ClusterCAD 2.0: an updated computational platform for chimeric type I polyketide synthase and nonribosomal peptide synthetase design.

Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical 'starters' and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

Development of Corynebacterium glutamicum as a monoterpene production platform.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Delayed Surgical Intervention After Chemoradiotherapy in Esophageal Cancer: (DICE) Study.

OBJECTIVE: To determine the impact of delayed surgical intervention following chemoradiotherapy (CRT) on survival from esophageal cancer. BACKGROUND: CRT is a core component of multimodality treatment for locally advanced esophageal cancer. The timing of surgery following CRT may influence the probability of performing an oncological resection and the associated operative morbidity. METHODS: This was an international, multicenter, cohort study, including patients from 17 centers who received CRT followed by surgery between 2010 and 2020. In the main analysis, patients were divided into 4 groups based upon the interval between CRT and surgery (0-50, 51-100, 101-200, and >200 days) to assess the impact upon 90-day mortality and 5-year overall survival. Multivariable logistic and Cox regression provided hazard ratios (HRs) with 95% CIs adjusted for relevant patient, oncological, and pathologic confounding factors. RESULTS: A total of 2867 patients who underwent esophagectomy after CRT were included. After adjustment for relevant confounders, prolonged interval following CRT was associated with an increased 90-day mortality compared with 0 to 50 days (reference): 51 to 100 days (HR=1.54, 95% CI: 1.04-2.29), 101 to 200 days (HR=2.14, 95% CI: 1.37-3.35), and >200 days (HR=3.06, 95% CI: 1.64-5.69). Similarly, a poorer 5-year overall survival was also observed with prolonged interval following CRT compared with 0 to 50 days (reference): 101 to 200 days (HR=1.41, 95% CI: 1.17-1.70), and >200 days (HR=1.64, 95% CI: 1.24-2.17). CONCLUSIONS: Prolonged interval following CRT before esophagectomy is associated with increased 90-day mortality and poorer long-term survival. Further investigation is needed to understand the mechanism that underpins these adverse outcomes observed with a prolonged interval to surgery.

Hybrid Minimally Invasive Esophagectomy for Esophageal Cancer.

BACKGROUND: Postoperative complications, especially pulmonary complications, affect more than half the patients who undergo open esophagectomy for esophageal cancer. Whether hybrid minimally invasive esophagectomy results in lower morbidity than open esophagectomy is unclear. METHODS: We performed a multicenter, open-label, randomized, controlled trial involving patients 18 to 75 years of age with resectable cancer of the middle or lower third of the esophagus. Patients were randomly assigned to undergo transthoracic open esophagectomy (open procedure) or hybrid minimally invasive esophagectomy (hybrid procedure). Surgical quality assurance was implemented by the credentialing of surgeons, standardization of technique, and monitoring of performance. Hybrid surgery comprised a two-field abdominal-thoracic operation (also called an Ivor-Lewis procedure) with laparoscopic gastric mobilization and open right thoracotomy. The primary end point was intraoperative or postoperative complication of grade II or higher according to the Clavien-Dindo classification (indicating major complication leading to intervention) within 30 days. Analyses were done according to the intention-to-treat principle. RESULTS: From October 2009 through April 2012, we randomly assigned 103 patients to the hybrid-procedure group and 104 to the open-procedure group. A total of 312 serious adverse events were recorded in 110 patients. A total of 37 patients (36%) in the hybrid-procedure group had a major intraoperative or postoperative complication, as compared with 67 (64%) in the open-procedure group (odds ratio, 0.31; 95% confidence interval [CI], 0.18 to 0.55; P<0.001). A total of 18 of 102 patients (18%) in the hybrid-procedure group had a major pulmonary complication, as compared with 31 of 103 (30%) in the open-procedure group. At 3 years, overall survival was 67% (95% CI, 57 to 75) in the hybrid-procedure group, as compared with 55% (95% CI, 45 to 64) in the open-procedure group; disease-free survival was 57% (95% CI, 47 to 66) and 48% (95% CI, 38 to 57), respectively. CONCLUSIONS: We found that hybrid minimally invasive esophagectomy resulted in a lower incidence of intraoperative and postoperative major complications, specifically pulmonary complications, than open esophagectomy, without compromising overall and disease-free survival over a period of 3 years. (Funded by the French National Cancer Institute; ClinicalTrials.gov number, NCT00937456 .).

