Cellular proliferation relies on the successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilize regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In Escherichia coli, it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism Bacillus subtilis and the pathogen Staphylococcus aureus. In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in B. subtilis, temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarization and cell lysis. This phenotype could be suppressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In S. aureus, cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in S. aureus was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.

Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD and DnaA, thereby restricting helicase recruitment from the origin during sporulation to inhibit further initiation events. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin ( oriC ) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaA ATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

AbstractBidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD and DnaA, thereby restricting helicase recruitment from the origin during sporulation to inhibit further initiation events. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.

ABSTRACT The mechanisms responsible for helicase loading during the initiation of chromosome replication in bacteria are unclear. Here we report both a positive and a negative mechanism for directing helicase recruitment in the model organism Bacillus subtilis . Systematic mutagenesis of the essential replication initiation gene dnaD and characterization of DnaD variants revealed protein interfaces required for interacting with the master initiator DnaA and with a specific single-stranded DNA (ssDNA) sequence located in the chromosome origin ( D naD R ecognition E lement, “DRE”). We propose that the location of the DRE within the replication origin orchestrates recruitment of helicase to achieve bidirectional DNA replication. We also report that the developmentally expressed repressor of DNA replication initiation, SirA, acts by blocking the interaction of DnaD with DnaA, thereby inhibiting helicase recruitment to the origin. These findings significantly advance our mechanistic understanding of helicase recruitment and regulation during bacterial DNA replication initiation. Because DnaD is essential for the viability of clinically relevant Gram-positive pathogens, DnaD is an attractive target for drug development.

The motivation behind this paper was to report the commonness of overweight/ stoutness by region, age, and sex in the year 2000 and to investigate at what age adiposity bounce back as estimated by BMI happens for Chinese youngsters. Techniques: A cross-sectional investigation was completed among 262,738 kids matured 3.5-6.4 years in 26 regions/urban communities in China. Weight and tallness were estimated from March 2000 through July 2000 by neighbourhood prepared wellbeing labourers. Worldwide age-and sex explicit BMI slice off focuses were utilized to get tantamount pervasiveness paces of overweight/stoutness. Various calculated relapse was utilized to assess chances proportions (OR) of being overweight/weight for various examinations.

496 Objectives: Gliomas are the most common primary brain tumor in the US.1 Currently, magnetic resonance imaging (MRI) is used to image gliomas, both for diagnosis and to monitor response to therapy. However, its utility is limited due to non-enhancing low-grade gliomas, and similar imaging findings with pseudo-progression and true recurrence on the initiation of a new treatment.2 The amino acid positron emission tomography (PET) tracer, 18F-fluciclovine, has Orphan Drug designation in the USA and Europe for the imaging of gliomas. Here, we report the safety and effectiveness of 18F-fluciclovine in the detection of recurrent gliomas using data from a retrospective observational study that analyzed results from multiple research sites. Methods: Data from two investigator-sponsored studies in the USA and a compassionate use program in Norway from adults who received 18F-fluciclovine PET for the detection of glioma were collected. The primary objective was to determine the positive predictive value (PPV) of 18F-fluciclovine PET to detect glioma in comparison to a histopathological truth standard. Secondary objectives included determination of the detection rate, sensitivity, specificity, and negative predictive value (NPV). A further objective was to evaluate adverse events (AEs) in any patient who received 18F-fluciclovine; any serious AE that occurred up to 35 days after injection of 18F-fluciclovine was reviewed.18F-Fluciclovine PET images that were evaluated by experienced readers prior to data collection, with lesions classified as either ‘positive’ or ‘negative’ for malignancy on the basis of visual assessment against an appropriate background were eligible for inclusion. Lesions with indeterminate results were excluded from analyses. A further requirement for the 18F-fluciclovine PET to have been conducted within 30 days of the histological truth standard being collected was set. Results: Between August 2004 and May 2015, data were collected from 82 patients. Of these, 18 scans met the diagnostic analysis criteria, 17 of which were from patients with recurrent glioma. These 17 patients, 71% of whom were male, 71% of whom had high grade tumors, and who had a median age of 55.0 years form the effectiveness cohort for the current analysis.Among the 17 patients with recurrent glioma, 18F-fluciclovine PET showed a PPV of 88.2%, a detection rate of 100% and sensitivity of 100%. In patients with recurrent high-grade glioma (n = 12), the PPV, detection rate and sensitivity were 83.3%, 100% and 100%, respectively, while in patients with recurrent low-grade glioma (n = 5) these were 100%, 100% and 100%, respectively. The specificity and NPV could not be calculated as no patients had a negative 18F-fluciclovine PET scan.In total, 3/82 (3.7%) patients experienced at least one treatment-emergent AE during the safety monitoring period. There was one fatal event of worsening glioblastoma which was considered unrelated to 18F-fluciclovine. All other treatment-emergent AE were considered TCAE Grade 1 in severity and were unrelated to 18F-fluciclovine. Conclusions: 18F-Fluciclovine PET is well tolerated and able to effectively detect the presence of tumors in adults with recurrent gliomas.

