PPB (≥ grade 3) was similar between the groups. Total procedure time was significantly shorter in the non-PC group than in the PC group (31 vs 36 min, P = 0.0002). Post-procedural abdominal fullness was less common in the non-PC group than in the PC group (20.8% vs 25.6%, P = 0.0833). CONCLUSION: Prophylactic clipping is not effective in preventing PBB for < 20-mm colon polyps (UMIN000012163).

X-ray Diffraction Analysis to Explore Molecular Traces of Eccentric Contraction on Rat Skeletal Muscle Parallelly Evaluated with Signal Protein Phosphorylation Levels.

We performed X-ray diffraction analyses on rat plantaris muscle to determine if there are strain-specific structural changes at the molecular level after eccentric contraction (ECC). ECC was elicited in situ by supramaximal electrical stimulation through the tibial nerve. One hour after a series of ECC sessions, the structural changes that remained in the sarcomere were evaluated using X-ray diffraction. Proteins involved in cell signaling pathways in the muscle were also examined. ECC elicited by 100, 75, and 50 Hz stimulation respectively developed peak tension of 1.34, 1.12 and 0.79 times the isometric maximal tetanus tension. The series of ECC sessions phosphorylated the forkhead box O proteins (FoxO) in a tension-time integral-dependent manner, as well as phosphorylated the mitogen-activated protein kinases (MAPK) and a protein in the mammalian target of rapamycin (mTOR) pathway in a maximal tension dependent manner. Compared to isometric contractions, ECC was more efficient in phosphorylating the signaling proteins. X-ray diffraction revealed that the myofilament lattice was preserved even after intense ECC stimulation at 100 Hz. Additionally, ECC < 75 Hz preserved the molecular alignment of myoproteins along the myofilaments, while 75-Hz stimulation induced a slight but significant decrease in the intensity of meridional troponin reflection at 1/38 nm-1, and of myosin reflection at 1/14.4 nm-1. These two reflections demonstrated no appreciable decrease with triple repetitions of the standard series of ECC sessions at 50 Hz, suggesting that the intensity decrease depended on the instantaneous maximal tension development rather than the total load of contraction, and was more likely linked with the phosphorylation of MAPK and mTOR signaling proteins.

Comparison of performances of artificial intelligence versus expert endoscopists for real-time assisted diagnosis of esophageal squamous cell carcinoma (with video).

BACKGROUND AND AIMS: Narrow-band imaging (NBI) is currently regarded as the standard modality for diagnosing esophageal squamous cell carcinoma (SCC). We developed a computerized image-analysis system for diagnosing esophageal SCC by NBI and estimated its performance with video images. METHODS: Altogether, 23,746 images from 1544 pathologically proven superficial esophageal SCCs and 4587 images from 458 noncancerous and normal tissue were used to construct an artificial intelligence (AI) system. Five- to 9-second video clips from 144 patients captured by NBI or blue-light imaging were used as the validation dataset. These video images were diagnosed by the AI system and 13 board-certified specialists (experts). RESULTS: The diagnostic process was divided into 2 parts: detection (identify suspicious lesions) and characterization (differentiate cancer from noncancer). The sensitivities, specificities, and accuracies for the detection of SCC were, respectively, 91%, 51%, and 63% for the AI system and 79%, 72%, and 75% for the experts. The sensitivity of the AI system was significantly higher than that of the experts, but its specificity was significantly lower. Sensitivities, specificities, and accuracy for the characterization of SCC were, respectively, 86%, 89%, and 88% for the AI system and 74%, 76%, and 75% for the experts. The receiver operating characteristic curve showed that the AI system had significantly better diagnostic performance than the experts. CONCLUSIONS: Our AI system showed significantly higher sensitivity for detecting SCC and higher accuracy for characterizing SCC from noncancerous tissue than endoscopic experts.

Efficient Induction of Ig Gene Hypermutation in Ex Vivo-Activated Primary B Cells.

Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. How AID is targeted to the Ig V gene and switch region to trigger SHM and CSR remains elusive. Primary B cells stimulated with CD40L plus IL-4 or LPS plus IL-4 undergo efficient CSR, but it has been difficult to induce SHM in these cells. In the current study, we used B cells from B1-8hi mice carrying a prerecombined VH186.2DFL16.1JH2 Ab gene to investigate the induction of SHM under in vitro culture conditions. B1-8hi splenic B cells stimulated with CD40L plus IL-4 or LPS plus IL-4 underwent robust CSR to IgG1, but failed to generate SHM in the VH186.2 gene. Remarkably, ectopic expression of AID in AID-deficient, but not wild-type, B1-8hi B cells induced efficient SHM at a rate close to that observed in germinal center B cells. We further established an AID-deficient CH12 B lymphoma line and found that ectopic expression of AID in the mutant line, but not in AID-sufficient CH12 cells, induced efficient point mutations and deletions in the V gene. These results demonstrate that the endogenous AID in ex vivo-activated primary B and B lymphoma cells not only cannot induce SHM but also inhibit the induction of SHM by the exogenous AID. Our results further suggest that the spatiotemporal distribution and/or posttranslational modification of AID strongly affects the induction of SHM in ex vivo-activated primary B cells.

TGF-ß suppression of HBV RNA through AID-dependent recruitment of an RNA exosome complex.

Transforming growth factor (TGF)-ß inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-ß is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-ß was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-ß mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-ß causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner.

Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut.

Immunoglobulin A (IgA) is generated in the gut by both T cell-dependent and T cell-independent processes. The sites and the mechanisms for T cell-independent IgA synthesis remain elusive. Here we show that isolated lymphoid follicles (ILFs) were sites where induction of activation-induced cytidine deaminase (AID) and IgA class switching of B cells took place in the absence of T cells. We also show that formation of ILFs was regulated by interactions between lymphoid tissue-inducer cells expressing the nuclear receptor ROR gamma t (ROR gamma t(+)LTi cells) and stromal cells (SCs). Activation of SCs by ROR gamma t(+)LTi cells through lymphotoxin (LT)-beta receptor (LT beta R) and simultaneously by bacteria through TLRs induced recruitment of dendritic cells (DCs) and B cells and formation of ILFs. These findings provide insight into the crosstalk between bacteria, ROR gamma t(+)LTi cells, SCs, DCs, and B cells required for ILF formation and establish a critical role of ILFs in T cell-independent IgA synthesis in gut.

Activation-induced cytidine deaminase is dispensable for virus-mediated liver and skin tumor development in mouse models.

Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID (-/-) mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID (+/+) and HCV-Tg/AID (-/-) mice at 20 months of age although the AID (+/+) mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID (+/+) liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID (-/-) and HPV8-Tg/AID (+/+) groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis.

Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium.

Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IkappaB kinase-dependent nuclear factor-kappaB activation pathway. H. pylori-mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori-associated gastric carcinogenesis.

Scheduled endoscopic surveillance controls secondary cancer after curative endoscopic resection for early gastric cancer: a multicentre retrospective cohort study by Osaka University ESD study group.

BACKGROUND: After endoscopic submucosal dissection (ESD) of early gastric cancer (EGC), patients are at high risk for synchronous or metachronous multiple gastric cancers. OBJECTIVE: To elucidate the time at which multiple cancers develop and to determine whether scheduled endoscopic surveillance might control their development. DESIGN: A multicentre retrospective cohort study from 12 hospitals was conducted. Patients with EGC who underwent ESD with en bloc margin-negative curative resection were included. Synchronous cancer was classified as concomitant cancer or missed cancer. The cumulative incidence of metachronous cancers and overall survival rate were calculated using the Kaplan-Meier method. RESULTS: From April 1999 to December 2010, 1258 patients met the inclusion criteria. Synchronous or metachronous multiple cancers were detected in 175 patients (13.9%) during a mean of 26.8 months. Among the 110 synchronous cancers, 21 were missed at the time of the initial ESD. Many of the missed lesions existed in the upper third of the stomach and the miss rate was associated with the endoscopist's inexperience (<500 oesophagogastroduodenoscopy cases). The cumulative incidence of metachronous cancers increased linearly and the mean annual incidence rate was 3.5%. The incidence rate did not differ between patients with or without Helicobacter pylori eradication. Four lesions (0.32%) were detected as massively invading cancers during the follow-up. CONCLUSIONS: Nineteen per cent of synchronous cancers were not detected until the initial ESD. The incidence rate of metachronous cancer after ESD was constant. Scheduled endoscopic surveillance showed that almost all recurrent lesions were treatable by endoscopic resection.

Non-Pure Intestinal Phenotype as an Indicator of Progression in Sporadic Nonampullary Duodenal Adenomas: A Multicenter Retrospective Cohort Study.