Chicken liver morphology as affected by feed withdrawal time and dietary aflatoxins.

1. An investigation was conducted on the effects of aflatoxin (AFL)-contaminated diets and feed withdrawal periods from 0 to 12 h in broiler chickens at 28 d of age. Both factors can potentially affect liver colour and can cause failure at veterinary inspection in the slaughterhouse.2. A total of 240, one-d-old female Cobb 500 broiler chickens were fed a common corn-soy pre-starters (d 1-7) and then either a non-contaminated control (CON) or feed with 1 ppm AFL (AFL) from d 8 to 28. The inoculum of AFL had 792 ppb of aflatoxin B1, 35 ppb of aflatoxin B2 and 219 ppb of aflatoxin G1. On d 28, all broilers were weighed and euthanised for necropsy following three different feed withdrawal time periods (0, 6 or 12 h), in a 2 × 3 factorial arrangement.3. Body weight gain, liver weight and liver fat content decreased as feed withdrawal lengthened, whereas FCR and gallbladder weight increased (P ≤ 0.05). AFL-fed birds had reduced body weight and proportion of liver fat and increased FCR, liver and gallbladder weights (P ≤ 0.05).4. Livers from fed broilers (0 h withdrawal) showed more lightness (L*) and yellowness (b*) than livers of broilers from 6 or 12 h withdrawal (P ≤ 0.05). The L* and redness (a*) values of livers from broilers fed diets COB were lower than those from AFL fed broilers (P ≤ 0.05).5. Prolonging pre-slaughter feed withdrawal decreased liver L*, whereas feeding AFL increased liver b*. These findings can be used to support veterinary assessment in slaughterhouses as shackled birds move on line through the inspection site. Since chicken liver is a valuable organ and an indicator of animal health, attention must be paid to these differences to ensure consumer safety.

Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae.

BACKGROUND: The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. OBJECTIVES: To characterize the genetic pathways leading to high-level azithromycin resistance. METHODS: A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. RESULTS: Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. CONCLUSIONS: This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

Early diagnosis is associated with improved clinical outcomes in benign esophageal perforation: an individual patient data meta-analysis.

BACKGROUND: Time of diagnosis (TOD) of benign esophageal perforation is regarded as an important risk factor for clinical outcome, although convincing evidence is lacking. The aim of this study is to assess whether time between onset of perforation and diagnosis is associated with clinical outcome in patients with iatrogenic esophageal perforation (IEP) and Boerhaave's syndrome (BS). METHODS: We searched MEDLINE, Embase and Cochrane library through June 2018 to identify studies. Authors were invited to share individual patient data and a meta-analysis was performed (PROSPERO: CRD42018093473). Patients were subdivided in early (≤ 24 h) and late (> 24 h) TOD and compared with mixed effects multivariable analysis while adjusting age, gender, location of perforation, initial treatment and center. Primary outcome was overall mortality. Secondary outcomes were length of hospital stay, re-interventions and ICU admission. RESULTS: Our meta-analysis included IPD of 25 studies including 576 patients with IEP and 384 with BS. In IEP, early TOD was not associated with overall mortality (8% vs. 13%, OR 2.1, 95% CI 0.8-5.1), but was associated with a 23% decrease in ICU admissions (46% vs. 69%, OR 3.0, 95% CI 1.2-7.2), a 22% decrease in re-interventions (23% vs. 45%, OR 2.8, 95% CI 1.2-6.7) and a 36% decrease in length of hospital stay (14 vs. 22 days, p < 0.001), compared with late TOD. In BS, no associations between TOD and outcomes were found. When combining IEP and BS, early TOD was associated with a 6% decrease in overall mortality (10% vs. 16%, OR 2.1, 95% CI 1.1-3.9), a 19% decrease in re-interventions (26% vs. 45%, OR 1.9, 95% CI 1.1-3.2) and a 35% decrease in mean length of hospital stay (16 vs. 22 days, p = 0.001), compared with late TOD. CONCLUSIONS: This individual patient data meta-analysis confirms the general opinion that an early (≤ 24 h) compared to a late diagnosis (> 24 h) in benign esophageal perforations, particularly in IEP, is associated with improved clinical outcome.

An in vivo evaluation of the effects of feed restriction regimens on wooden breast using ultrasound images as a predictive tool.

