BRCA-mutated (BRCAm) ovarian cancer. PARP inhibitors may benefit patients whose tumours are dysfunctional in DNA repair mechanisms unrelated to BRCA1/2. We report exploratory analyses, including the long-term outcome of candidate biomarkers of sensitivity to olaparib in BRCA wild-type (BRCAwt) tumours.Olaparib (Lynparza™) is a PARP inhibitor approved for advanced BRCA1 promoter methylation status, measurement of BRCA1 protein and Myriad HRD score.Tumour samples from an olaparib maintenance monotherapy trial  were analysed. Analyses included classification of mutations in genes involved in homologous recombination repair (HRR), BRCAm tumours gained most benefit from olaparib; a similar treatment benefit was also observed in 21/95 patients whose tumours were BRCAwt but had loss-of-function HRR mutations compared to patients with no detectable HRR mutations (58/95). A higher median Myriad MyChoice® HRD score was observed in BRCAm and BRCAwt tumours with BRCA1 methylation. Patients without BRCAm tumours derived benefit from olaparib treatment vs placebo although to a lesser extent than BRCAm patients.Patients with BRCA1/2 may constitute a small, molecularly identifiable and clinically relevant population who derive treatment benefit from olaparib similar to patients with BRCAm.Ovarian cancer patients with tumours harbouring loss-of-function mutations in HRR genes other than  In these tumours, DNA repair by low fidelity repair mechanisms such as non-homologous end joining can cause the accumulation of genomic instability, ultimately resulting in cell death; a concept referred to as synthetic lethality.3 Additionally, preclinical data suggest that PARP inhibitors may also benefit patients whose tumours are sensitive to platinum-based chemotherapy and who have an HRR deficiency caused by mutations other than those in the BRCA1/2 genes.5 Hence, the clinical and molecular profiles of high-grade serous ovarian cancer (SOC) appeared well matched to PARP inhibitor biology, as this cancer is noted for genome instability thought to be driven by HRR deficiency, repeated and prolonged platinum sensitivity and a high frequency of deficiency in BRCA and other candidate HRR proteins.6Poly(ADP-ribose) polymerase (PARP) plays a vital role in the repair of single-strand DNA breaks through the base excision repair pathway. PARP inhibitors are thought to become trapped at the sites of single-strand DNA breaks leading to double-strand DNA breaks when DNA replication is attempted.BRCA1/2-mutated tumours .7 This subgroup included patients with inherited and somatic (tumour only) mutations.7 Recent analyses strongly suggest that somatic loss-of-function mutations in BRCA1 and BRCA2 are an early event in the development of high-grade SOC and that tumours with somatic BRCA mutations phenocopy tumours in patients with inherited germline BRCA mutations in terms of genetic epidemiology, natural history and response to platinum chemotherapy, and response to olaparib and other PARP inhibitors.13Maintenance treatment with the PARP inhibitor olaparib (Lynparza™) in an AstraZeneca-sponsored, randomised, phase II trial of 265 patients with platinum-sensitive relapsed high-grade SOC  led to a significant increase in progression-free survival (PFS) vs placebo, which was greatest in the subgroup of 136 patients with BRCA tumours, although the treatment benefit was less  than in those patients with BRCA-mutated tumours.7 A similar trend of broad activity in ovarian cancer with a stronger effect in tumours with BRCA mutations has been noted for other PARP inhibitors.13 Hence, there is considerable interest in understanding the molecular basis of sensitivity to PARP inhibitors in patients whose tumours do not have mutations in the BRCA genes and in tumour tests that may aid in the identification of patients who will benefit most from treatment. In particular, an important question to address is whether in BRCA wild-type tumours, mutations in other HRR genes account for the benefit observed. To further characterise genetic changes in SOC tumours, we conducted exploratory candidate biomarker analyses on BRCA wild-type tumour samples from Study 19 and investigated the possible relationship of HRR deficiencies and clinical benefit.Of interest, in Study 19 a significant PFS benefit for olaparib vs placebo was also observed in the subgroup of 118 patients with wild-type 14 In brief, eligible patients were aged 18 years or older and had relapsed SOC (Grade 2 or 3) that was platinum sensitive. Patients entering the study were required to have received two or more previous courses of platinum-based chemotherapy and to have demonstrated an objective response  according to Response Evaluation Criteria in Solid Tumors (RECIST) or Gynecologic Cancer InterGroup criteria. Patients were randomised 1:1 to receive either olaparib 400 mg twice daily (b.i.d.) capsules or matching placebo. Study treatment was continued until progression in the absence of unacceptable toxicity. The primary endpoint was PFS, as determined by RECIST v1.0, and overall survival (OS) was a secondary endpoint.Study 19 was a phase II, randomised, double-blind, multicentre trial, undertaken at 82 sites in 16 countries. The study design, patient population and statistical analyses have previously been published in detail.BRCA mutations, exploratory biomarker analyses were conducted on tumour samples from Study 19 including BRCA1 promoter methylation, BRCA1 protein expression, HRR gene mutation, and Myriad homologous recombination deficiency (HRD) testing (which includes a tumour test for BRCA mutations).To identify patients that benefit from olaparib that do not have BRCA mutation status of patients was prespecified in the Study 19 statistical analysis plan. Molecular analyses to define the BRCA status of patients in Study 19 were completed retrospectively and were blinded to clinical outcomes.7 In brief, the BRCA-mutated population comprised patients with a mutation in their tumour and/or blood sample. Germline BRCA mutation status was either reported on case report forms after local testing or was established retrospectively using the Integrated BRACAnalysis® assay , with DNA extracted from blood samples obtained prior to randomisation.15Analysis of the BRCA mutation status subgroups defined retrospectively, which were blind to clinical outcomes but were not prespecified in the Study 19 statistical analysis plan.The analyses presented here are based on The provision of an archival tumour sample (blocks or sections) was mandatory for participation in Study 19. Samples received as blocks were made into tumour microarrays with two 0.6 mm cores from each tumour at the University of British Columbia.BRCA1, BRCA2 and other key HRR-related genes was established using the same DNA sequencing analysis performed to determine tumour BRCA mutation status as previously described.7 In brief, DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) archival tumour samples using a cancer gene panel enrichment procedure and deep resequencing performed with Illumina technology. Specifically, analysis was not performed with the commercially available Foundation Focus diagnostic test but with the Foundation Medicine T5 panel  at Foundation Medicine .16 Tumour analysis was performed on coded tumour samples and results were returned blind to the original Study 19 data set. The classification of variants was based on the American College of Medical Genetics recommendations. Patients with no known BRCA mutation and patients with a BRCA mutation classed as a variant of unknown significance (VUS) were included in the BRCA wild-type group as previously described.7Tumour mutation status in BRCA wild-type group were further subdivided into three groups: BRCA wild-type HRR-mutated, patients whose tumours had a loss-of-function mutation in a high-confidence HRR gene; HRR status unknown, patients with a potential loss-of-function mutation in any gene associated with DNA repair; and BRCA and HRR wild type, patients with no potential loss-of-function mutations in any gene involved in DNA repair. The HRR genes interrogated are listed in Supplementary Table Patients in the 17 Next-generation sequencing-based assays were used to generate genome-wide single-nucleotide polymorphism profiles, BRCA1/2 mutation screening and BRCA1 promoter methylation data. These analyses separately reported tumour BRCA mutation (tBRCAm) status, tumour BRCA1 methylation status and tumour HRD score. An HRD score ≥42 was considered positive. The reported results are based on a research assay performed at Myriad Genetics and not upon the commercially available test.18Tumour samples were analysed as described by Timms et al. (2014).FFPE sections of 4 µm thickness were cut and mounted on SuperFrost Plus™ electrostatically charged glass slides and dried overnight at 37 °C in an incubator. Sections were dewaxed in xylene, passed through a graded alcohol series and optimal antigen retrieval obtained using Dako pH 9 antigen retrieval buffer , heated in the RHS Microwave Histo-processor  to 110 °C with full pressure sustained for 2 min. The pressure vessel was then cooled under running tap water, the lid removed, and the slides washed.2O2 to quench endogenous peroxidase activity and then blocked with serum-free protein block (Dako). Following blocking, sections were incubated for 60 min with anti-BRCA1 (Ab-1) mouse monoclonal antibody (MS110) at a concentration of 1 µg/ml . Visualisation was achieved using Dako REAL™ Envision™, HRP rabbit/mouse (Dako), applied for 30 min, followed by 10 min incubation in 3,3'-diaminobenzidine. Sections were lightly counterstained in Carazzi’s haematoxylin, dehydrated through alcohol grades and mounted under a glass coverslip with Histomount .Immunohistochemical staining was performed at room temperature on a Lab Vision 720 Autostainer. Sections were first treated with 3% HStaining was negatively controlled by substituting mouse immunoglobulin fraction, diluted to the same concentration as that for the primary antibody. Immunostained