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We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14.3-3σ, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x). 14-3-3σ, cydinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours. [ABSTRACT FROM AUTHOR]

Treatment of phthalan derivatives with p -chloranil in dodecane in the presence of molecular sieves at 160-200 °C allowed the generation of unstabilized isobenzofurans, which underwent intramolecular Diels-Alder reaction to give endo cycloadducts exclusively. The cycloaddition turned out to be reversible, providing an equilibrium mixture of endo adducts when heating a substrate with a stereocenter on the tether. We also demonstrated the regioselective allylation of an oxygen-bridged cycloadduct upon exposure to EtAlCl 2 in the presence of allyltrimethylsilane, and the conversion to Rice's intermediate completed a formal synthesis of (±)-morphine., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Growing evidence from dogs and humans supports the abundance of mutation-based biomarkers in tumors of dogs. Increasing the use of clinical genomic diagnostic testing now provides another powerful data source for biomarker discovery., Hypothesis: Analyzed clinical outcomes in dogs with cancer profiled using SearchLight DNA, a cancer gene panel for dogs, to identify mutations with prognostic value., Animals: A total of 127 cases of cancer in dogs were analyzed using SearchLight DNA and for which clinical outcome information was available., Methods: Clinical data points were collected by medical record review. Variables including mutated genes, mutations, signalment, and treatment were fitted using Cox proportional hazard models to identify factors associated with progression-free survival (PFS). The log-rank test was used to compare PFS between patients receiving and not receiving targeted treatment before first progression., Results: Combined genomic and outcomes analysis identified 336 unique mutations in 89 genes across 26 cancer types. Mutations in 6 genes (CCND1, CCND3, SMARCB1, FANCG, CDKN2A/B, and MSH6) were significantly associated with shorter PFS. Dogs that received targeted treatment before first progression (n = 45) experienced significantly longer PFS compared with those that did not (n = 82, P = .01). This significance held true for 29 dogs that received genomically informed targeted treatment compared with those that did not (P = .05)., Conclusion and Clinical Importance: We identified novel mutations with prognostic value and demonstrate the benefit of targeted treatment across multiple cancer types. These results provide clinical evidence of the potential for genomics and precision medicine in dogs with cancer., (© 2023 Vidium Animal Health and The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Background: Longstanding overt ventriculomegaly in adults (LOVA) is a new form of progressive hydrocephalus characterized by onset in early childhood and gradual progression into adulthood. Patients with LOVA are usually asymptomatic in childhood. The diagnosis of LOVA in adolescence has not been reported., Case Report: A patient with macrocephaly and mild ventriculomegaly from infancy developed headache exacerbation and cognitive dysfunction at the age of 11 years. Brain magnetic resonance imaging showed mild tri-ventriculomegaly with no radiological aggravation compared to imaging at the age of 8 years. No papilledema was observed. Drainage of 15 ml of spinal fluid via a lumbar puncture relieved the headache and cognitive dysfunction. Based on repeated improvements in cognitive function and headaches after spinal fluid drainage, we diagnosed the patient with LOVA with symptom onset in early adolescence. A ventriculoperitoneal shunt was placed, and the headaches disappeared completely. The full-scale intellectual quotient, verbal comprehension, and working memory improved significantly., Conclusions: LOVA may manifest as early as adolescence. The clinical presentation, age, clinical, radiological features, and management vary, and a spinal tap exam is useful for diagnosing LOVA, even in children. The spinal tap exam may be indicated in children with longstanding ventriculomegaly and deteriorating neurological symptoms to diagnose this "treatable intellectual disability.", (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We describe the development of the practical manufacturing of Ensitrelvir, which was discovered as a SARS-CoV-2 antiviral candidate. Scalable synthetic methods of indazole, 1,2,4-triazole and 1,3,5-triazinone structures were established, and convergent couplings of these fragments enabled the development of a concise and efficient scale-up process to Ensitrelvir. In this process, introducing a meta -cresolyl moiety successfully enhanced the stability of intermediates. Compared to the initial route at the early research and development stage, the overall yield of the longest linear sequence (6 steps) was improved by approximately 7-fold. Furthermore, 9 out of the 12 isolated intermediates were crystallized directly from each reaction mixture without any extractive workup (direct isolation). This led to an efficient and environmentally friendly manufacturing process that minimizes waste of organic solvents, reagents, and processing time. This practical process for manufacturing Ensitrelvir should contribute to protection against COVID-19., Competing Interests: The authors declare the following competing financial interest(s): All authors were full-time employees of Shionogi & Co., Ltd. at the time when this study was performed., (© 2023 The Authors. Published by American Chemical Society.)