INTRODUCTION: We aimed to evaluate the natural course of sporadic nonampullary duodenal adenomas (SNDAs) and determine the risk factors of progression. METHODS: We retrospectively analyzed the follow-up outcomes of patients with biopsy-diagnosed SNDA between April 2010 and March 2016 at 13 institutions. All initial biopsy specimens were centrally evaluated. Only those diagnosed with adenomas were included. Mucinous phenotypes were classified into pure intestinal and non-pure intestinal phenotypes. Cumulative incidence rates of carcinoma and tumor enlargement were evaluated. Tumor enlargement was defined as a ≥25% or 5-mm increase in tumor size. RESULTS: Overall, 121 lesions were analyzed. Within a median observation period of 32.7 months, 5 lesions were diagnosed as carcinomas; the cumulative 5-year incidence of carcinoma was 9.5%. Male sex ( P = 0.046), initial lesion size ≥10 mm ( P = 0.044), and non-pure intestinal phenotype ( P = 0.019) were significantly associated with progression to carcinoma. Tumor enlargement was observed in 22 lesions, with a cumulative 5-year incidence of 33.9%. Initial lesion size ≥10 mm ( P < 0.001), erythematous lesion ( P = 0.002), high-grade adenoma ( P = 0.002), Ki67 negative ( P = 0.007), and non-pure intestinal phenotype ( P = 0.001) were risk factors of tumor enlargement. In a multivariate analysis, an initial lesion size ≥10 mm ( P = 0.010) and non-pure intestinal phenotype ( P = 0.046) were independent and significant risk factors of tumor enlargement. DISCUSSION: Lesion size ≥10 mm and non-pure intestinal phenotype on initial biopsy are risk factors of cancer progression and tumor enlargement in cases with SNDA. Thus, management effectiveness may be improved by focusing on lesion size and the mucinous phenotype.

Adiponectin negatively correlates with alcoholic and non-alcoholic liver dysfunction: Health check-up study of Japanese men.

AIM: Central obesity, insulin resistance and alcohol consumption are thought to be major risk factors for fatty liver formation. Adiponectin (APN) prevents fatty liver formation, and its serum levels are lower in subjects with central obesity and/or insulin resistance. The aim of this study was to explore the association among serum APN levels, central obesity, insulin resistance and liver dysfunction with or without fatty liver classified by alcohol consumption in healthy subjects. METHODS: A total of 5588 Japanese male subjects who underwent a health check-up were classified into three groups according to alcohol consumption: non- or light drinkers (15 g/day ≥ ethanol); mild drinkers (15 g/day < ethanol ≤ 30 g/day); and moderate- or heavy drinkers (30 g/day < ethanol). Central obesity and insulin resistance were assessed by waist circumference (WC) and Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR), respectively. RESULTS: WC was significantly increased, while HOMA-IR was significantly decreased according to the extent of alcohol consumption. Serum alanine aminotransferase levels were significantly lower and serum APN levels were significantly higher in mild drinkers than in the other two groups. Multiple linear regression analysis showed that serum APN level served as the significant and independent determinant for liver dysfunction in the subjects with fatty liver, irrespective of alcohol consumption. However, WC became a non-significant determinant of liver dysfunction as alcohol consumption increased. CONCLUSION: Hypoadiponectinemia is a significant determinant for steatotic dysfunction for all levels of alcohol consumption, but central obesity was not a significant determinant for alcoholic fatty liver-induced liver dysfunction.

Foot pressure pattern and its correlation with knee range of motion limitations for individuals with medial knee osteoarthritis.

OBJECTIVE: To determine the foot pressure pattern of individuals with medial knee osteoarthritis (OA) and to analyze its relation with knee flexion/extension range of motion. DESIGN: Descriptive. SETTING: Rehabilitation center. PARTICIPANTS: Individuals with unilateral, painful medial knee OA (n=50; mean age, 75y; OA group) were enrolled as subjects, and young, healthy persons (n=50; mean age, 28y; young group) and elderly persons (n=44; mean age, 74y; elderly group) without any pain, deformity, or apparent OA changes in either knee were enrolled as controls. INTERVENTION: Walking 10m at a comfortable speed. MAIN OUTCOME MEASURES: Partial foot pressures as percentages of body weight (%PFP), anteroposterior length of the center of pressure (COP) path as a percentage of foot length (%Long), transverse width of the COP path as a percentage of foot width (%Trans), knee flexion/extension range of motion (in the OA group). RESULTS: The %PFP values for the heel and hallux, %Long, and %Trans were all significantly lower in the OA group than in controls (P<.001, all). Within the OA group, a limited range of knee extension was significantly associated with a short %Long (P<.001) but not with %Trans. CONCLUSIONS: Individuals with medial knee OA exhibited low pressure on the heel and hallux and short %Long of the COP path, and limitation of knee extension was associated with shortness of %Long. The shortness of %Long in the OA group likely resulted from insufficient knee extension during the heel-contact phase associated with low pressure on the heel. %Long is a useful parameter in gait analysis by using a foot pressure measurement system.