slides were evaluated with light microscopy.The primary endpoint for Study 19 was PFS, which was reported for the full analysis population following a data cutoff (DCO) of 30 June 2010. PFS data were not collected following this DCO. Collection of time to first subsequent therapy or death (TFST), time to second subsequent therapy or death (TSST) and OS data continued, and the interim OS data reported here have a DCO of May 2016.14Analyses of PFS, TFST, TSST and OS use the same methodology; namely a Cox proportional hazards model adjusted for treatment, ethnic descent (Jewish vs non-Jewish), time to progression on penultimate platinum therapy (6–12 vs >12 months), and response to platinum therapy before randomisation . This methodology was used for the primary analyses of the study.Where there were fewer than 15 events formal statistical tests were not performed.7 111/209 patients were classified as having deleterious or suspected deleterious mutations present in BRCA1 and/or BRCA2 in their tumour samples and 98 patients had BRCA wild-type tumours; three further patients were known to have single exon indels in blood sample DNA but these were below the detection limit in tumour samples.7 Therefore, 95/209 patients (45.5%) were classified as having BRCA wild-type tumours. Twenty-one of these patients (22%) had at least one loss-of-function mutation in a candidate HRR gene, of whom 12 received olaparib and 9 received placebo. The specific HRR mutations identified in the 21 tumour samples were: BRIP1 (n = 5), CDK12 (n = 3), RAD54L (n = 2), RAD51B (n = 2), RAD54L rearr (n = 1), ATM rearr (n = 1), FANCA rearr (n = 1), FANCD2 (n = 1), FANCL rearr (n = 1), FANCL (n = 1), RAD51C (n = 1), RAD52 del (n = 1), XRCC3 rearr (n = 1)  patients and tumour sequencing was completed for 209/265 (79%) patients. Forty-four samples failed due to insufficient tumour cells, DNA quantity, sequencing library quality or sequence read coverage. As previously described,BRCA-mutated or HRR-mutated as a group containing a loss-of-function mutation in at least one pathway gene.For exploratory purposes, we defined a subset of patients as MSH2, MUTYH), or in genes where a postulated role in DNA repair has been the subject of significant controversy ; these patients were classified as HRR unknown. The remaining 58 patients’ tumours did not have a candidate loss-of-function mutation in a relevant gene. Twenty-five of these patients were randomised to olaparib and 33 to placebo.Of the remaining 74 patients, 16 were found to have mutations in genes that are involved more broadly in DNA damage repair  and BRCA wild-type populations   Fig. .7Fig. 2PBRCA mutation  than in patients with no detectable BRCA or HRR mutation  who received olaparib  was 0.20, 95% CI 0.12–0.33. It should be noted that PFS in the ITT population was the primary endpoint of the study and hence other analyses are exploratory subgroups of secondary or exploratory endpoints.The PFS hazard ratio for the exploratory subgroup of patients with a mutation in a BRCA mutation testing, BRCA1 methylation testing and determining HRD score, was performed in 219 tumour samples.Myriad tumour testing, which comprised BRCA status was reported for 212 patients, of whom 118 (56%) had a BRCA mutation. In total, BRCA status was determined for tumours from 235 patients using either the Myriad and/or Foundation Medicine analyses. Of the 193 patients for whom tumour BRCA status was available at both laboratories, tumour samples from 106 patients were classified as mutated and 84 cases were classed as wt/VUS by both laboratories (VUS samples were predominantly missense variants: impact on gene function is unknown). The concordance of tumour BRCA testing between the two laboratories was 98.5% for sequence analysis  had no BRCA mutation and BRCA analysis failed in two patients (1%). Of the 60 patients (30%) with low HRD scores 8 patients (13%) had a BRCA mutation, 51 patients (85%) had no BRCA mutation and 1 (2%) had a failed analysis. Overall, 156 patients would be classified as Myriad HRD positive with a BRCA mutation in the tumour or a Myriad HRD score ≥42.HRD scores were determined for 199 patients, of whom 139 (70%) were classified as HRD score high (HRD scores ≥42). Of the 139 tumours with high HRD scores, 101 patients (73%) had a BRCA-mutated tumours had the highest median HRD score, HRR-mutated tumours had an intermediate median HRD score and tumours without mutations in BRCA or other HRR genes had the lowest median HRD score  and in PSROC patients with a BRCA mutation in the EU (capsule formulation). We therefore performed exploratory analyses to examine separately the impact of HRD scores within patient groups with BRCA wild-type and BRCA-mutated tumours defined by Myriad tumour BRCA testing. There were only eight patients with BRCA mutations and HRD scores <42; this subgroup was not analysed further. When the prespecified threshold of ≥42 was used to define a BRCA wild-type HRD high group the PFS hazard ratio  was numerically superior to the PFS hazard ratio for the BRCA wild-type HRD low group  but was very similar to that for the overall BRCA wild-type population evaluable for HRD scores  . However, the relevant extremes of the HRD score distribution were only populated by small numbers of patients such that very few tumours with BRCA mutations had scores below 30 (n = 1) and conversely few tumours without BRCA mutations had scores greater than 50 (n = 30). Similar marginal differences in the hazard ratios observed for PFS between the BRCA wild-type HRD high group and the BRCA wild-type HRD low group were broadly observed for TFST, TSST and OS (Supplementary Table Olaparib is currently approved for use in patients with platinum-sensitive relapsed ovarian cancer (PSROC), regardless of n = 156) was 0.24, 95% CI 0.15–0.39. It should be noted that PFS in the ITT population was the primary endpoint of the study and hence other analyses are exploratory subgroups of secondary or exploratory endpoints.The PFS hazard ratio for the exploratory subgroup of patients with HRD-positive status  than the HRD-negative group  or the BRCA-mutated and/or HRD ≥33 group .A recent study suggested patients with tumours with HRD scores <33 are less likely to benefit from platinum chemotherapy.BRCA1. Of the 15 patients on olaparib for more than 6 years, 8 were originally reported as having BRCA mutations.7 One further patient was found to have somatic mutations in both BRCA1 and BRCA2 on Myriad blood and tumour testing (no Foundation Medicine tumour testing data were available); hence, 9/15 patients are known to have a BRCA mutation  in their tumour or blood sample, with 3/9 being a somatic mutation. This enrichment for BRCA2 mutation and presence of somatic cases in long-term responders has been noted previously.20 One of the remaining six patients was not evaluable for tBRCA status at either Myriad or Foundation Medicine, hence 5/15 patients treated with olaparib for more than 6 years had no detectable BRCA mutation. Three out of five patients had Myriad HRD scores available (2x HRD positive and 1x HRD negative) and 5/5 had HRRm status available . Of the two HRRwt tumours, one was Myriad HRD negative (Myriad HRD score 15) and the other was Myriad HRD status unknown. Therefore, patients without BRCA-mutated tumours and whose tumours tested negative for additional candidate biomarkers (HRRwt or BRCAwt HRD negative) were found among the patients who remained on olaparib for over 6 years.In Study 19 there were 16 patients treated for 6 years or longer, of whom one was on placebo and had a germline mutation in BRCA1-mutated cases was observed . In support of this theory, a non-randomised study of olaparib monotherapy in ovarian cancer subsequently found response rates of 41% and 24% in the germline BRCA-mutated and non-mutated carrier patient populations, respectively.25 Therefore, it was expected that the non-BRCA-mutated carrier high-grade SOC patient population included a significant number of patients whose tumours could be susceptible to PARP inhibition and Study 19 was a placebo-controlled study in the maintenance setting designed to test this hypothesis.7 Study 19 was a positive study overall and in a preplanned subgroup analysis the magnitude of PFS effect was greater in patients who had a BRCA mutation than those without.7 Tumour testing in Study 19 successfully identified the vast majority of germline BRCA-mutated cases and identified a further subset of patients with somatic or tumour only mutations.7 Subsequent exploratory analyses suggested that tumours with somatic BRCA mutations phenocopy those from germline carriers, both in terms of tumour genetics and in clinical benefit on olaparib10 and a clinical trial is under way to confirm this observation.26 Hence, BRCA mutation testing of tumour samples is the clearest indicator of tumour responsiveness to olaparib treatment in current clinical practice. Importantly, the data presented here confirm a very high level of consistency in research testing for tumour BRCA mutations in two independent laboratories and therefore confirms our previous observation that tumour testing accurately detects germline mutations. The number of mutations not captured by the Foundation Medicine assay was relatively small . Nonetheless, it is important that all tumour testing laboratories further develop their methodologies to accurately detect rare variants to allow tumour testing to detect all clinically relevant mutations.Elegant preclinical experiments led to the concept of synthetic lethality, which suggested that tumours without functional BRCA1 or BRCA2 protein would be more susceptible to PARP inhibitors compared with non-tumour tissues in the same patients.BRCA1 or BRCA2 who gain a PFS benefit from olaparib treatment. It is important that we determine whether there is a molecularly identifiable subgroup of patients with BRCA wild-type tumours that is differentiated from patients solely selected on available clinical parameters of platinum sensitivity and high-grade serous