394 Objectives: Gliomas are the most common primary brain tumor in the US and account for approximately 25% of all brain tumors, and 80% of all malignant brain tumors.1 The amino acid positron emission tomography (PET) tracer, 18F-fluciclovine, has Orphan Drug designation in the US and Europe for imaging glioma and has the potential to address the limited utility of the current standard of care, magnetic resonance imaging (MRI).2 However, in order to facilitate the successful migration of this technology from expert sites into general clinical use an understanding of the reproducibility of interpretation of 18F-fluciclovine images by naive users is required. Here, we explore 3D Volumes of Interest (VOI) derived from 18F-fluciclovine imaging of suspected glioma and report the reproducibility of 18F-fluciclovine PET image interpretation among naive users. Data were captured as part of a Phase 3, blinded image evaluation of the diagnostic performance and reproducibility of interpretation of 18F-fluciclovine images when interpreted in combination with contrast-enhanced T1-weighted (CE-T1W) MRI for suspected glioma (abbreviated as 18F-fluciclovine + CE-T1W). Methods: Thirty-five 18F-fluciclovine PET and MRI (CE-T1W and fluid-attenuated inversion recovery [FLAIR] [or T2W]) datasets with corresponding histopathological truth standards collected as part of a previous prospective Phase 2 trial (JapicCTI-132289) were evaluated. A neuroradiologist read the MRI images and 3 blinded nuclear medicine physicians who were naive to 18F-fluciclovine PET independently read each 18F-fluciclovine image in combination with CE-T1W MRI. Diagnostic performance parameters were determined for 18F-fluciclovine + CE-T1W and compared with those determined for CE-T1W and FLAIR (or T2W) alone. Readers delineated the extent of any malignancy, recording the VOI. Inter- and intra-reader reproducibility were assessed for both the diagnostic performance parameters and the VOI (using intersections and similarity metrics such as dice coefficient [DC] and concordance index [CI]). Results: The positive predictive value for 18F-fluciclovine PET + CE-T1W MRI was > 90% for all 3 readers, similar to CE-T1W MRI alone (94%), but higher than that of FLAIR (85%). The sensitivity of 18F-fluciclovine PET + CE-T1W MRI (66-71%) was higher than that of CE-T1W MRI alone (42%) and the specificity (89%) was higher than that of FLAIR (or T2W) MRI alone (33%). Inter‑reader concordance based on the binary interpretation of the data between the 3 readers for 18F-fluciclovine PET + CE‑T1W was 89% (Fleiss’ Kappa, 0.86). Inter‑reader VOI agreement was also high (median DC, 0.78−0.89; median CI, 0.64−0.80 between comparisons). Intra‑reader concordance was 83.3−100% (Cohen’s Kappa, 0.66−1.00) for the three readers interpreting an image for the second time, with the DC for second VOI recordings ranging from 0.90 to 0.91 and the median CI ranging from 0.81 to 0.84 across the 3 readers. When compared with the VOI identified on CE-T1W MRI alone, a median increase in VOI of 28−60% was recorded with 18F-Fluciclovine PET + CE-T1W MRI. Comparisons with FLAIR imaging showed a median additional volume identified on FLAIR (or T2W) MRI, but not on 18F-fluciclovine PET + CE-T1W MRI, of 67−68% of the total volume identified by FLAIR (or T2W) MRI. Conclusions: 18F-Fluciclovine PET + CE-T1W MRI shows good diagnostic performance, with consistent image interpretation demonstrated across 3 naive readers. 18F-Fluciclovine PET + CE-T1W MRI accurately identified higher tumor volumes than CE-T1W MRI alone and showed greater specificity than FLAIR (or T2W) MRI. The addition of 18F-fluciclovine PET to MRI has potential clinical value in delineating which part of the additional volume of abnormality shown on FLAIR is not malignant, thus more accurately identifying the extent of glioma prior to surgery or radiotherapy.

