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Objective: To evaluate trends in outpatient versus inpatient hysterectomy for endometrial cancer and assess enabling factors, cost and safety., Methods: In this retrospective cohort study, patients aged 18 years or older who underwent hysterectomy for endometrial cancer between January 2008 and September 2015 were identified in the Premier Healthcare Database. The surgical approach for hysterectomy was classified as open/abdominal, vaginal, laparoscopic or robotic assisted. We described trends in surgical setting, perioperative costs and safety. The impact of patient, provider and hospital characteristics on outpatient migration was assessed using multivariate logistic regression., Results: We identified 41 246 patients who met inclusion criteria. During the time period studied, we observed a 41.3% shift from inpatient to outpatient hysterectomy (p<0.0001), an increase in robotic hysterectomy, and a decrease in abdominal hysterectomy. The robotic hysterectomy approach, more recent procedure (year), and mid-sized hospital were factors that enabled outpatient hysterectomies; while abdominal hysterectomy, older age, Medicare insurance, black ethnicity, higher number of comorbidities, and concomitant procedures were associated with an inpatient setting. The shift towards outpatient hysterectomy led to a $2500 savings per case during the study period, in parallel to the increased robotic hysterectomy rates (p<0.001). The post-discharge 30-day readmission and complications rate after outpatient hysterectomy remained stable at around 2%., Conclusions: A significant shift from inpatient to outpatient setting was observed for hysterectomies performed for endometrial cancer over time. Minimally invasive surgery, particularly the robotic approach, facilitated this migration, preserving clinical outcomes and leading to reduction in costs., Competing Interests: Competing interests: CS reports personal fees from Intuitive Surgical, outside the submitted work. YL is a full-time employee and stock holder of Intuitive Surgical., (© IGCS and ESGO 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

We sought to determine the incidence and location of human papillomavirus (HPV)-associated anogenital disease in women with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) via a retrospective cohort study with prospective contact to update history at Mayo Clinic in Rochester, Minnesota. Females undergoing treatment for nonmetastatic HPV-positive OPSCC from 2011 to 2019 were identified. Clinical history and outcomes were abstracted from medical records. Patients without documented anogenital history were contacted, consented, and administered a survey, and external records were requested and reviewed. Seventeen of 46 patients (37.0%) had a history of anogenital HPV-associated disease, and 16 of 17 (94.1%) required procedures to diagnose or treat HPV lesions. The cervix was the most common site (16/17, 94.1%). Procedures included colposcopy (n = 6), cervical excision (n = 3), cryotherapy (n = 4), and hysterectomy (n = 3). One case of fatal cervical carcinoma was noted, diagnosed 1 year following OPSCC. Three of 17 (17.6%) had HPV-related vulvovaginal disease, and 1 of 17 had anal disease. Patients with a history of HPV-positive OPSCC may be at elevated risk for HPV-associated anogenital disease.

Objective: Endometrial cancer surgical staging includes lymph node assessment which can lead to lower extremity lymphedema. The aim of this study was to estimate prevalence after sentinel lymph node biopsy versus lymphadenectomy., Methods: Consecutive patients who underwent minimally invasive surgery at the Mayo Clinic, Rochester, Minnesota, USA, between January 2009 and June 2016 for newly diagnosed endometrial cancer were mailed our validated 13 item lower extremity lymphedema screening questionnaire. We also ascertained via questionnaire whether the patient was ever diagnosed with lower extremity lymphedema., Results: Among 378 patients included in the analysis, 127 (33.5%) had sentinel lymph node biopsy with or without side specific lymphadenectomy (sentinel lymph node cohort) and 251 (66.4%) underwent bilateral lymphadenectomy prior to sentinel lymph node biopsy implementation at our institution or as 'backup' after sentinel lymph node mapping (lymphadenectomy cohort). The prevalence of lower extremity lymphedema was 41.5% (157/378), with 69 patients (18.3%) self-reporting a lower extremity lymphedema diagnosis after their endometrial cancer surgery at a median of 54.3 months (interquartile range 31.2-70.1 months), and an additional 88 patients (23.3%) identified by the screening questionnaire. The prevalence of lower extremity lymphedema was significantly higher in the lymphadenectomy cohort compared with the sentinel lymph node group (49.4% (124/251) vs 26.0% (33/127); p<0.001). When the cohorts were restricted to patients surgically managed after the introduction of sentinel lymph node, the prevalence of lower extremity lymphedema was still significantly higher in the lymphadenectomy cohort compared with the sentinel lymph node cohort (39.0% (41/105) vs 26.0% (33/127); p=0.03). In a multivariable analysis adjusted for body mass index, receipt of adjuvant external beam radiation, diabetes, congestive heart failure, and International Federation of Gynecology and Obstetrics grade, the adjusted odds ratio for the association between type of nodal sampling (lymphadenectomy cohort vs sentinel lymph node cohort) and lower extremity lymphedema was 2.75 (95% confidence interval 1.69 to 4.47, p<0.001)., Conclusions: Sentinel lymph node biopsy was associated with a decreased risk of post-treatment lymphedema compared with lymphadenectomy in patients who underwent surgical staging for endometrial carcinoma., Competing Interests: Competing interests: None declared., (© IGCS and ESGO 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Burnout in health care is a public health crisis. Burnout is a triad of emotional exhaustion, depersonalization, and feelings of reduced personal accomplishment. More than half of practicing physicians and trainees experience burnout, and the rates are increasing. This review highlights the current prevalence of burnout among U.S. physicians, especially obstetrician-gynecologists. We review personal and systemic risk factors for burnout, consequences of burnout, and proven interventions, especially at the systems level, to treat and prevent burnout., Competing Interests: Financial Disclosure The authors did not report any potential conflicts of interest., (Copyright © 2020 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.)

One of the most important stress responses in bacteria is the stringent response. The main player in this response is the signal molecule (p)ppGpp, which is synthesized by a Rel family protein. In Escherichia coli , RelA is the main synthetase of (p)ppGpp in response to amino acid starvation. Although the synthetic activity of RelA is well-understood, its regulation is not yet fully characterized. The C-terminus domain (CTD) of the E. coli RelA is responsible for the regulation of the protein and for its complete dependency on wild-type (WT) ribosome. The CTD contains three Cysteine residues, positioned in a very conserved order. Together with our previous results, we show in vitro the negative dominant effect of a part of the WT CTD (AA 564-744) named YG4 on RelA synthetic activity. This effect is abolished using mutated YG4 (YG4-638). In vitro and mass spectrometry (MS)-MS analysis of the native RelA and the mutated RelA in Cys-638 (Rel638) in the presence of the native and mutated YG4 (YG4-638) reveals that RelA forms a homodimer via its CTD by the formation of a disulfide bond between the two Cys-638 residues. This supports our previous data which showed, using a two-hybrid system, interactions between RelA proteins via the CTD. Finally, we show in vitro that excess of the native YG4 inhibited RelA synthetic activity but did not affect the amount of RelA bound to the ribosome. Our results suggest that the regulatory mechanism of RelA is by the dimerization of the protein via disulfide bonds in the CTD. Upon amino-acid starvation, the dimer changes its conformation, thus activating the stringent response in the cell., (Copyright © 2020 Kaspy and Glaser.)