1. Gradual feed restriction was applied to broilers in order to reduce growth rate and, as a consequence, gradually impacts wooden breast myopathy occurrence. Ultrasound (US) images of breast muscle in live birds were correlated with breast fillets presenting wooden breast characteristics (WB). 2. A total of 1800 Cobb × Cobb 500 slow-feathering male chicks were fed one of the six feed restriction treatments with 12 replicates of 25 birds each, in a completely randomised design. Birds were fed ad libitum or were pair-fed to 50%, 60%, 70%, 80% or 90% of normal ad libitum intakes from 8 to 49 d to provide a gradual reduction in growth rate. Ultrasound images were obtained weekly from all birds and, in parallel, one bird per pen was weekly slaughtered and the major breast muscle was weighed and WB graded as 0 (normal), 1 (mild hardening in the upper), 2 (moderate hardening in the upper and/or lower), 3 (severe hardening) and 4 (severe hardening with haemorrhagic lesions and yellow fluid). Blood was taken for analysis of enzymes related to muscle cell breakdown. 3. Feed restriction applied at 50%, 60%, 70%, 80% and 90% of the ad libitum feed intake (FI) resulted in decreased body weight gain (BWG; P ≤ 0.05). 4. From 21 to 49 d, the increasing feed restriction led to linear increases (P ≤ 0.05) in WB scores, fibre density as well as breast depth and breast echogenicity. Creatine kinase, lactate dehydrogenase and aspartate aminotransferase concentration decreased linearly when broilers were feed restricted (P ≤ 0.05). 5. Wooden breast was positively correlated with echogenicity at 21 d (r = 0.510), 28 (r = 0.531), 35 (r = 0.470), 42 (r = 0.430) and 49 d (r = 0.548) (P ≤ 0.001). The use of breast echogenicity can be an additional tool to early detect alterations related to wooden breast.

Trastuzumab emtansine with or without pertuzumab versus trastuzumab with taxane for human epidermal growth factor receptor 2-positive advanced breast cancer: Final results from MARIANNE.

BACKGROUND: In the phase 3 MARIANNE trial, trastuzumab emtansine (T-DM1) with or without pertuzumab showed noninferior progression-free survival and better tolerability than trastuzumab plus a taxane (HT) for the first-line treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer. This article reports the final descriptive overall survival (OS) analysis, updated safety data, and additional patient-reported outcomes and biomarker analyses. METHODS: OS was assessed in 1095 patients with HER2-positive breast cancer and no prior therapy for advanced disease who had been randomized to HT, T-DM1 plus a placebo (hereafter T-DM1), or T-DM1 plus pertuzumab (T-DM1+pertuzumab). A post hoc exploratory landmark analysis of OS, baseline patient and disease characteristics, and tumor biomarkers in patients with and without an objective tumor response (OR) according to the Response Evaluation Criteria in Solid Tumors within 6.5 months of randomization was conducted. RESULTS: The median OS was similar across groups (50.9, 53.7, and 51.8 months for the HT, T-DM1, and T-DM1+pertuzumab groups, respectively). Among patients with an OR, the median OS was longer with T-DM1 (64.4 months) and T-DM1+pertuzumab (not reached) versus HT (56.3 months). No baseline characteristics or biomarkers were strongly associated with OR. The incidence of grade 3 or higher adverse events was greater with HT (55.8%) than T-DM1 (47.1%) or T-DM1+pertuzumab (48.6%). The median time to clinically meaningful deterioration (a 3-point or greater change) in neurotoxicity symptoms was shorter with HT (2.1 months) and T-DM1+pertuzumab (4.2 months) than T-DM1 (6.2 months). Fewer patients reported alopecia and diarrhea and were bothered by treatment side effects in the T-DM1 arm. CONCLUSIONS: These results support T-DM1 as a first-line treatment for patients with HER2-positive metastatic breast cancer who are deemed unsuitable for taxane-based therapy.

Correction to: Relationship between tumor biomarkers and efficacy in MARIANNE, a phase III study of trastuzumab emtansine ± pertuzumab versus trastuzumab plus taxane in HER2-positive advanced breast cancer.

Following publication of the original article [1], the authors reported the following errors in the article.

Relationship between tumor biomarkers and efficacy in MARIANNE, a phase III study of trastuzumab emtansine ± pertuzumab versus trastuzumab plus taxane in HER2-positive advanced breast cancer.