The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 5' recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1 Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pigmentary mosaicism of the Ito type, also known as hypomelanosis of Ito, is a neurocutaneous syndrome considered to be predominantly caused by somatic chromosomal mosaicism. However, a few monogenic causes of pigmentary mosaicism have been recently reported. Eleven unrelated individuals with pigmentary mosaicism (mostly hypopigmented skin) were recruited for this study. Skin punch biopsies of the probands and trio-based blood samples (from probands and both biological parents) were collected, and genomic DNA was extracted and analyzed by exome sequencing. In all patients, plausible monogenic causes were detected with somatic and germline variants identified in five and six patients, respectively. Among the somatic variants, four patients had MTOR variant (36%) and another had an RHOA variant. De novo germline variants in USP9X, TFE3, and KCNQ5 were detected in two, one, and one patients, respectively. A maternally inherited PHF6 variant was detected in one patient with hyperpigmented skin. Compound heterozygous GTF3C5 variants were highlighted as strong candidates in the remaining patient. Exome sequencing, using patients' blood and skin samples is highly recommended as the first choice for detecting causative genetic variants of pigmentary mosaicism., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The first total syntheses of two 4,10-dihydroxy-8,12-guaianolides that were reported to be natural products were achieved. Toward the syntheses of a collection of related guaianolides, the typical 5,7-fused system of 8,12-guaianolides was constructed by a ring expansion reaction of a hydroxylated coronafacic acid analogue that can be practically synthesized and optically resolved. The total syntheses of these compounds revealed that the previously reported structures of both natural products were incorrect.

Probst bundles are selectively seen in patients with agenesis of the corpus callosum (CC) and are thought to be homologous to the CC. We herein report a 19-year-old woman with partial agenesis of the CC. She developed acute encephalopathy during Bordetella pertussis infection. Brain magnetic resonance imaging (MRI) showed restricted diffusion of bilateral Probst bundles. She was treated with anti-epileptics and azithromycin and recovered with no neurological sequelae. Follow-up MRI showed the resolution of the diffusion abnormality. The characteristics of diffusion-weighted images on brain MRI and clinical course mimicked those in cases of clinically mild encephalopathy/encephalitis with reversible splenial lesion.

Palau'amine has received a great deal of attention as an attractive synthetic target due to its intriguing molecular architecture and significant immunosuppressive activity, and we achieved its total synthesis in 2015. However, the synthesized palau'amine has not been readily applicable to the mechanistic study of immunosuppressive activity, because it requires 45 longest linear steps from a commercially available compound. Here, we report the short-step construction of the ABCDEF hexacyclic ring core of palau'amine. The construction of the CDE tricyclic ring core in a single step is achieved by our p K a concept for proceeding with unfavorable equilibrium reactions, and a palau'amine analog without the aminomethyl and chloride groups is synthesized in 20 longest linear steps from the same starting material. The palau'amine analog is confirmed to retain the immunosuppressive activity. The present synthetic approach for a palau'amine analog has the potential for use in the development of palau'amine probes for mechanistic elucidation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The aims of studies were to estimate the withdrawal period of antibiotic from milk after the intramammary infusion of cefazolin sodium (CEZ) in cows with difficulties in frequent milk discharge due to disease such as teat injury. The period was compared among cows milked twice a day after 150 or 450 mg of CEZ were administered to all quarters (Study 1, 2) and the cows in which milking of front-right quarter was ceased for five days after administration of these infusions to only that quarter (Study 3). In Studies 1 and 2, the median of 17.66 µg/ml and 83.18 µg/ml of CEZ were detected in the samples of first milking after intramammary administration, respectively; however, there was no residual antibiotic by 72 hr in all cows. In Study 3, the median of 1.96 µg/ml of CEZ was detected in the sample after the resumption of milking at 120 hr, and the residual was eliminated by 174 hr. The withdrawal period may be prolonged by the cessation of milking after administration, and the period is the total time from cessation to 72 hr after the resumption of milking.

DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp., (© 2020 Hara et al.)

Replication initiation at specific genomic loci dictates precise duplication and inheritance of genetic information. In eukaryotic cells, ATP-bound origin recognition complexes (ORCs) stably bind to double-stranded (ds) DNA origins to recruit the replicative helicase onto the origin DNA. To achieve these processes, an essential region of the origin DNA must be recognized by the eukaryotic origin sensor (EOS) basic patch within the disordered domain of the largest ORC subunit, Orc1. Although ORC also binds single-stranded (ss) DNA in an EOS-independent manner, it is unknown whether EOS regulates ORC on ssDNA. We found that, in budding yeast, ORC multimerizes on ssDNA in vitro independently of adenine nucleotides. We also found that the ORC multimers form in an EOS-dependent manner and stimulate the ORC ATPase activity. An analysis of genomics data supported the idea that ORC-ssDNA binding occurs in vivo at specific genomic loci outside of replication origins. These results suggest that EOS function is differentiated by ORC-bound ssDNA, which promotes ORC self-assembly and ATP hydrolysis. These mechanisms could modulate ORC activity at specific genomic loci and could be conserved among eukaryotes., (© 2019 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Background: Parkinson's disease is a progressive, disabling neurodegenerative disorder associated with significant economic burden for patients and caregivers. The objective of this study was to compare the direct and indirect economic burden of Parkinson's patients' caregivers with demographically matched controls in the United States, in the 5 years after first diagnosis of Parkinson's disease., Methods: Policyholders (18-64 years old) linked to a Parkinson's disease patient (≥2 diagnoses of Parkinson's disease; first diagnosis is the index date) from January 1, 1998 to March 31, 2014, were selected from a private-insurer claims database and categorized as Parkinson's caregivers. Eligible Parkinson's caregivers were matched 1:5 to policyholders with a non-Parkinson's dependent (controls). Multivariable regression adjusted for baseline characteristics estimated direct costs (all-cause insurer cost [medical and prescription] and comorbidity-related medical costs; patient out-of-pocket costs) and indirect costs (disability and medically related absenteeism costs). Income progression was also compared between cohorts., Results: A total of 1211 eligible Parkinson's caregivers (mean age, 56 years; 54% female) were matched to 6055 controls. In adjusted analyses, Parkinson's caregivers incurred significantly higher year 1 total all-cause insurer costs ($8999 vs $7117) and medical costs ($7081 vs $5568) (both P < 0.01) and higher prescription costs (range for years 1-5, $2506-2573 vs $1405-$1687) and total out-of-pocket costs ($1259-1585 vs $902-$1192) in years 1-5 (all P < 0.01). Parkinson's caregivers had significantly higher adjusted indirect costs in years 1-3 (range for years 1-3, $2054-$2464 vs $1681-$1857; all P < 0.05) and higher cumulative income loss over 5 years ($5967 vs $2634 by year 5; P for interaction = 0.03)., Conclusions: Parkinson's caregivers exhibited higher direct and indirect costs and greater income loss compared with matched controls. © 2018 International Parkinson and Movement Disorder Society © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society., (© 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.)