histology used in Study 19. Furthermore, if such a subgroup exists it will be important to determine whether the differential in benefit is clinically meaningful. Several candidate molecular measures have been suggested to fulfil this role including a continuous measure of genomic instability or HRR deficiency ‘scar’, BRCA1 methylation and loss-of-function mutations in other homologous recombination pathway genes. The supporting data behind the candidate biomarkers investigated are summarised in Table We have shown that Study 19 contained a readily identifiable, biologically coherent subpopulation of patients with inherited or acquired mutations in BRCA wild-type patient population may include a majority of patients whose tumours lack DNA repair gene loss-of-function mutations and who gain less benefit from PARP olaparib treatment than patients with candidate loss-of-function mutations in genes involved in HRR.Here, we report that the ovarian BRCA mutations .8First, we attempted to identify patients with loss-of-function mutations in candidate HRR genes in their tumours. Clearly this approach is limited by the genes sequenced by the Foundation Medicine panel and by the relative absence of relevant preclinical and clinical data. We therefore chose a pragmatic approach to include all genes with a DNA repair pathway role and some supportive preclinical data. Subsequently, we defined a higher confidence patient population by differentiating between candidate genes in which the primary role is related to mismatch repair or where, despite a purported role in double-strand DNA repair, either the genetic mechanism is distinct and/or the phenotypic impact on platinum sensitivity and prognosis in ovarian cancer is directionally opposed to that of BRIP1 (n = 5), CDK12 (n = 3), RAD54L (n = 2) and RAD51B (n = 2). BRIP1  is a protein that works with BRCA1 to repair damaged DNA and the inheritance of two mutated copies of BRIP1 causes Fanconi anaemia. Women who inherit one mutated copy of BRIP1 are likely to have an increased risk of ovarian cancer; however, the implication of this for breast cancer risk is inconclusive.29We identified 37 patients with mutations in any gene involved in DNA repair, of these patients 21 had mutations in the (relatively) higher confidence gene set. Genes involved in more than one patient were CDK12 is the least validated gene included in our HRR gene list. It is biallelically inactivated in ovarian cancer; hypothesis-directed experiments have found that mutations or depletion are associated with sensitivity to radiation, platinum or PARP inhibitor treatment and it was also highlighted as a candidate gene in a hypothesis-free synthetic lethal screen approach.31 However, mechanistically it has been proposed to have an indirect effect on DNA repair via transcriptional regulation32 and it was recently suggested to be associated with a different pattern of genome instability than that seen in patients with BRCA mutations or BRCA1 methylation.33BRCA1 or BRCA2) in Study 19 is too small to derive definitive conclusions. Furthermore, because of the low prevalence of mutations in any one gene a similarly sized trial could easily result in a different profile of mutated genes. However, it was possible to define a subpopulation of 58 patients without a candidate loss-of-function mutation in any measured relevant HRR gene. While it is acknowledged that mutations or alterations in other unmeasured genes may not have been detected in these 58 patients, it is notable that for all efficacy measures there was marked attenuation of the treatment benefit from olaparib. Our data are consistent with those of another study,8 suggesting that tumours with mutations in HRR genes may be associated with increased platinum sensitivity and prolonged survival.The number of patients with mutations in any HRR gene , whereas 14 of the 15 patients with BRCA1 methylation who were evaluable for HRD score were HRD positive. BRCA1-methylated cases accounted for at least 39% of the BRCA wild-type HRD-positive population in Study 19. An exploratory analysis of the 36 BRCA wild-type Myriad HRD-positive cases suggested that these patients were only marginally more likely to benefit from olaparib than selection based on study entrance criteria (response to platinum and high-grade serous histology); it may be that this is largely because of an inability of BRCA1 methylation to phenocopy BRCA1 mutation in terms of olaparib sensitivity/ease of reversibility as has also been reported for platinum sensitivity.8 The BRCA wild-type HRD scores ≥42 analysis reported here is an exploratory analysis of a small number of patients (n = 36) and aligns with the recently reported data for the NOVA and ARIEL3 trials which used the Myriad HRD (42) score and Foundation Medicine LOH score, respectively.13 In further exploratory analysis of Study 19, we show that relaxing the HRD threshold to a value of 33 differentiates a potential population receiving enhanced benefit compared to patients below the new threshold (hazard ratio 0.71 in HRD score <33 vs hazard ratio = 0.25 in HRD score ≥33). Particularly in a setting where the majority of patients appear to derive some level of benefit from PARP inhibitors, considering a biomarker that aims to exclude those patients least likely to benefit, rather than identifying those that are most likely to benefit, might be an attractive strategy. Compared to the HRD threshold of 42, this modified HRD scar approach, using a threshold <33, may have the potential to better identify a patient population that would derive least benefit from PARP inhibitors, but requires further study.The ability to avoid these issues for causal genetic changes and use an HRD score or genomic read-out of the effects of HRD, regardless of causal mechanism, is inherently attractive in theory. In Study 19 there were only 36 patients with BRCA2 mutations in the long-term responders, neither an HRRm or HRD approach was able to exclude patients who remained on treatment for 6 years or more. It is likely that additional mechanisms, such as those relating to the immune system, are important in determining which patients may receive long-term benefit from olaparib and potentially also from chemotherapy.35Importantly, although we observed an enrichment for BRCA mutations in clinically available FFPE samples can be highly reproducible and may identify a subpopulation of patients with the greatest PFS benefit from olaparib. A measure of HRD or genomic instability (Myriad MyChoice HRD) identified most patients with BRCA mutations and nearly all patients with BRCA1 methylation; however, only ~50% of a small number of patients with mutations in other candidate HRR genes were identified as HRR deficient. In underpowered exploratory analyses (i) patients with BRCA wild-type HRD-positive tumours did not achieve as high a treatment benefit from olaparib as patients with BRCA-mutated tumours and (ii) patients with tumours without mutations in candidate HRR genes, which comprised the majority of the BRCA wild-type subpopulation, received the least benefit from olaparib treatment, an observation which may be particularly important in the maintenance setting in the absence of any acute indicator of tumour response to olaparib treatment.In summary, the analyses reported here demonstrate that next-generation sequence-based tumour testing for BRCA wild-type high-grade SOCs.Further clinical studies examining all candidate tumour DNA-based measures of cause and effect deficiencies in HRR pathways are required to confirm that these additional measurements can provide clinically relevant information and treatment benefits for patients with https://astrazenecagrouptrials.pharmacm.com/ST/Submission/SearchAstraZeneca’s policy on clinical trial data, results and other information from or regarding AstraZeneca-sponsored clinical trials are described in full in the AstraZeneca website: Supplementary material


Triticum aestivum L.) was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs). In the present study, TaAGPS1b was fused with green fluorescent protein (GFP) in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains.ADP-glucose pyrophosphorylase (AGPase), the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b) of AGPase in grains of bread wheat ( Starch is one of the primary plant carbohydrate reserves in higher plants and it is composed of two different types of glucose (Glc) polymers: amylose  and amylopectin  ,3. DisprArabidopsis thaliana plants expressing a bacterial β-glucuronidase (GUS) gene under the control of the AGPS gene promoter, the expression patterns of GUS gene followed closely the AGPS protein location and starch production in plant tissues/cells, indicating the essential function of AGPase subunits in starch synthesis in higher plants . One unit was defined as amounts of AGPase that catalyzed production of 1 nmol NADPH per min.Activities of AGPase enzyme were assayed for the same transgenic wheat lines for analysis of the above transcript levels of eviously . Embryo 3 transgenic wheat lines were used for analysis on starch content and yield characteristics. Harvested grains were weighted after drying to equivalent moisture content. The starch contents were measured and calculated according to methods of Zhao and his colleagues [Mature grain harvested from wild and Tlleagues . The yielleagues .p < 0.05 level by SPSS (version 20.0) statistical software. Three biological replications were performed, and all data are the mean ± standard deviation (SD) of three independent experimental replicates. Data were statistically analyzed using one-way analysis of variance (ANOVA), and means were compared by using the Duncan's multiple range test at TaAGPS1b (EU586278), a wheat AGPase plastidial small subunit with a truncated transit peptide, was proved to be localized in chloroplasts and its overexpression transgenic wheat plants exhibited the improved AGPase activities, starch contents, and grain weights, indicating its important role in starch synthesis.


