Plant PAXX has an XLF-like function and stimulates DNA end joining by the Ku-DNA ligase IV/XRCC4 complex.

Non-homologous end joining (NHEJ) plays a major role in repairing DNA double-strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals.

Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites.

BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.

Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).

Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency.

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase µ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.

Impact of early tumor reduction on outcome differs by histological subtype in stage III non-small-cell lung cancer treated with definitive radiotherapy.

BACKGROUND: We retrospectively investigated the impact on survival of early tumor reduction during definitive radiotherapy for inoperable stage III non-small cell lung cancer (NSCLC) patients, according to their histological subtypes. METHODS: Between November 2006 and December 2012, 152 consecutive patients with inoperable stage III NSCLC who underwent definitive radiotherapy were reviewed retrospectively. Forty-one patients were excluded for not satisfying the inclusion criteria. Forty-five (40.5 %) and 48 (43.2 %) patients were diagnosed with squamous cell carcinoma (SQC) and adenocarcinoma (ADC), respectively. The tumor reduction rate (TRR) was defined as follows: TRR = 1-[gross tumor volume (GTV) on computed tomography at shrinking irradiation field planning]/(GTV on computed tomography at the initial treatment planning). The Cox proportional hazard model was used to identify significant prognostic factors for overall survival (OS) and progression-free survival (PFS). RESULTS: We evaluated 111 patients, with a median follow-up time of 52.2 months in surviving patients. The median TRR was 45.9 %. In all patients, there were significant associations between TRR and PFS (P = 0.036) on multivariate analysis, although TRR had no correlation with OS (P = 0.141). With respect to histological subtype, multivariate analyses revealed that a higher TRR showed significant associations with better OS and PFS in the SQC group (P = 0.013 and 0.040, respectively). In contrast, a higher TRR was associated with poorer OS in the ADC group (P = 0.030); there was no association between TRR and PFS. CONCLUSION: We found that a higher TRR is a promising prognostic factor for better survival and disease control in SQC patients.

[Endogenous neural stem cell mobilization with intraventricular growth factor administration].

It is now well recognized that new neurons are continuously generated in the specific regions of the adult brain across all mammalian species including humans. Now the cumulative findings demonstrate that this capacity for neurogenesis at the adult stage is associated with self-repair mechanisms in the damaged brain. Then, one of the main reasons that in vivo growth factor treatment is worth studying is the expectation that such treatment would promote the regeneration of neurons after stroke. Here we show the cumulative evidence that this approach is feasible as one of the brain repair therapies.

Proteomic profiling of centrosomes across multiple mammalian cell and tissue types by an affinity capture method.

Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.

Cartwheels create symmetry and asymmetry.

In this issue of Structure, Kantsadi et al. (2022) present the crystal structures of coiled-coil bundles from SAS-6, a core component of the centriole cartwheel, and reveal that two coiled-coil domains interact asymmetrically. This work provides insights into how the polarity of centrioles is established.

Non-homologous end-joining partners in a helical dance: structural studies of XLF-XRCC4 interactions.

XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double-strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF-XRCC4 complex. An 8.5 Å (1 Å = 0.1 nm) resolution crystal structure of XLF-XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)-MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.

Relationship between pretreatment FDG uptake and local control after stereotactic body radiotherapy in stage I non-small-cell lung cancer: the preliminary results.

OBJECTIVE: Relationship between pretreatment uptake of (18)F-fluoro-2-deoxy-d-glucose and local control after stereotactic body radiotherapy in stage I non-small-cell lung cancer was examined. METHODS: Between June 2006 and June 2009, 90 clinically diagnosed stage I primary lung cancer in 86 patients were treated with stereotactic body radiotherapy in Shikoku Cancer Center. Among these, 51 tumors in 51 patients were evaluated by positron emission tomography using (18)F-fluoro-2-deoxy-d-glucose before treatment. Twenty-six tumors of histopathologically confirmed non-small-cell lung cancer were reviewed in this study. Tumors were divided into two groups by the threshold maximum standardized uptake value of 5.0 (high-uptake tumors, 9; low-uptake tumors, 17). One tumor with low uptake was pure ground-glass opacity. Typically, 48 Gy in four fractions was given at the isocenter. RESULTS: Follow-up time was 4-44 months (median, 21 months). Local failure-free rates at 15 months of the high-uptake group and the low-uptake group were 40% and 93% for all tumors (P= 0.0001), 0% and 91% for tumors 3 cm or less (P= 0.0004), 50% and 100% for tumors larger than 3 cm, and 40% and 89% for the mainly solid tumors (P= 0.0010). There were no statistically significant differences of local failure-free rates according to age, sex and tumor size (P= 0.4804, P= 0.4170 and P= 0.3638, respectively). CONCLUSIONS: High uptake of (18)F-fluoro-2-deoxy-d-glucose in a primary tumor was the significant unfavorable factor for local control after stereotactic body radiotherapy in stage I non-small-cell lung cancer.