BACKGROUND: The phase III EMILIA and TH3RESA trials demonstrated clinical benefits of trastuzumab emtansine (T-DM1) therapy in patients with previously treated HER2-positive metastatic breast cancer (MBC). Data from these and other trials showed that T-DM1-associated survival benefits were observed across biomarker subgroups tested in these trials. Prespecified, exploratory analyses of the phase III MARIANNE study examined the effects of HER2-related biomarkers on PFS in patients administered T-DM1 in the first-line MBC setting. METHODS: In MARIANNE, patients with previously untreated HER2-positive MBC were randomized (1:1:1) to trastuzumab plus taxane, T-DM1 plus placebo, or T-DM1 plus pertuzumab. Biomarker subgroups included HER2 and HER3 mRNA expression levels (≤median vs. >median), HER2 staining intensity (IHC 3+ vs. 2+ vs. 0/1+), PIK3CA status (mutated vs. non-mutated), PTEN H-score (≤median vs. >median), and PTEN protein expression level (0 vs. 1+ vs. 2+ vs. 3+ vs. 4+). PFS was analyzed descriptively for each subgroup using Kaplan-Meier methodology. Additional exploratory post-hoc analyses evaluated the effects of HER2 heterogeneity. Multivariate analyses were also performed. RESULTS: Median PFS was numerically longer for patients with HER2 mRNA levels >median versus ≤median across treatment arms. In general, there were no predictive biomarkers of benefit for either T-DM1 treatment arm; most hazard ratios were close to 1 with wide confidence intervals that included the value 1. Focal HER2 expression (IHC 3+ or IHC 2+) was present in 3.8% of patients and was associated with numerically shorter PFS in the T-DM1-containing treatment arms versus trastuzumab plus taxane. Compared with non-mutated PIK3CA, mutated PIK3CA was associated with numerically shorter median PFS across treatment groups. Post-hoc multivariate analysis showed HER2 mRNA expression and mutated PIK3CA were prognostic for PFS (P ≤ 0.001 for both biomarkers). CONCLUSIONS: In MARIANNE, biomarkers related to the HER2 pathway did not have predictive value for PFS when comparing T-DM1 (with or without pertuzumab) with trastuzumab plus taxane. However, HER2 mRNA level and PIK3CA mutation status showed prognostic value. Evaluation of other potential biomarkers, including immune markers, is ongoing. TRIAL REGISTRATION: Registration number: NCT01120184 . Date of registration: April 28, 2010 (registered prospectively).

Impact of Extracapsular Lymph Node Involvement After Neoadjuvant Chemoradiation Therapy Followed by Surgery in Carcinoma of the Esophagus: A Multicenter Study.

OBJECTIVES: The current study aims to examine the impact of extracapsular lymph node involvement (EC-LNI) on survival for both esophageal adenocarcinoma (AC) and squamous cell carcinoma (SCC) treated with neoadjuvant chemoradiation therapy (nCRT) followed by surgery. BACKGROUND: Studies have demonstrated the negative prognostic value of EC-LNI in primary surgery, but its impact after nCRT remains unclear. METHODS: From the databases of 6 European high-volume centers 1505 patients with R0 resections were withheld. Oncologic variables, including ypT, ypN, number of positive lymph nodes, and lymph node capsular status: EC-LNI and intracapsular lymph node involvement (IC-LNI), were examined. Statistical analysis was performed by Cox proportional hazards modeling. RESULTS: In SCC 182 patients (31.6%) had positive lymph nodes, of whom 60 (33.0%) showed EC-LNI. In AC 391 patients (42.1%) had positive lymph nodes, of whom 147 (37.6%) showed EC-LNI. Overall 5-year survival (O5YS) in SCC was 42.0%. Presence of EC-LNI meant a significantly worse O5YS than IC-LNI or pN0 (10.6%, 39.5%, and 47.4%, respectively; P < 0.05). O5YS in AC was 41.2%. No significant difference was observed between EC-LNI and IC-LNI (P = 0.322). In the multivariate analysis, among the examined possible prognosticators, presence of EC-LNI showed the highest hazard ratio (2.29, confidence interval: 1.52-3.47) as an independent prognosticator for overall survival in SCC, but it was not in AC. CONCLUSIONS: Based on this international multicenter study, the presence of EC-LNI after nCRT is at least as important as N-stage for survival and EC-LNI is the strongest prognosticator for overall survival in SCC but not in AC.