Cerebrovascular conductance index (CVCi) was calculated as MCAv/MAP. Central arterial stiffness was assessed using carotid–femoral pulse wave velocity (PWV). Older adults had higher PWV  compared with young adults. MCAv and CVCi reactivity to hypercapnia were not different between young and older adults ; however, older adults demonstrated higher MAP reactivity to hypercapnia . There were no associations between PWV and cerebrovascular reactivity . Our results demonstrate that cerebrovascular reactivity was not different between young and older adults who habitually exercise; however, MAP reactivity was augmented in older adults. This suggests an age-associated difference in the reliance on MAP to increase cerebral blood flow during hypercapnia.Reduced cerebrovascular reactivity to a vasoactive stimulus is associated with age-related diseases such as stroke and cognitive decline. Habitual exercise is protective against cognitive decline and is associated with reduced stiffness of the large central arteries that perfuse the brain. In this context, we evaluated the age-related differences in cerebrovascular reactivity in healthy adults who habitually exercise. In addition, we sought to determine the association between central arterial stiffness and cerebrovascular reactivity. We recruited 22 young  and 21 older  habitual exercisers who partake in at least 150 min of structured aerobic exercise each week. Middle cerebral artery velocity (MCAv) was recorded using transcranial Doppler ultrasound. In order to assess cerebrovascular reactivity, MCAv, end-tidal carbon dioxide (ETCO Measuriow (CBF) . Importaow (CBF) .As advancing age increases the risk for development of cerebrovascular disease and cognitive decline , it is i2 inhalation . Participants were not taking antihypertensive medication or any other vasoactive medications other than statins (older adults n = 2) or thyroid medication (older adults n = 4). Young women were studied during days 2–6 of their menstrual cycle or during the non-active pill phase of oral contraceptives (n = 7). Young women were not pregnant which was confirmed by a urine pregnancy test. Older women must have been postmenopausal for at least 1 year and not taking menopausal hormone therapy. All study procedures were approved by the Institutional Review Board of the Mayo Clinic and the University of Wisconsin–Madison and were performed according to the Declaration of Helsinki, including obtaining written informed consent from each participant.Habitually exercising adults were recruited via recruitment flyers and word of mouth. Forty-three adults including 21 older adults and 22 young adults participated in the study. All participants performed at least 150 min of structured moderate intensity aerobic exercise per week in their free time. Individuals who did not meet these criteria were excluded from the study. Participants were also non-smoking, had a body mass index (BMI) <34 kg/mn = 13) and the Bruno Balke Biodynamics Laboratory at the University of Wisconsin–Madison (n = 30). A mix of young and older participants were studied at each site. Study protocols were identical at the two sites. The tests were conducted in controlled ambient temperature between 22 and 24°C. Central arterial stiffness measurements and stepped hypercapnia trials were performed on a separate study visit from the O2max test. Prior to the study visits, participants were asked to fast overnight, to abstain from non-steroidal anti-inflammatory drugs (NSAIDs) for 5 days, and to abstain from caffeine, exercise, and alcohol for 24 h. Additionally, participants did not take any over the counter medications, vitamins or supplements on the day of the study visit. Upon arrival, height and weight were measured using a standard scale. BMI was calculated as kg/m2.Experimental study days were completed at Mayo Clinic (O2max) was performed on an incremental cycle ergometer test to exhaustion . During the exercise test, ventilation and gas exchange variables were measured utilizing a metabolic cart . Participants maintained a self-selected pace. After a 1 min warm up, the workload increased by 20–40 W  each minute until acceptable test criteria was met. The criteria for an acceptable test was failure to increase VO2 with an increasing workload, a respiratory exchange ratio (RER) of over 1.10, and a maximum heart rate (HR) value within 10 bpm of the age predicted maximum (220 – age). All participants met the criteria for an acceptable O2max test.Habitual aerobic exercise participation was assessed with a 7 day exercise log and the GODIN questionnaire. The GODIN questionnaire is a 4-item questionnaire that describes the number of times during a typical week one engages in mild, moderate, and strenuous leisure-time physical activity, as detailed in 2) . Breath-by-breath end-tidal carbon dioxide (ETCO2) was continually measured and recorded . A non-invasive finger blood pressure cuff placed around the middle finger continually measured and recorded beat-by-beat MAP . A height-correcting unit was used in order to account for any differences between the height of the finger and the height of the heart.After 10 min of supine rest, baseline MAP was taken in triplicate with a non-invasive brachial blood pressure cuff in the supine position . The average of the three measurements is reported. Participants were instrumented with a three-lead electrocardiogram to continually monitor HR and a pulse oximeter to monitor oxygen saturation  measurements were completed utilizing arterial tonometry as previously reported . BrieflyParticipants were imaged using a 2 MHz Transcranial Doppler (TCD) probe  to estimate right middle cerebral artery velocity (MCAv) . The 2 M2, 21% oxygen, and balanced nitrogen were administered. ETCO2 was elevated and maintained constant for 3 min at each level of inspired CO2.Stepped hypercapnia trials were performed as previously described using a steady-state, open-circuit technique , 2013. B2 during stepped hypercapnia. In order to account for changes in perfusion pressure that may affect flow, cerebrovascular conductance index (CVCi) was calculated as MCAv/MAP and also related to changes in ETCO2 during hypercapnia. MAP reactivity was calculated as the linear relationship between MAP and ETCO2. Cerebrovascular reactivity and MAP reactivity slopes were compared between young and older adults using a one-way ANOVA. A two-way repeated measures ANOVA compared the cerebral hemodynamic and gas exchange variables during stepped hypercapnia followed by the Holm-Sidak method to test pairwise comparisons. The associations between central arterial stiffness and cerebrovascular reactivity were evaluated utilizing a Spearman rank order correlation in separate groups (young and older participants). In a secondary analysis, we employed multivariable linear regression with MCAv as the dependent variable and MAP and ETCO2 as the independent variables in separate groups (young and older adults) in order to determine the contribution of the linear combination of MAP and ETCO2 to MCAv. Statistical significance was set a priori at p < 0.05. In our previous study, it was found that CVCi reactivity was statistically different between young (n = 12) and older (n = 10) adults  (n = 16 subjects (8 per group) would provide a statistical power of 80% to detect a difference at α = 0.05. However, because we included the potential association between central arterial stiffness and cerebrovascular reactivity, we recruited at least 20 subjects per group.Data were collected at 250 Hz and analyzed off-line using signal processing software . Participant demographics and baseline characteristics were compared using a one-way ANOVA. Beat-by-beat hemodynamic measurements were averaged over the final minute of room air breathing and at each level of hypercapnia. Cerebrovascular reactivity was quantified as the linear relationship between MCAv and ETCO < 0.05) . Based oTable 1. Young and older adults had a similar BMI and supine systolic blood pressures at baseline, yet older adults had higher supine diastolic blood pressures and MAP when compared with young adults. Additionally, older adults demonstrated higher carotid–femoral PWV indicating higher central arterial stiffness. The coefficient of variation for PWV trials was 9%. Young and older adults had similar aerobic exercise participation based on the GODIN questionnaire and MET minutes per week calculated from the 7-day recall of exercise (Table 1).Participant characteristics are displayed in Figure 1A. SpO2 was 99% during room air inhalation and did not change during the protocol or differ between groups (data not shown). MCAv was not different between groups at any level; however, older adults demonstrated lower CVCi at room air and during each level of stepped hypercapnia . Older adults also demonstrated higher MAP than young adults at 4 and 6% CO2 .Cerebral hemodynamic and gas exchange variables during the stepped hypercapnia protocol are displayed in Figure 1B. MCAv and CVCi reactivity to stepped hypercapnia were not different between young and older adults; however, older adults demonstrated a significantly higher MAP reactivity compared to young adults . Cerebrovascular reactivity did not differ by study site (data not shown).Cerebrovascular reactivity to stepped hypercapnia is displayed in Table 2). There was a trend for a positive correlation between carotid–femoral PWV and MAP reactivity in young adults such that young adults with higher central arterial stiffness demonstrated a greater MAP response to hypercapnia (p = 0.07). No such association was present in older adults (Table 2).When examining the association between central arterial stiffness and cerebrovascular reactivity, there were no correlations in young or older adults (2 was a significant predictor of MCAv in young adults (p < 0.001). In older adults, both MAP and ETCO2 were significant predictors of MCAv (p < 0.001).In our multivariable linear regression analysis, only ETCOThe most salient finding of the present study is that there were no age-associated differences in MCAv or CVCi reactivity to hypercapnia in healthy adults who habitually exercise. Importantly, despite no differences in MCAv responses to hypercapnia, MAP reactivity was augmented with aging. This suggests that healthy older adults may rely on a different mechanism  to sufficiently increase CBF during a vasodilatory stimulus such as hypercapnia. Additionally, we report that central arterial stiffness was elevated in healthy older adults who habitually exercise compared to young adults. Contrary to our hypothesis, central arterial stiffness was not associated with measures of cerebrovascular reactivity or MAP reactivity. This finding suggests that central arterial stiffness is not associated with cerebral vasodilatory responses in healthy habitual exercisers.2 inhalation.This study used a cross-sectional approach to determine the effects of primary aging on cerebrovascular reactivity and its association with central arterial stiffness. It addressed many limitations of previous studies by: (1) including participants with low cardiovascular risk and excluding individuals taking vasoactive medications, (2) recruiting young and older adults with similar BMI, systolic blood pressure, and habitual exercise habits, and (3) including approximately equal distribution of men and women. In addition, this study used standard techniques to non-invasively quantify central arterial stiffness at rest and characterize cerebrovascular reactivity and beat-by-beat MAP responses during COThe present finding that cerebrovascular reactivity is preserved with age conflicts our previous work in healthy sedentary and recreationally active adults . It is p2 threshold  healthy older adults may rely on a different mechanism  to sufficiently increase CBF during a vasodilatory stimulus such as hypercapnia; and (2) in healthy habitual exercisers, central arterial stiffness is not associated with cerebral vasodilatory responses. Our findings provide one potential explanation as to why habitual exercise is protective against certain cerebral pathologies and cognitive decline. Future studies should evaluate the mechanisms responsible for age-related differences in cerebral vasodilatory function, the effect of habitual aerobic exercise, and cerebrovascular reactivity in the context of disease etiology.KM and JB designed the experiments, analyzed and interpreted the data. KM, AH, RH, ME, and JB performed the data collection. KM wrote the initial draft of the manuscript. All authors edited and approved the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