Gene expression profiling and functional assays of activated hepatic stellate cells suggest that myocardin has a role in activation.

BACKGROUND: Myofibroblast-like cells derived from transdifferentiated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood. AIM: Our aim was to identify genes that are differentially regulated by HSC activation and to explore their function. METHODS: Using oligonucleotide microarrays, we performed transcriptional analysis of the human HSC cell line, LI90, cultured on Matrigel. Microarray data were validated by quantitative real-time polymerase chain reaction and Western blotting. The function of myocardin was assessed by myocardin RNAi and overexpression. RESULTS: Examination of Matrigel-induced deactivation of LI90 cells revealed marked downregulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Small interfering RNA-mediated suppression of myocardin expression in both activated LI90 and rat activated HSC resulted in loss of the phenotypic characteristics of myofibroblasts and significantly impaired the production of activated HSC markers, such as alpha-smooth muscle actin and extracellular matrix proteins like type I collagen. Overexpression of myocardin led to the upregulation of these marker genes. Myocardin was upregulated in rat primary HSC during in vitro activation and in the fibrotic liver of a dimethylnitrosamine-induced fibrosis rat model. CONCLUSIONS: This study demonstrates that myocardin is involved in the activation of HSC; myocardin may serve as a novel therapeutic target in the treatment of liver fibrosis.

Coil Embolization of VA-PICA Aneurysm Presenting with Hemifacial Spasm with Assistance of Abnormal Muscle Response Monitoring.

Objective: We report a rare case of symptomatic vertebral and posterior inferior cerebellar arteries (VA-PICA) aneurysm-caused ipsilateral hemifacial spasm (HFS) for which coil embolization of the aneurysm with the assistance of abnormal muscle response (AMR) monitoring was effective. Case Presentation: A 62-year-old woman presented with left HFS. Magnetic resonance imaging showed a saccular aneurysm of the left VA-PICA which compressed the seventh cranial nerve at its root exit zone (REZ). Stent-assisted coil embolization resulted in intraoperative disappearance of AMR in the intraoperative electrophysiological study and HFS was relieved temporally. One month after endovascular surgery, HFS slightly occurred again with the re-appearance of the AMR, although there was no recurrence of aneurysm. Thereafter, the frequency of her HFS markedly decreased to once per several days 1 year after the coiling. Conclusion: Although complete disappearance of symptoms was not obtained, it was suggested that coil embolization is one of the therapeutic options for HFS which is caused by aneurysmal compression of REZ and intraoperative AMR is useful for identification of responsible lesions and determination of therapeutic effects.

CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions.

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.

Selective CB1 cannabinoid receptor antagonist, SR141716A, attenuates liver injury induced by Concanavalin A.

AIM: The aim of this study was to investigate the hepatoprotective activity of a selective cannabinoid receptor 1 (CB1) antagonist, SR141716A, in a Concanavalin A (Con A)-induced mouse liver injury model and to determine whether SR141716A has an effect on the production of inflammatory cytokines and chemokines induced by Con A. RESULTS: Injection of Con A (20 mg/kg) to mice developed hepatitis determined by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation and necrosis in the liver. Pretreatment with SR141716A (30 mg/kg) significantly reduced plasma AST and ALT level, protected against necrosis in the liver, and significantly reduced plasma cytokine and chemokine levels, including TNFalpha, IFN-gamma, CXCL9, MIP1-alpha, and IL-10 and no change decreased in IL-4. CONCLUSIONS: The selective CB1 antagonist, SR141716A, exerts a hepatoprotective effect on Con A-induced liver injury in mice by attenuating the increase in cytokine and chemokine levels and inhibiting hepatocyte injury. These findings raise the possibility of using CB1 antagonists as anti-inflammatory drugs for treating hepatitis as well as other inflammatory diseases.

Understanding the structure and role of DNA-PK in NHEJ: How X-ray diffraction and cryo-EM contribute in complementary ways.

DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End Joining repairs DNA double-strand breaks directly without a template and so can take place at any point in the cell cycle. Ku70/80 heterodimers rapidly assemble around broken DNA ends, allowing DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to be recruited and facilitating synapsis of broken DNA ends. This then provides a stage for end-processing and ligation. Here we review progress leading in 2017 to the medium resolution X-ray structure of DNA-PKcs, a single polypeptide chain of 4128 amino acids. This was followed quickly by chain tracing of cryo-EM structures of DNA-PKcs in complex with Ku and DNA. We discuss how combination of structural information from X-ray and cryo-EM studies can produce a working model for complex multicomponent molecular assemblies such as those found in DNA-double-strand-break repair.

Utility of plasma circulating mRNA as a marker to detect hepatic injury.

Utility of plasma circulating mRNA as a molecular marker to detect hepatic injury was evaluated. Total RNA was isolated from plasma of the rat liver fibrosis models at various time points, and plasma circulating mRNAs of major liver-derived genes, albumin and haptoglobin, were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Sensitivity and kinetics of plasma circulating mRNA were compared with those of plasma alanine aminotransferase (ALT) activity. We have found that the measurement of plasma circulating mRNA is more sensitive than plasma ALT activity, and enables early detection of hepatic injury. The plasma circulating mRNA will serve as a novel and highly sensitive molecular marker for hepatic injury.

[Primary Sjögren's syndrome with lymphocytic interstitial pneumonia, autoimmune hemolytic anemia and an endobronchial amyloid nodule].

A 73-year-old woman was admitted to our hospital for examination of anemia, dry feeling of oral and eyes with chest abnormal shadow in October, 1999. Chest radiograph showed interstitial shadows difference and CT showed small nodular opacities in both lung fields. Laboratory examination showed direct Coombs test was positive, and the serum levels of indirect bilirubin and haptoglobin were elevated. Anti-SS-A antibody and anti-SS-B antibody were positive with hypergammaglobulinemia. The serum levels of SP-D and KL-6 were elevated. Lip biopsy showed atrophy and lymphocyte infiltration of the salivary glands. According to these findings, she was diagnosed as primary Sjögren's syndrome with autoimmune hemolytic anemia. In addition, bronchoscopic examination showed an endobronchial polyp in the right B8, and the biopsy specimen showed AL type amyloid deposits. In order to obtain pathological diagnosis of lung lesions, we performed lung biopsy by video-assisted thoracoscopic surgery. The biopsy specimen showed severe infiltration of lymphocytes and the plasma cells around peribronchiole with lymphoepithelial lesions, suggesting malignant lymphoma. However, immunohistochemistry did not show monoclonal profile and Southern blot hybridization assay demonstrated no rearrangement of JH gene. Lung lesions were diagnosed as lymphocytic interstitial pneumonia due to Sjögren's syndrome. Steroid therapy was performed and followed by improvement of anemia and pulmonary lesions. Malignant lymphoma has not been involved for eight years after a diagnosis.

Gene Signature-Based Approach Identified MEK1/2 as a Potential Target Associated With Relapse After Anti-TNFα Treatment for Crohn's Disease.

Background: Anti-tumor necrosis factor alpha (anti-TNFα) therapy has become the mainstay of therapy for Crohn's disease (CD). However, post-therapy, the recurrence rate is still high. The aim of this study was to dissect the molecular mechanism for recurrence of CD treated with anti-TNFα therapy and investigate novel therapeutic options that could induce complete remission. Methods: We re-analyzed publicly available mucosal gene expression data from CD patients pre- and post-infliximab therapy to extract the transcriptional differences between responders and healthy controls. We used a systematic computational approach based on identified differences to discover novel therapies and validated this prediction through in vitro and in vivo experimentation. Results: We identified a set of 3545 anti-TNFα therapy-untreatable genes (TUGs) that are significantly regulated in intestinal epithelial cells, which remain altered during remission. Pathway enrichment analysis of these genes clearly showed excessive growth state and suppressed terminal differentiation, whereas immune components were clearly resolved. Through in silico screening strategy, we observed that MEK inhibitors were predicted to revert expression of genes dysregulated in infliximab responders. In vitro transcriptome analysis demonstrated that selective MEK1/2 inhibitor significantly normalized reference genes from TUGs. In addition, in vitro functional study proved that MEK1/2 inhibitor facilitated intestinal epithelial differentiation. Finally, using murine colitis model, administration of MEK1/2 inhibitor significantly improved diarrhea and histological score. Conclusions: Our data revealed the abnormalities in anti-TNFα responders' CD colons that would be cause of recurrence of CD. Also, we provided evidence regarding MEK1/2 inhibitor as a potential treatment against CD to achieve sustainable remission.