Enrichment of Immune-Related Genes in Aggressive Primary Breast Angiosarcoma: A Case Report.

Primary breast angiosarcoma is an extremely rare disease with a poor prognosis. Primary angiosarcoma is distinct from secondary angiosarcoma, which usually occurs in patients who have been previously treated for breast cancer. The low incidence of primary breast angiosarcoma has hindered the elucidation of its etiology and potential therapies. Here, we report a case of a patient with primary breast angiosarcoma who experienced recurrence after surgery. The tumor was refractory to systemic treatments, and the patient died 18 months after the surgery. We used RNA sequencing for gene expression profiling of the tumor. A high tumor inflammation signature score indicated enrichment in immune-related signaling. CIBERSORTx, a tool used to characterize the cellular composition of complex tissues based on gene expression, indicated that the immune cells in the tumor were predominantly macrophages, and this was confirmed using immunohistochemical analysis. These findings indicate the possible use of checkpoint immunotherapy for the treatment of primary breast angiosarcoma.

Carboxy-terminal domain of AID required for its mRNA complex formation in vivo.

Activation-induced cytidine deaminase (AID) is essential for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. Originally, AID was postulated to be an RNA-editing enzyme, because of its structural homology with a known RNA-editing enzyme, APOBEC1. In support of this idea, AID shares many of the properties of RNA-editing enzymes, including nucleocytoplasmic shuttling and a dependency on de novo protein synthesis. However, it has not been shown whether AID recognizes a specific mRNA and edits it to generate an enzyme involved in CSR or SHM. Here, we examined the association between AID and polyadenylated [poly(A)(+)] RNA in vivo, using UV cross-linking coupled with a poly(A) capture method that relies on biotinylated oligo(dT) and streptavidin-conjugated beads. We found that both exogenous AID expressed in transfected CH12 cells and endogenous AID expressed in BL2 cells were associated with poly(A)(+) RNA. Similar protein-poly(A)(+) RNA complexes were formed by APOBEC1 and APOBEC3G. However, the interactions of all of these cytidine deaminase family members, including AID, with poly(A)(+) RNA were indirect. This was expected for APOBEC1, which is known to act through an RNA-interacting cofactor, APOBEC1 complementation factor (ACF). In addition, the carboxy-terminal region of AID, which is essential for class switching, was also required for its interaction with poly(A)(+) RNA. These results suggest that the CSR activity of AID requires an ACF-like cofactor that specifically interacts with the carboxy-terminal domain of AID.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Assessment of central aponeurosis curvature as an index of rectus femoris muscle overstrain in individuals with knee osteoarthritis.

BACKGROUND: In medial knee osteoarthritis (knee OA), compensatory overstrain of the rectus femoris (RF) muscle leads to its hypertrophy. We hypothesize that besides hypertrophy of the RF, a prominent flattening of the central aponeurosis (CA) curvature is also indicative of RF. This study aims to evaluate the structural changes in the CA and clarify the conditions associated with RF overstrain in knee OA. OBJECTIVE: Twenty-three legs of 20 elderly without knee OA (elderly group) and 26 legs of 20 individuals with K-L grade II knee OA (knee OA group) with typical "comma"-shaped CA participated in this study. METHODS: The knee extension torque (Nm/kg) in the sitting position, the thickness of the RF and vastus intermedius (VI) muscles (VI), and change in CA curvature (%Curvature) were measured at the mid-thigh by ultrasonography. RESULTS: The knee extension torque was not significantly different between the two groups. Compared to the elderly group, the knee OA group had significantly thicker RF at rest, while the VI thickness during contraction was significantly smaller. The %Curvature was significantly higher in the knee OA group than in the elderly group. CONCLUSIONS: In the knee OA group, the RF was hypertrophic with a more pronounced CA flattening during muscle contraction, although the other quadriceps muscles were atrophic, suggesting an overstrained RF. Assessing thickness and CA curvature of the RF is, therefore, useful and simple for evaluating overstrain caused by RF compensation.