G12D. Arid1a cooperates with Kras in the adult pancreas as postnatal silencing of Arid1a following sustained KrasG12D expression induces rapid and irreversible reprogramming of acinar cells into mucinous PDAC precursor lesions. In contrast, Arid1a silencing during embryogenesis, concurrent with KrasG12D activation, leads to retention of acinar cell fate. Together, our results demonstrate Arid1a as a critical modulator of Kras-dependent changes in acinar cell identity, and underscore an unanticipated influence of timing and genetic context on the effects of SWI/SNF complex alterations in epithelial tumorigenesis.Mutations in members of the SWI/SNF chromatin remodeling family are common events in cancer, but the mechanisms whereby disruption of SWI/SNF components alters tumorigenesis remain poorly understood. To model the effect of loss of function mutations in the SWI/SNF subunit Arid1a in pancreatic ductal adenocarcinoma (PDAC) initiation, we directed shRNA triggered, inducible and reversible suppression of Arid1a to the mouse pancreas in the setting of oncogenic Kras The pancreas produces many different hormones, as well as several substances important for digestion. To perform these roles, the pancreas contains different types of cells; for example, acinar cells make digestive enzymes that help to break down food. But, like other cells in the body, pancreatic cells can accumulate mutations in their DNA that cause them to divide, acquire an altered identity and form a cancerous tumor.The DNA of cells is packed into a structure called chromatin. While the DNA sequence is essentially the same across all normal cells of a given individual, chromatin can be more or less compacted in the different cell types that comprise our body tissues. A collection of proteins called the SWI/SNF complex can reorganize the chromatin to change how tightly the DNA is packed. This determines which genes in the DNA are accessible and can be activated, and which ones cannot.Around 25% of pancreatic cancers contain mutations in genes that produce proteins of the SWI/SNF complex. These mutations normally occur with an additional mutation that over-activates the gene that produces a potentially cancer-causing protein called Kras. Livshits et al. have now genetically engineered mice to investigate how one such SWI/SNF complex protein, called Arid1a, affects how pancreatic cancer develops using a genetic approach that made possible to temporarily halt the production of Arid1a in acinar cells by feeding these mice an antibiotic. The gene that produces Kras was also over-activated in the pancreases of the mice, making them more likely to develop cancer.Within just two weeks of stopping the production of Arid1a, the acinar cells stopped producing digestive enzymes and started making other proteins that are typically found in cancerous cells, indicating that Arid1a is involved in maintaining the normal identity and activity of these cells. Restoring the ability of altered acinar cells to produce normal levels of Arid1a (by removing the mice from the antibiotic diet) did not reverse these changes. Biochemical experiments showed that acinar cells with reduced levels of Arid1a have altered chromatin. In particular, the genes that produce digestive enzymes, which are normally active in healthy pancreases, were less accessible in mice who had over-active Kras and reduced levels of Arid1a.The results presented by Livshits et al. provide the first evidence of how alterations to Arid1a can lead to irreversible changes in the identity and activity of pancreatic acinar cells. These results will need to be carefully considered by researchers who are developing treatments for cancer patients with mutations in Arid1a and other SWI/SNF proteins. In particular, methods that attempt to restore the functions of absent SWI/SNF proteins to cancer cells are unlikely to treat the cancer successfully. Aberrant chromatin regulation is a hallmark of cancer . NumerouSWI/SNF complexes regulate chromatin accessibility and thus gene expression by re-positioning nucleosomes and interacting with available transcription factors (TFs) at enhancer regions . While tAs the SWI/SNF complex dictates chromatin accessibility by available TFs, such context dependencies might broadly relate to development or pathological cell state. Indeed, in drosophila and mammalian systems, SWI/SNF complex dependent regulation of gene expression controls cell identity and differentiation in developmental as well as regenerative contexts . How losARID1A or other SWI/SNF components occur in up to 25% of cancer patients , a setting in which mutations in patients . PDAC isoncogene , while aoncogene . PDAC cac stages .Ptf1a, Nr5a2 and Pdx1, when combined with oncogenic Kras, accelerates ADM and PanIN development  technology with embryonic stem (ES) cell genetic engineering (G12D (Col1a1 homing cassette that enables insertion of a single copy of a construct into the Col1a1 locus via recombinase-mediated cassette exchange (RMCE)  . The Ptfe (RMCE) . Thus ine (RMCE) .Col1A1 targeting cassette downstream of a tetracycline-response element (TRE) driven promoter; this configuration enables inducible and reversible expression of a downstream transgene in cells expressing the rtTA and supplied with the tetracycline analog doxycycline (dox)  . KC-RIK Kras (after completion of pancreas development (We placed KC-RIK-shArid1a mice on a doxycycline (dox) diet at five weeks of age. This enabled us to suppress Arid1a in the presence of existing oncogenic Kras  but afteelopment , mimickielopment . KC-RIK-elopment . Consistelopment .Surprisingly, a substantial portion of shArid1a mice became moribund within two weeks of enrollment on dox chow, while shRen mice remained healthy . Acute mKras mutation rapidly perturbs the identity and function of pancreatic acinar cells, enhancing their sensitivity to metaplastic and PanIN-inducing signals stemming from oncogenic Kras.The phenotypes described above were on target, as the same changes in histology and cell fate markers were observed in mice carrying two distinct Arid1a shRNAs . FurtherG12D allele from ES cell-derived KC-RIK-shArid1a and KC-RIK-shRen mice though one round of breeding. These C-RIK-shRNA mice were then placed on dox at five weeks of age, after the completion of pancreas development, as in the KC-RIK experiments. In contrast to settings involving oncogenic Kras, these mice did not become moribund following dox administration, and pancreata from both shArid1a and shRen mice appeared normal  or proliferation (Ki67) were noted  and a concomitant decrease in expression of markers of acinar differentiation (Cpa1) . ImportaG12D, CAGS-LSL-RIK, and TRE-shRNA alleles are activated upon Cre expression, leading to concomitant activation of oncogenic Kras and rtTA, allowing simultaneous silencing of Arid1a in the embryos of pregnant mice treated with dox  whereas, when occurring concomitant with KrasG12D activation, it paradoxically promotes retention of the acinar cell phenotype (E7 cohort). Taken together, these data reveal a temporal, context-dependent role for Arid1a in regulating acinar identity in response to oncogenic Kras.In both E7 and P5 cohorts, mKate2 positive pancreas tissue was readily observed in highly chimeric on dox mice at 7 weeks of age, indicating that Arid1a silencing during pancreas morphogenesis does not in and of itself impair pancreatic cell fitness . Howeverr tissue , a featur tissue , the retr tissue . Of noter tissue . Thus, AIn addition to uncoupling the timing of oncogene activation and tumor suppressor gene loss, our system is reversible, and as such enables assessment of whether ongoing gene suppression is needed to sustain pathological phenotypes. To test the stability of the acinar-to-mucinous reprogramming triggered by Arid1a depletion, we placed cohorts of KC-RIK-shRen and KC-RIK-shArid1a mice on dox at 5 weeks of age for two weeks to establish the acute Arid1a phenotype. Mice were then withdrawn from doxycycline to restore Arid1a expression .non-reversible reprogramming of cell fate that is dependent on cellular and genetic context.Remarkably, Arid1a restoration did not reverse the acinar reprogramming phenotype. As expected, pancreata from mice withdrawn from dox retained expression of the mKate2 lineage marker, but lost the shRNA-linked GFP fluorescence signal . Stainin+GFP+ epithelial cells sorted from 3 KC-RIK-shRen, or shArid1a and mice after 5 days of dox treatment. This timepoint occurs after maximal Arid1a suppression but before any observed histological changes, thus reducing the confounding effect of secondary changes in gene expression resulting from tissue inflammation (We next sought to understand the mechanism(s) whereby Arid1a regulates pancreas cell identity. Perturbation of the SWI/SNF chromatin-remodeling complex is expected to impact the expression of SWI/SNF target genes . In ordeammation . Principammation . Unsuperammation .Further analysis of the RNAseq data using an FDR threshold of 0.05 detected 711 downregulated and 505 upregulated genes . Among tIn parallel with mucin upregulation characteristic of PanIN development in KC-RIK-shArid1a mice, GSEA identified downregulation of genes associated with acinar function, such as those encoding the digestive enzymes Cel, Klk1 and Amy1 , which w+GFP+ cells sorted from KC-RIK-shRen and shArid1a mice after 5 days on dox, as in our transcriptional profiling experiments. ATAC-seq assesses chromatin accessibility genome-wide, and therefore provides a direct readout of the chromatin remodeling activity of SWI/SNF complexes. Importantly, ATAC-seq requires substantially fewer cells than current ChIP-seq protocols and, as such, is appropriate for analyzing chromatin states in the limited cell numbers that can be recovered from the preneoplastic murine pancreas.Building on recent observations , we hypoWe found that knockdown of Arid1a resulted in striking changes in chromatin accessibility, with shArid1a chromatin profiles that clearly separated from shRen by PCA . 