Previously, we reported that adenosine N1-oxide (ANO), which is found in royal jelly, inhibited the secretion of inflammatory mediators by activated macrophages and reduced lethality in lipopolysaccharide (LPS)-induced endotoxin shock. Here, we examined the regulatory mechanisms of ANO on the release of pro-inflammatory cytokines, with a focus on the signaling pathways activated by toll-like receptor (TLR)4 in response to LPS. ANO inhibited both tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion from LPS-stimulated RAW264.7 cells without affecting cell proliferation. In this response, phosphorylation of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK)1/2, p38 and SAPK/c-Jun N-terminal kinase (JNK)) and nuclear factor-κB (NF-κB) p65 was not affected by treatment with ANO. In contrast, phosphorylation of Akt (Ser473) and its downstream molecule glycogen synthase kinase-3β (GSK-3β) (Ser9) was up-regulated by ANO, suggesting that ANO stimulated GSK-3β phosphorylation via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The phosphorylation of GSK-3β on Ser9 has been shown to negatively regulate the LPS-induced inflammatory response. Activation of PI3K/Akt signaling pathway has also been implicated in differentiation of mesenchymal stem cells into osteoblasts and adipocytes. As expected, ANO induced alkaline phosphatase activity and promoted calcium deposition in a mouse pre-osteoblastic MC3T3-E1 cell line. The ANO-induced differentiation into osteoblasts was abrogated by coincubation with Wortmannin. Furthermore, ANO promoted insulin/dexamethasone-induced differentiation of mouse 3T3-L1 preadipocytes into adipocytes at much lower concentrations than adenosine. The protective roles of PI3K/Akt/GSK-3β signaling pathway in inflammatory disorders have been well documented. Our data suggest that ANO may serve as a potential candidate for the treatment of inflammatory disorders. Promotion of osteogenic and adipocyte differentiation further suggests its application for regenerative medicine.

A method of synthesizing a directly connected 1,3a,6a-triazapentalene (TAP) ring system as a linearly bonded aromatic system with a planar form was established. Various TAP-dimers and a 2-alkyl-TAP-trimer were synthesized and their fluorescence properties were evaluated. Although the direct connection of the TAP ring with other TAP rings did not affect the fluorescence properties in diluted solvent, TAP-dimers showed unique fluorescence properties derived from the aggregation state under highly concentrated conditions. In particular, TAP-dimer 5 f showed aggregation-induced emission in highly concentrated solution, and 5 b showed typical mechanochromic fluorescence in the solid state despite their compact molecular size., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nanoparticles capable of accumulating in tumor tissues are promising materials for tumor imaging and therapy. In this study, two radical nanoparticles (RNPs), denoted as 1 and 2, composed of self-assembled ureabenzene derivatives possessing one or two amphiphilic side chains were demonstrated to be candidates for metal-free functional magnetic resonance imaging (MRI) contrast agents (CAs). Because of the self-assembly behavior of 1 and 2 in a saline solution, spherical RNPs of sizes ∼50-90 and ∼30-100 nm were detected. In a highly concentrated solution, RNP 1 showed considerably small water-proton relaxivity values (r 1 and r 2 ), whereas RNP 2 showed an r 1 value that was around 5 times larger than that of RNP 1. These distinct r 1 values might be caused by differences in the self-assembly behavior by a hydration or dehydration process. In vivo studies with RNP 2 demonstrated a slightly enhanced T 1 -weighted image in mice, suggesting that the RNPs can potentially be used as metal-free functional MRI CAs for T 1 -weighted imaging.

The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer-template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer-template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer-template junction. Furthermore, PCNA and CTF18-RFC-Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) were identified in the late 1980s as essential factors for replication of simian virus 40 DNA in human cells, by reconstitution of the reaction in vitro. Initially, they were only thought to be involved in the elongation stage of DNA replication. Subsequent studies have demonstrated that PCNA functions as more than a replication factor, through its involvement in multiple protein-protein interactions. PCNA appears as a functional hub on replicating and replicated chromosomal DNA and has an essential role in the maintenance genome integrity in proliferating cells.Eukaryotes have multiple paralogues of sliding clamp, PCNA and its loader, RFC. The PCNA paralogues, RAD9, HUS1, and RAD1 form the heterotrimeric 9-1-1 ring that is similar to the PCNA homotrimeric ring, and the 9-1-1 clamp complex is loaded onto sites of DNA damage by its specific loader RAD17-RFC. This alternative clamp-loader system transmits DNA-damage signals in genomic DNA to the checkpoint-activation network and the DNA-repair apparatus.Another two alternative loader complexes, CTF18-RFC and ELG1-RFC, have roles that are distinguishable from the role of the canonical loader, RFC. CTF18-RFC interacts with one of the replicative DNA polymerases, Polε, and loads PCNA onto leading-strand DNA, and ELG1-RFC unloads PCNA after ligation of lagging-strand DNA. In the progression of S phase, these alternative PCNA loaders maintain appropriate amounts of PCNA on the replicating sister DNAs to ensure that specific enzymes are tethered at specific chromosomal locations.