1281 Objectives: Neurotensin receptor 1 (NTSR1) is overexpressed in a range of solid tumors including pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). The receptor is therefore a promising target for systemic radiation therapy (SRT). 177Lu-IPN01087 (also called 177Lu-3BP-227) is a DOTA (tetraxetan)-conjugated NTSR1 antagonist that has demonstrated anti-tumor activity in vitro and in vivo. This study (NCT03525392; EudraCT: 2017-001263-20) will evaluate 177Lu-IPN01087 safety and efficacy, including objective response rate (ORR), in patients with metastatic or locally advanced solid tumors expressing NTSR1 who have not responded to or are unsuitable for standard-of-care treatments. Methods: This is a phase 1/2, multicenter, open-label study. Tumor types may include PDAC, CRC, gastric cancer, gastrointestinal stromal tumor, squamous-cell carcinoma of head and neck, and Ewing sarcoma. Phase 1 will have a dose-escalation design, with potential for expansion cohorts; phase 2 will further explore safety and efficacy and will be conducted in specific indications in which suitable safety and anti-tumor activity have been demonstrated in phase 1. Phase 2 will have a basket design or optimal Simon’s two-stage design. Phase 1: approximately 30 patients will receive 177Lu-IPN01087. There will be up to six dose-escalation steps, starting with 5 GBq fractionated in two administrations (2 x 2.5 GBq) at least 4 weeks apart, up to a maximum of 15 GBq (2 x 7.5GBq). Three to five patients will be treated at each dose level to establish the maximum tolerated cumulative activity (MTCA). Patients with PDAC and CRC may be most likely to proceed to phase 2; other cohorts may also be included. Approximately 125 patients (PDAC, n = 55; CRC, n = 70) will be enrolled. Phase 2 will investigate whether 177Lu-IPN01087 produces a better ORR than the accepted threshold of current standard-of-care treatment for metastatic or locally advanced disease. Results: The phase 1 primary endpoint is MTCA determined using dose-limiting toxicities and organ exposure to radiation, or the maximum administered cumulative activity. The phase 2 primary endpoint is ORR assessed every 8 weeks or at disease progression, evaluated using Response Evaluation Criteria in Solid Tumors. Secondary endpoints include: phase 1 - pharmacokinetics, biodistribution, dosimetry, pharmacodynamics and efficacy; and phase 2 - efficacy, quality of life, safety, pharmacokinetics, biodistribution and dosimetry. Conclusion: This ongoing phase 1/2 study will provide safety, efficacy and pharmacokinetics data for 177Lu-IPN01087 SRT in patients with NTSR1-positive solid tumors, including PDAC and CRC. Research support: Research funding for this study and medical writing support for the abstract were provided by Ipsen.

Genome duplication is essential for cell proliferation, and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. In bacteria, the master initiator DnaA binds the chromosome origin (oriC) and unwinds the DNA duplex to permit helicase loading. However, despite decades of research it remained unclear how the information encoded within oriC guides DnaA‐dependent strand separation. To address this fundamental question, we took a systematic genetic approach in vivo and identified the core set of essential sequence elements within the Bacillus subtilis chromosome origin unwinding region. Using this information, we then show in vitro that the minimal replication origin sequence elements are necessary and sufficient to promote the mechanical functions of DNA duplex unwinding by DnaA. Because the basal DNA unwinding system characterized here appears to be conserved throughout the bacterial domain, this discovery provides a framework for understanding oriC architecture, activity, regulation and diversity. Bacterial replication origin sequences are complex and diverse. In vivo dissection of a basal bacterial chromosome unwinding mechanism in Bacillus subtilis provides a framework for understanding oriC architecture, function and diversity. Systematic in vivo dissection of sequence elements essential for DnaA‐dependent strand separation in Bacillus subtilis provides a framework for understanding bacterial replication origin architecture and diversity.