Objective: The choice of adjuvant treatment for women with stage II endometrial cancer is challenging, given the known increase in morbidity with external beam radiation compared with vaginal brachytherapy, and the lack of consensus on its benefits. We summarized the evidence on survival and recurrence for stage II endometrial cancer, defined as cervical stromal invasion, after adjuvant postoperative external beam radiotherapy and vaginal brachytherapy., Methods: We searched the MEDLINE, EMBASE, CENTRAL, and Scopus databases from inception to January 2019 to identify studies that compared adjuvant postoperative external beam radiotherapy with or without vaginal brachytherapy and vaginal brachytherapy alone in stage II endometrial cancer. Our primary outcome was the locoregional recurrence rate, defined as recurrence in the pelvis or vagina. Secondary outcomes included the rate of recurrence at any site, distant recurrence rate, vaginal recurrence rate, pelvic recurrence rate, and 5 year overall survival. Study selection, assessment, and data abstraction were performed by an independent set of reviewers. Random effects models were used to synthesize quantitative data., Results: We included 15 cohort studies reporting data on 1070 women. Most women with stage II endometrial cancer (848/1070, 79.3%) were treated with external beam radiotherapy with or without vaginal brachytherapy. Subgroup analysis was stratified by whether >90% of the women included underwent pelvic lymph node assessment (sampling or full dissection). Locoregional recurrence (pelvic and vaginal recurrence) was significantly reduced with external beam radiotherapy with or without vaginal brachytherapy compared with vaginal brachytherapy alone (14 studies (n=1057); odds ratio (OR) 0.33 (95% confidence interval (CI) 0.16 to 0.68); I 2 =5%) regardless of pelvic lymph node assessment. Most women (81.8%) who recurred locoregionally had a least one uterine risk factor (grade 3 tumor, myometrial invasion >50%, or lymphovascular invasion). There was no difference in overall survival with external beam radiotherapy with or without vaginal brachytherapy compared with vaginal brachytherapy alone (five studies (n=463); OR 0.78 (95% CI 0.34 to 1.80); I 2 =48%)., Conclusions: External beam radiotherapy with or without vaginal brachytherapy decreased the locoregional recurrence threefold for stage II endometrial cancer, regardless of pelvic lymph node assessment. Most women who suffered recurrence locoregionally had a least one high risk factor. Vaginal brachytherapy alone may be sufficient therapy for node negative stage II endometrial cancer without uterine risk factors, while those with uterine risk factors should be considered for external beam radiotherapy with or without vaginal brachytherapy to improve locoregional control., Competing Interests: Competing interests: None declared., (© IGCS and ESGO 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Brewers' spent grain (BSG) is a low-cost by-product of the brewing process. BSG liquor names the liquid components of BSG, mainly glucose, maltose, and long-chain α-1,4-glycosidic bond glucose oligomers. These substances should be separated in existing BSG biorefineries, as they might lead to an increased formation of microbe-inhibiting compounds in well-established hydrothermal/enzymatic saccharification processes. In most cases, this liquid fraction is discarded. The present study presents for the first time an optimized process with BSG liquor for the purpose of producing bulk chemicals (e.g., lactate) in relevant concentrations. The process comprises the application of yeast extract, produced from own brewing processes, as the sole supplemented complex constituent in a simultaneous fermentation and saccharification process. Kinetic parameters for the final optimized process conditions with the organism Lactobacillus delbrueckii  subsp.  lactis were: maximum specific growth rate µ max   =  0.47 h -1 , maximum lactate concentration c Lac, max   =  79.06 g L -1 , process yield Y PS   =  0.89 g Lac  g Sugar -1   =  4.18 g P  g Lac  g CDW -1 =  4.93 g -1 , and productivity P  15 h   =  4.93 g Lac  L -1  h -1 . BSG liquor, linked with yeast extract from Brewers' yeast, can be a promising substrate for further bioprocess engineering tasks and contribute to a holistic and sustainable usage of Brewers' spent grain., Competing Interests: The authors have declared no conflict of interest., (© 2020 The Authors. Engineering in Life Sciences published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: It is unclear if sentinel lymph node biopsy is associated with improved surgical outcomes compared with lymphadenectomy in patients with endometrial cancer. In this study we aimed to compare peri-operative surgical outcomes and treatment-related morbidity in patients who underwent robotic-assisted sentinel lymph node biopsy versus systematic pelvic lymphadenectomy or hysterectomy alone for apparent early-stage endometrial cancer., Methods: Records were reviewed of consecutive patients with International Federation of Gynecology and Obstetrics stages I-III endometrial cancer undergoing robotic-assisted staging from January 1, 