Awareness and Misconceptions of Breast Cancer Risk Factors Among Laypersons and Physicians.

BACKGROUND: Primary prevention of cancer relies on awareness of and consequent identification of risk factors. We investigated knowledge of breast cancer risk factors not only among laywomen but also among female physicians. METHODS: The EDIFICE 4 nationwide observational survey was conducted by phone interviews of a representative female population (737 laywomen and 105 female physicians) aged 40-75 years, using the quota method. This analysis focuses on spontaneous replies to the question "In your opinion, what are the five main risk factors that increase the risk of breast cancer?". RESULTS: Heredity/Family history of breast cancer was the most widely recognized risk factor in both study populations (98.1% physicians vs. 54.2% laywomen; P ≤ 0.01). Smoking (19.0 and 17.5%) and alcohol consumption (3.8 and 5.5%) were among the lifestyle risk factors that were cited by similar proportions of physicians and laywomen, respectively. Other established risk factors were however very rarely cited by either physicians or laywomen, e.g., Exposure to medical radiation (4.8 vs. 0.4%, respectively; P ≤ 0.05) or not cited at all, i.e., Benign mastopathy and Personal history of breast cancer. CONCLUSION: This survey highlights a number of misconceptions relating to behavioral risk factors for breast cancer, including the relative impact of alcohol and tobacco consumption and the importance of menopausal status. The limited awareness of the risk related to Exposure to medical radiation, Benign mastopathy, or Personal history raises concern regarding compliance with national screening recommendations.

Airway stenting in the management of iatrogenic tracheal injuries: 10-Year experience.

BACKGROUND AND OBJECTIVE: Iatrogenic tracheal injury (ITI) is a rare yet severe complication of endotracheal tube (ETT) placement or tracheostomy. ITI is suspected in patients with clinical and/or radiographic signs or inefficient mechanical ventilation (MV) following these procedures. Bronchoscopy is used to establish a definitive diagnosis. METHODS: We conducted a retrospective, single-centre chart review of 35 patients between 2004 and 2014. Depending on the nature and location of ITI and need for MV, patients were triaged to surgical repair, endoscopic management with airway stents or conservative treatment consisting of ETT or tracheotomy cannula (TC) placement distal to the wound and bronchoscopic surveillance. RESULTS: Three of the four patients (11.43%) presenting with tracheoesophageal fistula (TEF) underwent surgery. Seven patients (20%) who did not require MV underwent endoscopic surveillance. Of the 24 ventilated patients (68.57%), 7 with ITI in the lower trachea were treated with silicone Y-stent (ETT or TC was placed inside the stent) and 17 patients with ITI in the upper trachea were managed by placing ETT or TC cuff distal to the injury. Overall management success, defined as complete healing of the ITI, was seen in 88.57% of patients. Four patients (11.43%) died of non-ITI-related comorbidities. CONCLUSION: Conservative management should be considered in non-ventilated patients with ITI and when ITI is located in the upper trachea of ventilated patients where ETT or TC bypasses the injury. Airway stenting should be considered in ventilated patients with ITI located in the lower trachea. Surgery should be reserved for TEF and conservative and endoscopic management failure.

Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. METHODS: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). RESULTS: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). CONCLUSIONS: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

International Multicenter Study on the Impact of Extracapsular Lymph Node Involvement in Primary Surgery Adenocarcinoma of the Esophagus on Overall Survival and Staging Systems.

OBJECTIVE: The current pathological lymph node (pN) staging is based on the number of positive lymph nodes but does not take into consideration characteristics of the involved lymph nodes itself. The current study aims to examine the prognostic value of extracapsular lymph node involvement (EC-LNI) and intracapsular lymph node involvement (IC-LNI) for esophageal adenocarcinoma treated by primary surgery. METHODS: From the databases of five European high volume centers, 1639 adenocarcinoma patients with primary R0-resection were withheld after excluding 90-day mortality. Oncologic variables, including number of resected lymph nodes, number of resected positive lymph nodes, and EC-LNI/IC-LNI were examined. The Union Internationale contre le Cancer (UICC) 7th edition prognostic staging was used as baseline staging system. Statistical analysis was performed by Cox proportional hazards modeling and verified using the Random Survival Forest technique. RESULTS: EC-LNI showed significantly worse overall 5-year survival compared with IC-LNI overall (13.4% vs 37.2%, P < 0.0001), including in each pN-category [16.4% vs 45.6% in pN1 (P < 0.0001), 16.1% vs 23.8% (P = 0.047) in pN2 (P = 0.065), and 8.7% vs 26.3% in pN3 categories, respectively]. pN1 IC-LNI patients show a 5-year overall survival comparable (P = 0.92) with stage IIB (ie, pT3N0). Reclassifying the UICC prognostic stages according to these findings into an adapted staging model showed a significant (P < 0.0001) increase in homogeneity, discriminatory ability, and monotonicity compared with the original UICC TNM 7th edition prognostic staging. CONCLUSIONS: These data suggest that lymph node capsular status is an important prognostic factor and should be considered for the future edition of the TNM staging system for esophageal cancer.