50,000–To define the TFs that might be responsible for these changes, we searched for transcription factor motifs enriched in regions genomic regions showing reduced accessibility following Arid1a suppression using the HOMER suite of tools for motif discovery . SeveralCela2a). Increased chromatin closing observed upon Arid1a knockdown at these TF target sites was associated with reduced gene expression at adjacent genes. As shown in To further connect these chromatin changes with the transcriptional repression of the acinar identity genes identified above , we perfKras mutations accelerates acinar to ductal reprogramming and specification of mucinous, pancreatic cancer precursor lesions that cannot be reversed by restoring endogenous Arid1a expression. Thus, our data suggest that Arid1a can provide a potent barrier to Kras driven acinar to ductal metaplasia and, consequently, early stage pancreatic neoplasia. Once disrupted by Arid1a mutation, these altered cells are reprogrammed in such a manner that Arid1a is no longer needed to sustain the aberrant cell fate.Mutations in chromatin remodelers are prevalent across the human cancer spectrum, making identification of tissue specific relationships between chromatin regulation and tumorigenesis an important goal in understanding neoplastic progression. In this study, we used a rapid and robust mouse-modeling platform to study the consequences of inducible and reversible suppression of the Arid1a SWI/SNF subunit in a mouse model of pancreatic cancer. Suppression of Arid1a in adult acinar cells harboring oncogenic Kras mutation is characterized by reduced chromatin accessibility accompanied by loss of acinar morphology, reduced expression of acinar genes, and upregulation of a signature characteristic of mucinous lesions. These features imply that genomic areas rendered accessible by Arid1a harbor elements involved in preventing acinar to duct like trans-differentiation in the context of mutant Kras, a critical rate-limiting step in Kras driven pancreatic neoplasia. Indeed, our transcriptional and chromatin profiling data imply that Arid1a disruption, in the presence of oncogenic Kras, reduces the chromatin accessibility of key TFs to the enhancers of genes needed to sustain acinar cell fate. At least one such factor, Nr5a2, can physically interact with the SWI/SNF complex via the Baf60 subunit  that co-occur with KRAS and ARID1A can then contribute to further PanIN progression. While studies to explore the role of ARID1A in later stage PDAC progression are in progress, the irreversibility of the Kras-induced PanIN phenotype indicates that impaired SWI/SNF function may not be required for tumor maintenance. If confirmed, the identification of genotype-specific dependencies produced by SWI/SNF dysfunction rather than restoration of its normal activity may be a more effective way to exploit SWI/SNF mutations for new therapeutic approaches. While some proposed vulnerabilities, such as ARID1B, extend across multiple tissues and mutational landscapes  was changed twice weekly. Chimeric mouse cohorts were generated through the Mouse Genetics core and Center for Pancreatic Cancer Research (CPCR) at MSKCC. For caerulein experiments, mice were given eight hourly intraperitoneal injections of PBS or 50 μg/kg caerulein one day apart. shRNA and genotyping primer sequences shRNAs targeting the following sequences were cloned into mir30-based targeting constructs as described previously . shRen: Col1a1 primer paired with a transgene specific primer:Genotyping of chimeric mice and their progeny was performed as previously described using a common Common: TTCAGACAGTGACTCTTCTGC shRen.713: GTATAGATAAGCATTATAATTCCTA shArid1a.6421: TATACTATACAACTTCTCCAGGCG shArid1a.1803: TATGAATCTTGAGAAAGCTCCTGTAB_300798 and Cell Signaling Technology Cat# 2956S, RRID:AB_1196615), mKate2 , Arid1a , Ki67 (BD BD55609), Cpa1 (R and D AF2765), Clusterin , Sox9 , CK19 Troma III , Cleaved-Caspase 3 , CK8 , E-cadherin (BD BD610181). For immunofluorescence, the following secondary antibodies were used: donkey-anti-chicken CF488 , goat-anti-chicken AF488 and AF647 , donkey anti-rabbit AF594 , goat anti-rabbit AF594 (Life Technologies A11037). Slides were counterstained with DAPI and mounted in ProLong Gold (Life Technologies). For Arid1a, pERK and Mesothelin immunohistochemistry, Vector ImmPress HRP kits were used for secondary detection. Tissues were counterstained with haematoxylin or Alcian blue, dehydrated and mounted with Permount (Fisher). Images were acquired on a Zeiss AxioImager microscope using Axiovision software. CD45 Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with heat mediated antigen retrieval using citrate buffer. Antibodies used were rat monoclonal CD45 primary antibody (Abcam 25386). Rabbit anti rat  was used as secondary and Bond Polymer Refine Detection (Leica Biosystems) was used according to manufacturer’s protocol. Sections were then counterstained with hematoxycilin, dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems). Quantification of GFP, Cpa1, Ki67 and c-Casp3 was performed using FIJI on sections from >3 animals per condition. For histological analysis of mucinous lesions, PanINs were classified and graded by a veterinary pathologist blinded to genotype, using established criteria , Arid1a , Cpa1 (R and D AF2765), Amylase (Sigma A8273) and Actin-HRP (Sigma A3854). Blots were developed with SuperSignal West Femto substrate (ThermoFisher).2+ and Ca2+ supplemented with 0.1 mg/ml DNase I  and 0.1 mg/ml Soybean Trypsin Inhibitor (Sigma T9003-250mg) for 30 min while rotating at 37°C. After washing with PBS, samples were further digested with 0.05% Trypsin-EDTA at 37°C. Trypsin digestion was neutralized with FACS buffer (10 mM EGTA and 2% FBS in Ca2+ Mg2+in PBS) containing STI. After an additional wash, samples were subsequently digested with 2 U/ml dispase (Roche) in PBS containing DNase I and STI for 20 min at 37°C. Samples were then washed in FACS buffer containing DNase I and STI, filtered through a 40 μm strainer and subjected to FACS. Samples were collected in FACS buffer with an additional 10% FBS for ATAC-seq or sorted directly into TRIzolLS for RNAseq. Each sample was used for either RNAseq or ATAC-seq.Pancreata from KC-RIK-shRNA mice were chopped with scissors, finely minced with a blade and dissociated with digestion buffer: 1 mg/ml Collagenase V dissolved in HBSS with Mghttp://software.broadinstitute.org/gsea/msigdb) and published expression signatures in organoid and mouse models . Sequencing and library preparation was performed at the Integrated Genomics Operation (IGO) at MSKCC. Ribosomal RNA was depleted using RiboZero (Illumina) and stranded KAPA RNA libraries were prepared from 1 ug RNA. Approximately 40 million paired-end 50 bp reads were sequenced per replicate on a HiSeq 2000. Resulting RNA-Seq data was analyzed by removing adaptor sequences using Trimmomatic . Reads we models .Total RNA was isolated using the TRIZOL (Thermo Fisher Scientific), and cDNA was obtained from 500 ng of RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) after treatment with DNAse I (Invitrogen) and according to manufacturer’s instructions. The following gene-specific primer sets for mouse sequences were used: Arid1a_F TCCCAGCAAACTGCCTATTC, Arid1a_R CATATCTTCTTGCCCTCCCTTAC, Agr2_F ACAACTGACAAGCACCTTTCTC, Agr2_R GTTTGAGTATCGTCCAGTGATGT, Klk1_F GCCCTGGCAAGTGGCTGTGT, Klk1_R AAGCCGGTGTTGGGCAGAGG, Msl1_F CCCATCGAAGTGGTCAGTCTC, Msln_R GGTGTATGACGGTCAGCTTAGA, Cel_F AAGTTGCCCGTGAAAAAGCAG, Cel_R ATGGTAGCAAATAGGTGGCCG, Amy1_F TCACACGGGTGATGTCAAGTT, Amy1_R GTCTGGGTTAATGCTCACTTCTT, Hprt_F TCAGTCAACGGGGGACATAAA and Hprt_R GGGGCTGTACTGCTTAACCAG. qRT-PCR was carried out in triplicate (5 cDNA ng/reaction) using SYBR Green PCR Master Mix (Applied Biosystems) on the ViiA 7 Real-Time PCR System (Life technologies). Hprt served as endogenous normalization control.+ cells isolated by FACS were lysed in 50 ul lysis buffer (http://broadinstitute.github.io/picard/). Density profiles for genome browser tracks were created using the BEDTools suite (http://bedtools.readthedocs.io). Enriched regions were discovered using MACS2 and scored against input sequence . Dynamic regions between two conditions were discovered using a similar method, with the second ATAC library replacing input. Peaks were then filtered against genomic ‘blacklisted’ regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/mm9-mouse/mm9-blacklist.bed.gz) and those within 500 bp were merged. All genome browser tracks and read density tables were normalized to a sequencing depth of ten million mapped reads. K-means clustering was performed on all dynamic peaks, with the optimal k determined by silhouette analysis. Dynamic peaks were annotated using linear genomic distance and motif signatures were obtained using the ‘de novo’ approach with Homer v4.5 (http://homer.ucsd.edu/). For analyses of TF density, transcription factor ChIP-seq enriched regions were discovered by comparison with matched input using MACS2, as with the ATAC-seq data. TF peaks overlapping sites decreasing in accessibility were classified using 'bedtools intersect' with an overlap of 1 bp. Gene density of these peaks was calculated using RefSeq annotated gene boundaries ± 30 kb of gene start or end, and these gene names were then matched with either differentially expressed genes, or genes with expression not changing by more than 10%. ATACseq data have been deposited to GEO under accession GSE114567.50,000 mKate2s buffer  and subj In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Fiona Watt as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Arid1a restrains Kras-dependent changes in acinar cell identity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In their manuscript, Livshits et al. investigate the role of the chromatin remodeling protein Arid1a in pancreatic carcinogenesis and acinar cell identity. To study the consequences of Arid1a-deficieny on acinar fate in the presence and absence of oncogenic Kras activation the authors utilize a very elegant ES cell RNAi based mouse model that facilitates inducible and reversible targeting of Arid1a independent on the Cre-mediated activation of oncogenic Kras during embryogenesis. In addition to phenotypic analyses using the described mouse model system the manuscript provides first mechanistic insights into pancreatic carcinogenesis in the context of Arid1a deficiency using RNA-Seq and ATAC-Seq analysis. Together, the authors demonstrate that Arid1a can function as a barrier to Kras-driven pancreatic cancer initiation, albeit the impact of Arid1a-deficiency underlies a significant context-dependency. The manuscript significantly extends our understanding of the involvement of chromatin remodeling processes in general and the function of Arid1A in particular in pancreatic cancer development. All together the manuscript is perfectly written and follows a logical order and the conclusions drawn from the data are largely justified.1) Please provide more information about the lesions in the mice. The authors state that the ductal lesions found in the Arid1a KD mice can be described as PanIN or IPMN-lesions. In Figure 2D only PanINs have been quantified. How have the lesions been distinguished from IPMNs so that only PanINs were counted? Do the Arid1a KD mice give rise to both, PanINs and IPMN? As BRG1-deficiency in the context of oncogenic Kras activation in the pancreas results to development of IMPN lesions , formation of IPMN might be a likely phenotype in Arid1A kd mice. As the severity of PanINs can be more important than their total numbers, it is important to illustrate the PanIN quantification in Figure 2D for the different PanIN stages separately. Please state how many mice have been observed until an age of app. 1 year ? Was there a significant number of mice to justify the authors´ statement that Arid1a kd animals do not progress to PDAC? Can the authors show that Arid1a is still absent after one year of Dox treatment? How exactly have those lesions been quantified? Counting the lesions per field might lead to misleading numbers, if the Arid1 kd pancreata suffer from pancreatic atrophy. How is the number of ADMs normalized to pancreatic size?Figure 3J: Arid1a kd and control mice are challenged with caerulein to induce pancreatitis. The authors state that they observe comparable ADM formation and pancreatic inflammation in both genotypes. Could the authors please quantify the inflammatory response  to objective the statement of comparable inflammation? In the same line: is the caerulein-induced damage comparable in both models? If blood samples from the mice were available, lipase and amylase serum levels could be assessed. Comparable damage in both models is important to justify the statement of equal regenerative potential in both models.2) Please provide more information about the transcriptional analysis. The unsupervised hierarchical clustering depicted in Figure 6B shows 3 samples for each Arid1a shRNA. In contrast, the PCA in Figure 6A only shows two dots for A1A.1803. Is the dot hidden behind another one or have the authors excluded one sample? If so, what has been the rationale for excluding the sample? In the same line: in ATAC-Seq, there are