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we show that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail by NMR. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

165 Background: Detection of the extent of local recurrence and of metastases in biochemical recurrence (BCR) of prostate cancer facilitates selection of appropriate treatment. The FALCON trial (NCT02578940) assessed the impact of 18F-fluciclovine PET/CT on the clinical management of men with BCR of prostate cancer following initial radical therapy. Methods: Men being considered for curative-intent salvage therapy following first BCR were recruited at 6 UK sites. Management plans were documented prior to and following 18F-fluciclovine PET/CT imaging. Post-scan changes to treatment modality such as salvage radiotherapy [RT] to systemic therapy were classed as ‘major’, while changes within a modality (e.g. modified RT fields) were classed as ‘other’. A pre-planned interim analysis of the first 85 patients was conducted; recruitment was to be stopped for efficacy if the number of treatment changes was > 45 (52.9%; 97.5% CI: 40.3–62.3%), or for futility if ≤ 8 (9.4%, 97.5% CI: 3.6–18.9%). Results: The 85 enrolled patients were a mean 4.8 y post-initial diagnosis, with a median age of 67 y and median PSA of 0.63ng/mL. Twelve (14.1%) had a Gleason score ≤ 6, 60 (70.6%) had a score of 7 and 13 (15.3%) had a score ≥ 8. Most (56; 65.9%) had previously undergone radical prostatectomy (RP), with 27 having received RT (± other therapy). The majority of those imaged (52; 61.2%) had a change in management (CIM) post-scan (Table). Recruitment was subsequently stopped as the pre-specified condition defining overwhelming efficacy was met. Conclusions: This prospective trial shows 18F-fluciclovine PET/CT has substantial impact on clinical decisions for men with a first BCR of prostate cancer after curative-intent primary therapy. Clinical trial information: NCT02578940. [Table: see text]

When biochemical recurrence (BCR) of prostate cancer is suspected, early and accurate localization of metastases facilitates treatment when tumors are small and most amenable to localized therapy, and may guide clinicians in making management plans regarding salvage therapy. Here, we present results of a preplanned analysis of the FALCON trial (NCT02578940), which assessed the impact of PET/CT imaging with 18F-fluciclovine on clinical management choices for men with BCR of prostate cancer.

Prostate cancer is the commonest solid-organ cancer diagnosed in males and represents an important source of morbidity and mortality worldwide. Imaging plays a crucial role in diagnosing prostate cancer and informs the ongoing management of the disease at all stages. Several novel molecular imaging technologies have been developed recently that have the potential to revolutionise disease diagnosis and the surveillance of patients living with prostate cancer. These innovations include hyperpolarised MRI, choline PET/CT and PSMA PET/CT. The major utility of choline and PSMA PET/CT currently lies in their sensitivity for detecting early recurrence after radical treatment for prostate cancer and identifying discrete lesions that may be amenable to salvage therapy. Molecular imaging is likely to play a future role in characterising genetic and biochemical signatures in individual tumours, which may be of particular significance as cancer therapies move into an era of precision medicine.

The mainstay of treatment for men with three or fewer non-castrate metastatic lesions outside of the prostate remains morbid palliative androgen deprivation therapy. We believe there is now a significant body of retrospective literature to suggest a survival benefit if these men have radical treatment to their primary tumour alongside 'metastasis-directed therapy' to the metastatic deposits. However, this regimen should be reserved to high-volume centres with quality assurance programmes and excellent outcomes. Patients should be made clear as to the uncertainty of benefit for this multi-site treatment strategy, and we await the publication of randomised controlled trials reporting in the next 5 years.