Prospective Clinical Utility Study of the Use of the 21-Gene Assay in Adjuvant Clinical Decision Making in Women With Estrogen Receptor-Positive Early Invasive Breast Cancer: Results From the SWITCH Study.

BACKGROUND: The 21-gene Oncotype DX Recurrence Score assay is a validated assay to help decide the appropriate treatment for estrogen receptor-positive (ER+), early-stage breast cancer (EBC) in the adjuvant setting. The choice of adjuvant treatments might vary considerably in different countries according to various treatment guidelines. This prospective multicenter study is the first to assess the impact of the Oncotype DX assay in the French clinical setting. METHODS: A total of 100 patients with ER+, human epidermal growth factor receptor 2-negative EBC, and node-negative (pN0) disease or micrometastases in up to 3 lymph nodes (pN1mi) were enrolled. Treatment recommendations, physicians' confidence before and after knowing the Recurrence Score value, and physicians' perception of the assay were recorded. RESULTS: Of the 100 patients, 95 were evaluable (83 pN0, 12 pN1mi). Treatment recommendations changed in 37% of patients, predominantly from chemoendocrine to endocrine treatment alone. The proportion of patients recommended chemotherapy decreased from 52% pretest to 25% post-test. Of patients originally recommended chemotherapy, 61% were recommended endocrine treatment alone after receiving the Recurrence Score result. For both pN0 and pN1mi patients, post-test recommendations appeared to follow the Recurrence Score result for low and high values. Physicians' confidence improved significantly. CONCLUSION: These are the first prospective data on the impact of the Oncotype DX assay on adjuvant treatment decisions in France. Using the assay was associated with a significant change in treatment decisions and an overall reduction in chemotherapy use. These data are consistent with those presented from European and non-European studies.

The effect of daily usage of Listerine Cool Mint mouthwash on the oropharyngeal microbiome: a substudy of the PReGo trial.

Introduction. ListerineÒ is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear.Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome.Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome.Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a double-blind single-centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP). Fifty-nine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non-host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA-like differential expression analysis.Results. Streptococcus was the most abundant genus in most samples (n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6-44.8), followed by Prevotella [13.5% (IQR 4.8-22.6)] and Veillonella [10.0% (IQR 4.0-16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine (P = 0.006, pseudo-F = 2.29) or placebo (P = 0.003, pseudo-F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline.Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function.

Interferon Stimulated Gene (ISG) expression plays a key role in the control of viral replication and development of a robust adaptive response. Understanding this dynamic relationship between the pathogen and host is critical to our understanding of viral life-cycles and development of potential novel anti-viral strategies. Traditionally, plasmid based exogenous prompter driven expression of ISGs has been used to investigate anti-viral ISG function, however there are deficiencies in this approach. To overcome this, we investigated the utility of CRISPR activation (CRISPRa), which allows for targeted transcriptional activation of a gene from its endogenous promoter. Using the CRISPRa-SAM system to induce targeted expression of a panel of anti-viral ISGs we showed robust induction of mRNA and protein expression. We then employed our CRISPRa-SAM ISG panel in several antiviral screen formats to test for the ability of ISGs to prevent viral induced cytopathic cell death (CPE) and replication of Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus Kunjin (WNVKUN), Hepatitis A Virus (HAV) and Human Coronavirus 229E (HCoV-229E). Our CRISPRa approach confirmed the anti-viral activity of ISGs like IFI6, IFNß and IFNλ2 that prevented viral induced CPE, which was supported by high-content immunofluorescence imaging analysis. This work highlights CRISPRa as a rapid, agile, and powerful methodology to identify and characterise ISGs and viral restriction factors.