Human Ctf18-RFC, a PCNA loader complex, interacts with DNA polymerase ε (Polε) through a structure formed by the Ctf18, Dcc1 and Ctf8 subunits. The C-terminal stretch of Ctf18, which is highly conserved from yeast to human, is necessary to form the Polε-capturing structure. We found that in the budding yeast Saccharomyces cerevisiae, Ctf18, Dcc1 and Ctf8 formed the same structure through the conserved C-terminus and interacted specifically with Polε. Thus, the specific interaction of Ctf18-RFC with Polε is a conserved feature between these proteins. A C-terminal deletion mutant of Ctf18 (ctf18(ΔC) ) exhibited the same high sensitivity to hydroxyurea as the complete deletion strain (ctf18Δ) or ATPase-deficient mutant (ctf18(K189A) ), but was somewhat less sensitive to methyl methanesulfonate than either of them. These phenotypes were also observed in dcc1Δ and ctf8Δ, predicted to be deficient in the interaction with Polε. Furthermore, both plasmid loss and gross chromosomal rearrangement (GCR) rates were increased in ctf18(ΔC) cells to the same extent as in ctf18Δ cells. These results indicate that the Ctf18-RFC/Polε interaction plays a crucial role in maintaining genome stability in budding yeast, probably through recruitment of this PCNA loader to the replication fork., (© 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

Purpose: This review compared the real-world effectiveness of everolimus-based therapy versus endocrine monotherapy or chemotherapy in postmenopausal hormone receptor positive (HR+)/ human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC) patients with multiple metastatic sites., Methods: This retrospective chart review examined a nationwide sample of postmenopausal HR+/HER2- mBC women with ≥2 non-lymph-node metastatic sites. Patients must have initiated everolimus-based therapy (monotherapy or combination therapy including everolimus), endocrine monotherapy (any endocrine agent), or chemotherapy (monotherapy or combination with another chemotherapeutic or endocrine agent) for mBC between July 1, 2012 and August 15, 2013 after nonsteroidal aromatase inhibitor failure. Progression-free survival and time on treatment were compared using Kaplan-Meier analysis and Cox proportional hazard models, adjusting for line of therapy and baseline characteristics., Findings: One hundred patients received everolimus-based therapy, 79 received endocrine monotherapy, and 86 received chemotherapy. Everolimus-based therapy was associated with significantly longer progression-free survival and time on treatment than endocrine monotherapy and chemotherapy., Implications: Among HR+/HER2- mBC patients with multiple metastatic sites, everolimus-based therapy was associated with better real-world effectiveness than endocrine monotherapy or chemotherapy., (Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.)