four replicates for shRen, but 3 in shArid1A. Why? Has a shArid1A sample been excluded? The transcriptional analysis in cell extracts from KC-Ren and KC-shArid1a cells reveals genes and signatures associated with carcinogenesis and acinar cell fate. Could the authors please validate the distinct expression of some of these genes in their isolated cells and in the tissue of the mice? Have RNA-Seq and ATAC-Seq been performed using cells from identical animals?3) Please solidify the analysis of transcriptional programs and chromatin accessibility by demonstrating clearer relationships between the two. For example, the text states "gene sets comprising the targets of these transcription factors were enriched among the downregulated genes in GSEA analysis of transcriptional profiles derived from shArid1a animals ." However, Figure 7H only shows that genes whose chromatin accessibility decreased showed reduced expression. The authors should analyze the data to correlate expression and accessibility at target sites of key acinar transcription factor binding sites, and separately determine the accessibility at genes whose expression changes in shArid1a. […] 1) Please provide more information about the lesions in the mice. The authors state that the ductal lesions found in the Arid1a KD mice can be described as PanIN or IPMN-lesions. In Figure 2D only PanINs have been quantified. How have the lesions been distinguished from IPMNs so that only PanINs were counted? Do the Arid1a KD mice give rise to both, PanINs and IPMN? As BRG1-deficiency in the context of oncogenic Kras activation in the pancreas results to development of IMPN lesions , formation of IPMN might be a likely phenotype in Arid1A kd mice. As the severity of PanINs can be more important than their total numbers, it is important to illustrate the PanIN quantification in Figure 2D for the different PanIN stages separately.G12D”, second paragraph). Regarding PanIN grades, histological analysis 2 weeks after shArid1a inhibition in this adult cohort reveals the majority of lesions are consistent with PanIN-1, though a small number of PanIN-2 lesions were present as well. No PanIN-3 lesions were observed. Similarly, few IPMNs were identified. These data have been included in new Figure 2D. The differences between Arid1a KD and Brg1-deficiency mouse models are discussed in Results subsection “Temporal context dictates the outcome of Arid1a depletion” (first paragraph), and in the fourth and fifth paragraphs of the Discussion.We now quantify and classify ductal lesions as ADM, PanIN , or IPMN, confirming that Arid1a suppression in adult pancreatic cells following expression of mutant Kras during embryonic development predominantly drives development of low grade PanIN lesions ? Was there a significant number of mice to justify the authors´ statement that Arid1a kd animals do not progress to PDAC? Can the authors show that Arid1a is still absent after one year of Dox treatment?G12D”, last paragraph). This supports a role for Arid1a suppression constraining Kras driven preneoplastic development rather than malignant progression as addressed in the original and revised manuscript.A substantial portion of the 66 KC-shArid1a mice placed on dox as adults became morbid and required euthanasia prior to 1 year of age in the absence of PDAC, likely due to the exocrine dysfunction as described in the text. 11 shArid1a mice were analyzed at >1 year of age; of these, one had progressed to cancer, while the rest retained mucinous lesions as pictured. We have confirmed the stability of Arid1a knockdown in mucinous PanIN lesions in this aged cohort .This frequency of PDAC development after a year is consistent with historically reported rates in mouse models where mutant Kras is expressed in the developing pancreas in the absence of genetic modification of other tumor suppressors (subsection “Acute Arid1a knockdown induces rapid changes in adult acinar cell fate in the setting of KrasHow exactly have those lesions been quantified?Quantification was performed by a veterinary pathologist experienced in murine pancreas histology (J. Wilkinson) blinded to genotype. To clarify our methods to the reader, we added a supplementary figure  showing our scoring criteria and examples of the quantified lesions , and extended our Materials and method’s description.Counting the lesions per field might lead to misleading numbers, if the Arid1 kd pancreata suffer from pancreatic atrophy. How is the number of ADMs normalized to pancreatic size?increased pancreas:body weight ratio compared to controls at the time when mice were analyzed, likely due to the stromal expansion that accompanies preneoplastic initiation  . As suggested, we also updated the graphs that reported “lesion per field” to express lesions relative to mm2 of tissue area analyzed .While we agree that counting lesions per field can be misleading in the setting of pancreatic atrophy, shArid1a mice displayed a slightly Figure 3J: Arid1a kd and control mice are challenged with caerulein to induce pancreatitis. The authors state that they observe comparable ADM formation and pancreatic inflammation in both genotypes. Could the authors please quantify the inflammatory response  to objective the statement of comparable inflammation? In the same line: is the caerulein-induced damage comparable in both models? If blood samples from the mice were available, lipase and amylase serum levels could be assessed. Comparable damage in both models is important to justify the statement of equal regenerative potential in both models.To exclude the possibility that genotype specific differences in acute pancreatic damage in response to caerulein prevents accurate evaluation of the regenerative capacity of shArid1a expressing pancreatic cells, we have more fully characterized the time course of pancreatic inflammation, exocrine stress, and exocrine regeneration in shArid1a and shRen expressing mice. Scoring inflammation based on histological analysis and CD45 immunohistochemistry reveals similar levels of inflammation at D2 post caerulein in both shArid1a and shRen expressing mice, as well as comparable resolution at D9. .While no blood samples were available to measure serum pancreatic enzyme levels as proxy for pancreatic damage, we compared molecular markers of acinar stress and acinar differentiation after caerulein treatment. As shown in new Figure 3—figure supplement 2C, caerulein treatment induced a comparable increase in clusterin, a well-established anti-apoptotic gene that is transiently expressed in damaged acini following pancreatic damage and downregulated upon regeneration , in both genotypes at D2 that is markedly reduced upon regeneration (D9). Furthermore, we now show an inverse expression pattern of the differentiated acinar marker Cpa1 in both shRen and shArid1a acinar cells undergoing injury-induced regeneration . Importantly, as shown in Figure 3—figure supplement 3B, Arid1a levels remained suppressed at all stages of response and regeneration following acute pancreatitis, thus confirming the absence of counter selection against Arid1a suppressed cells and expansion of Arid1a expressing cells. These results provide strong evidence that Arid1a suppression does not alter the damage:regeneration capacity of adult acinar cells and that Arid1a is dispensable for restoring acinar fate after injury. These issues are now detailed fully in the results .2) Please provide more information about the transcriptional analysis. The unsupervised hierarchical clustering depicted in Figure 6B shows 3 samples for each Arid1a shRNA. In contrast, the PCA in Figure 6A only shows two dots for A1A.1803. Is the dot hidden behind another one or have the authors excluded one sample? If so, what has been the rationale for excluding the sample?There are 3 dots for A1A.1803 in the PCA plot in Figure 6A, with one inadvertently cropped during composition of the figure. We updated Figure 6A to correct this error. Importantly, the results are not affected by this correction.In the same line: in ATAC-Seq, there are four replicates for shRen, but 3 in shArid1A. Why? Has a shArid1A sample been excluded?No samples were excluded – we were only able to generate ATAC libraries of sufficient quality from 3 shArid1a and 4 control animals and all available results are presented.The transcriptional analysis in cell extracts from KC-Ren and KC-shArid1a cells reveals genes and signatures associated with carcinogenesis and acinar cell fate. Could the authors please validate the distinct expression of some of these genes in their isolated cells and in the tissue of the mice?We now include validation of the RNAseq data by qRT-PCR performed in RNA extracted from FACS-sorted shRNA expressing epithelial cells 5 days after dox administration in adult mice with histologically validated Arid1a knockdown . qRT-PCR analyses confirmed significant deregulation of the expression of both carcinogenesis-associated genes  and acinar identity genes  upon Arid1a knockdown . Moreover, histological validation of select up- and down-regulated genes at 2 weeks post dox treatment shows that early transcriptional changes induced by Arid1a suppression  are maintained in the resultant metaplastic pancreas .Have RNA-Seq and ATAC-Seq been performed using cells from identical animals?Technical challenges involved in dissociating epithelial cells from fibrotic pancreata undergoing Kras driven metaplasia precluded our ability to collect sufficient cells to perform both ATAC and RNA SEQ from the same animals. This is clarified in the revised Materials and methods section.3) Please solidify the analysis of transcriptional programs and chromatin accessibility by demonstrating clearer relationships between the two. For example, the text states "gene sets comprising the targets of these transcription factors were enriched among the downregulated genes in GSEA analysis of transcriptional profiles derived from shArid1a animals ." However, Figure 7H only shows that genes whose chromatin accessibility decreased showed reduced expression. The authors should analyze the data to correlate expression and accessibility at target sites of key acinar transcription factor binding sites, and separately determine the accessibility at genes whose expression changes in shArid1a.We performed a comprehensive analysis of gene expression and chromatin accessibility at targets of pancreatic TFs necessary for maintenance of acinar differentiation  and acinar regeneration (Pdx1) that previously demonstrated to constrain Kras driven preneoplastic development . We compared ATAC-SEQ profiles following Arid1a suppression to high confidence, experimentally-defined datasets of TF binding sites defined by ChIP-seq . This analysis showed a significant enrichment of TF binding sites at ATAC-seq peaks that lose accessibility upon Arid1a KD compared with sites not changing in accessibility . Composite plots of average ATAC-seq signal at these TF binding sites also demonstrated decreased accessibility in Arid1a KD compared with control . Importantly, increased chromatin closing observed upon Arid1a KD at these TF target sites is associated with reduced gene expression at target genes, now documented in two ways. First, by associating these TF-ATAC sites with genes by linear genomic distance and then using GSEA, we were able to find significant enrichment of these peaks in genes that become downregulated upon Arid1a KD as compared with control . Second, by computing the density of these TF-ATAC sites per gene, we found increased density in genes downregulated compared with genes whose expression is upregulated or unchanged . While the TF density was calculated per gene, we note that this is not simply an effect of underlying gene size discrepancies in the different expression categories . Lastly, we note that various intersections of ATAC-seq dynamics and TF binding sites failed to yield a correlation with genes that become upregulated upon Arid1a KD.