Background: Hormone receptor-positive, human epidermal growth factor receptor-2-negative (HR+/HER2-) is the most common type of metastatic breast cancer (mBC). While mBC patients generally have poor prognosis with limited progression-free survival (PFS) and overall survival (OS), those with multiple metastatic sites may have even worse clinical outcomes due to multiple organ involvement. This study aimed to compare clinical outcomes including PFS, time on treatment (TOT), and OS between HR+/HER2- mBC patients with multiple metastases versus those with a single metastasis in a real-world clinical setting., Methods: This was a retrospective chart review study of postmenopausal HR+/HER2- mBC women who had failed a non-steroidal aromatase inhibitor in the adjuvant or metastatic setting and initiated a new treatment for mBC between 07/01/2012 and 04/15/2013. Patients were classified to one of two study groups (multiple metastases or single metastasis) based on the number of non-lymph-node metastases at the initiation of the new treatment. PFS, TOT and OS were compared between the two groups using Kaplan-Meier analyses and multivariable Cox proportional hazard models adjusting for patient disease and treatment characteristics. Separate Cox models were conducted including models with an interaction term between line of therapy and study group to assess the impact of multiple metastases on clinical outcomes across different lines of therapy., Results: A total of 699 patient charts were collected, including 291 patients with multiple metastases and 408 single metastasis patients. Worse performance status and a higher proportion of prior chemotherapy for mBC were observed among patients with multiple metastases. Overall, patients with multiple metastases had significantly shorter PFS [adjusted hazard ratio (HR) = 1.55, 95 % confidence interval (CI) 1.21-1.98], TOT (adjusted HR = 1.33, 95 % CI 1.05-1.67), and OS (adjusted HR = 1.77, 95 % CI 1.15-2.74) than single metastasis patients. Similar outcomes were observed in each line of therapy., Conclusions: Among HR+/HER2- mBC patients, patients with multiple metastases had significantly shorter PFS, TOT, and OS than single metastasis patients, highlighting the substantial clinical burden and unmet need for more efficacious treatments for the former group of patients.

In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC.

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9(ΔC)-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9(ΔC)-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: Assessing real-world effectiveness of everolimus-based therapy (EVE) versus fulvestrant monotherapy (FUL) among postmenopausal women with hormone receptor-positive (HR(+))/HER2(-) metastatic breast cancer (mBC) after progression on nonsteroidal aromatase inhibitor (NSAI)., Data & Methods: Medical charts of community-based patients who received EVE or FUL for mBC after NSAI were examined. Progression-free survival (PFS), time on treatment and time to chemotherapy were compared using Kaplan-Meier curves and Cox proportional hazards models adjusting for line of therapy and patient characteristics., Results & Conclusion: 192 patients received EVE and 156 FUL. After adjusting for patient characteristics, EVE was associated with significantly longer PFS than FUL (hazard ratio: 0.71; p = 0.045). EVE was associated with better PFS than FUL among NSAI-refractory postmenopausal HR(+)/HER2(-) mBC patients.

Background: Everolimus-based therapy and endocrine monotherapy are used among postmenopausal women with hormone receptor-positive human epidermal growth factor receptor-2 negative (HR+/HER2-) metastatic breast cancer (mBC) whose disease progressed or recurred on a non-steroidal aromatase inhibitor (NSAI). However, limited evidence exists regarding the real-world comparative effectiveness of these agents., Methods: This retrospective chart review examined postmenopausal HR+/HER2- mBC patients in community-based oncology practices who received everolimus-based therapy or endocrine monotherapy (index therapy) as any line of therapy for mBC between 1 July 2012 and 15 April 2013 after NSAI failure. Time on treatment (TOT), progression-free survival (PFS), and time to chemotherapy (TTC) from index therapy initiation were compared using Kaplan-Meier analyses and Cox proportional hazards models adjusting for baseline characteristics., Results: A total of 243 and 270 patients received everolimus-based therapy or endocrine monotherapy in a quota-based sample. Patients treated with everolimus-based therapy had a higher proportion of visceral metastases, high tumor burden, and use of prior chemotherapies for mBC. After adjusting for baseline characteristics, everolimus-based therapy was associated with significantly longer TOT (HR = 0.67, 95% CI: 0.51-0.87) and PFS (HR = 0.75, 95% CI: 0.57-0.98) than endocrine monotherapy. No significant difference was found between everolimus-based therapy and endocrine monotherapy in TTC (HR = 0.81, 95% CI: 0.52-1.27). Results stratified by line of therapy were generally consistent with the overall results., Limitations: Limitations include recall and information bias with potentially absent or erroneous chart data, unobserved factors due to non-randomization, inability to measure outcome assessments paired with measuring outcomes prior to exposures, and potential patient selection bias associated with chart review., Conclusions: Among a nationwide sample of postmenopausal HR+/HER2- mBC patients treated in community oncology settings, treatment with everolimus-based therapy was associated with significantly longer TOT and PFS compared to endocrine monotherapy.