Listeria monocytogenes (Lm), generation of an individual isolate for sequencing typically takes about 6 days. Here we demonstrate that a hybrid, “quasimetagenomic” approach ie; direct sequencing of microbiological enrichments (first step in pathogen detection and recovery) can provide high resolution source tracking sequence data, 5 days earlier than response that focuses on culture and sequencing of an individual isolate. This expedited approach could save lives, prevent illnesses and potentially minimize unnecessary destruction of food.The more quickly bacterial pathogens responsible for foodborne illness outbreaks can be linked to a vehicle of transmission or a source, the more illnesses can be prevented. Whole genome sequencing (WGS) based approaches to source tracking have greatly increased the speed and resolution with which public health response can pinpoint the vehicle and source of outbreaks. Traditionally, WGS approaches have focused on the culture of an individual isolate before proceeding to DNA extraction and sequencing. For Listeria monocytogenes following protocols outlined in the Bacteriological Analytic Manual (BAM). DNA from enriching microbiota was extracted and sequenced at incremental time-points during the first 48 h of pre-enrichment using the Illumina MiSeq platform (2 by 250), to evaluate genomic coverage of target pathogen, Listeria monocytogenes.Naturally contaminated ice cream (from a 2015 outbreak) was enriched to recover Lm strain/s were part of the ongoing outbreak or not. Genomic data from hours 24, 28, 32, 36, 40, 44, and 48 of pre-enrichments all provided identical phylogenetic source tracking utility to the WGS of individual isolates .Quasimetagenomic sequence data acquired from hour 20 were sufficient to discern whether or not The speed of this approach (more than twice as fast as current methods) has the potential to reduce the number of illnesses associated with any given outbreak by as many as 75% percent of total cases and potentially with continued optimization of the entire chain of response, contribute to minimized food waste. Salmonella enterica [, Listeria monocytogenes [Escherichia coli [, and many others by facilitating the whole genome sequencing (WGS) of these small bacterial genomes in as little as 20h hours. Sequence data is organized using pipelines that generate matrices of single nucleotide polymorphisms (SNPs) that can be used with a variety of algorithms to infer phylogenetic relationships. This approach can differentiate highly clonal strains within serovars that differ by as few as 1 to 29 nucleotides [Lm from a food source, as described in the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) begins with 48 h of pre-enrichment, followed by incubation on selective agars (another 48 h), growth on nutrient agars (24 h) and then confirmation (3 to 24 h) of individually selected colonies [Next generation sequencing (NGS) technologies have revolutionized our ability to source track pathogens such as enterica , Listeriytogenes , Escherihia coli , and manleotides . GenomeTleotides . The bigcolonies . Time coLm genomic data with source tracking utility could be obtained from the quasimetagenomes. Because the word “metagenomics” refers to true culture independent (CI) genomics [To evaluate hybrid options for improving the speed of source tracking, we sequenced DNA from enrichment microbiomes every 4 hours) using short read (Illumina Miseq) sequencing. Using naturally contaminated ice cream previously linked to an outbreak of listeriosis that resulted in three deaths [ hours usgenomics , we use genomics  to descrLm recovery outlined in the BAM [Lm cells) were assayed at all time-points.Homogenized ice cream scoop samples were added to Buffered Listeria Enrichment Broth (BLEB) according to the specifications for  the BAM . Four reDNA was extracted using DNeasy Blood and Tissue kit (Qiagen) following the protocol for Gram-positive bacteria with minor modifications: 1.5 ml of the culture was pelleted  and the pellet resuspended in 200 μL of enzymatic lysis buffer containing 20 mM Tris-HCl (pH -8.0), 2 mM Sodium EDTA, 1.2% Triton X- 100, 20 mg/ml of lysozyme. The samples were incubated for 60 min at 37 °C.Libraries were prepared with Nextera XT (Illumina) according to the manufacturers specifications. For the first and second enrichments, approximately 16 libraries (independent replicates) were multiplexed per run  using Illumina MiSeq V2 (2 by 250).https://github.com/CFSAN-Biostatistics/snp-pipeline. Phylogenetic trees were created using the Genetic Algorithm for Rapid Likelihood Inference (GARLI) [WGS analyses were performed using the CFSAN SNP pipeline 0.6.1 , 10. Raw (GARLI) . TaxonomAll data is available at NCBI associated with BioProject PRJNA370011.Lm genome/s from ice-cream enrichments (Table Lm were part of the outbreak cluster or not. Determination of inclusion or exclusion after only 20 h (plus sequencing time) in cases where a reference genome is available facilitates a greatly expedited ability to remove contaminated commodities from the food supply and protect safe products from costly holds. High quality draft Lm genomes were achieved with data from h 24 through h 48 with coverage (12.74 to > 52.75x) -shown for hours 20–40 in Table Lm genomes from enrichments of all time-points from h 20 through h 48 were used with WGS genomes of environmental and clinical isolates from the full outbreak cluster (available at NCBI) , Anoxybacillus, and Geobacillus , and Lactococcus, Enterococcus and Streptococcus   play a role in inhibiting Listeria monocytogenes until it proliferates in h 20. This same phenomenon was also noted in previous work that focused on the description of the total ecology of enriching microbiota from naturally contaminated ice-cream using the three most commonly used enrichment protocols by; the International Organization for Standardization (ISO), the United States Department of Agriculture (USDA) and the FDA [As microbiota from the naturally contaminated ice cream grew in the FDA BAM delineated media (BLEB), the following predominant bacterial genera were observed; es) Fig. . Lm rema the FDA .Fig. 2RMetagenomic approaches have been accidentally and intentionally employed for decades to describe “black boxes” of human and environmental microbiota. These inquiries coupled with rapidly advancing NGS technologies have contributed to a renaissance in our understanding of numerous complex, previously poorly understood microbial ecologies. Focusing this approach on the “black box” of culture based pathogen recovery methods, many of which have not changed in 30+ years, provides novel insight to further expedite and optimize state of the art pathogen recovery methods. This applies both to the microbiological dynamics, ie; can we identify chemical, temperature or antibiotic inputs that would optimize recovery of targets by inhibiting competitors. Or, as presented here, by direct capture of genomic data of enriching targets from within the enrichment microbial community (quasimetagenome).E. coli O104: H4 linked to a 2011 outbreak that resulted in over 40 deaths [E. coli, Salmonella enterica and Listeria monocytogenes, when they occur as low abundant members of highly diverse ecologies, are still prohibitive for most operations. Thus, validation of the quasimetagenomic approach (culture and shotgun sequencing) presented here, offers a protocol that can be easily integrated into public health laboratories at no additional cost to current programs.A true CI metagenomic approach was used to successfully identify strains of Shigatoxigenic 0 deaths . This ponaturally contaminated food samples. With modeled (spiked in) experiments, it is difficult to understand if results represent “real life” contamination dynamics or not. Quasimetagenomic sequencing can be applied to all culturable pathogens from all types of matrices (food or other). Ultimately this approach might have incredibly significant utility in hospital situations for rapid identification of strains causing septicemia. Temporal parameters, enrichment schema, and quantity of data necessary for optimal genomic coverage will be different for every pathogen, substrate and matrix. As shown here for source tracking Listeria monocytogenes from ice-cream the qMGS approach provides the exact same answer as WGS in a timeframe that when leveraged efficiently, could potentially eliminate the majority of illnesses associated with many outbreaks and potentially reduce unnecessary destruction of food.The benefits achieved by the quasimetagenomic (qMGS) approach presented here, efficiently address previously described challenges of cost and speed, while maintaining diagnostic sensitivity. The source tracking phylogenetic success achieved by this experiment is particularly valuable because it validates the hybrid approach using Additional file 1. Annotation for phylogenetic trees of full and abbreviated NCBI 2015 listeriosis outbreak cluster linked to ice cream.