2009, through June 30, 2016. For the purpose of this analysis we focused on the actual patients who had sentinel node biopsy only (ie, excluding those who had an associated lymphadenectomy either for failed mapping or during the learning curve). We also excluded patients who had para-aortic lymph node dissection from the lymphadenectomy group. Perioperative and 30-day surgical outcomes were compared between patients who underwent sentinel lymph node assessment and those who had pelvic lymphadenectomy or hysterectomy alone, respectively. Inverse probability of treatment weighting derived from propensity scores was used to minimize allocation bias in the comparison of outcomes between groups., Results: A total of 621 patients were analyzed: 188 (30.3%) with sentinel lymph node biopsy, 198 (31.9%) with pelvic lymphadenectomy, and 235 (37.8%) with hysterectomy alone. Inverse probability of treatment weights analysis balanced for baseline characteristics (age, body mass index, American Society of Anesthesiologists score, Charlson co-morbidity index, parity, prior cesarean section, and previous abdominal operation) showed no significant differences in intra-operative and post-operative complications, re-admissions, and re-operations between the groups. Compared with pelvic lymphadenectomy, the sentinel lymph node biopsy group had a shorter mean operative time (138.0 vs 222.8 min, p<0.001) and less median blood loss (50 vs 100 mL, p<0.001). Sentinel lymph node biopsy also was not associated with worse morbidity compared with hysterectomy alone., Conclusions: Introduction of sentinel lymph node biopsy reduces operative times and improves peri-operative surgical outcomes of robotic-assisted staging for apparent early-stage endometrial cancer without worsening the morbidity of hysterectomy alone., Competing Interests: Competing interests: None declared., (© IGCS and ESGO 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: This is the first updated Enhanced Recovery After Surgery (ERAS) Society guideline presenting a consensus for optimal perioperative care in gynecologic/oncology surgery., Methods: A database search of publications using Embase and PubMed was performed. Studies on each item within the ERAS gynecologic/oncology protocol were selected with emphasis on meta-analyses, randomized controlled trials, and large prospective cohort studies. These studies were then reviewed and graded according to the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system., Results: All recommendations on ERAS protocol items are based on best available evidence. The level of evidence for each item is presented accordingly., Conclusions: The updated evidence base and recommendation for items within the ERAS gynecologic/oncology perioperative care pathway are presented by the ERAS® Society in this consensus review., Competing Interests: Competing interests: GN is the Secretary for the ERAS® Society. MS received honoraria for lecturing and travel expenses from Baxter Healthcare, Merck, and Deltex. He is an Executive Committee member of the ERAS® Society. AA has received speaker’s honoraria from Sanofi and AstraZeneca, and is on the Advisory Board of AstraZeneca. GM is a consultant for ConMed and Edwards Lifesciences and has stock options with Pacira Pharmaceutical. OL has an appointment with Nutricia Advisory Board, has given advice to MSD, Abbot and Advanced Medical Nutrition. He has received speaker’s honoraria from Nutricia, MSD, BBraun, Medtronic and Fresenius-Kabi. He is the current Chairman of the ERAS® Society. He founded, and owns stock in, Encare AB that runs the ERAS® Society Interactive Audit System (EIAS). SD is a member of the Board of Directors for ERAS® USA and a content expert for the AHRQ program for improving surgical care and recovery., (© IGCS and ESGO 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Pelvic exenteration (PE) is an extensive surgery associated with high rates of postoperative morbidity and mortality. The absence of well-defined preoperative selection criteria to identify patients eligible for PE prompted the assessment of pre-operative predictors of 30-day major surgical complications., Methods: Demographics and surgical characteristics of patients undergoing PE for gynecologic cancer in a single institution between 01/2004-12/2016 were reviewed. Postoperative complications within 30 days following surgery were graded using the Accordion grading system. Logistic regression was used to analyze potential risk factors for severe postoperative complications., Results: A total of 138 patients were included in the cohort. Forty-five patients underwent total PE, 52 anterior PE, and 41 posterior PE. Among the 137 patients with follow-up, a severe postoperative complication was experienced by 37 patients (27.0%) and 3 patients (2.2%) experienced death within 90 days. The most frequent grade 3 complications were complications of urinary reconstruction (n = 15), wound dehiscence (n = 9), and abdominal abscess requiring intervention with drain or return to the operating room (n = 6). On multivariable analysis, independent predictors of severe postoperative complications were anterior or total PE (adjusted odds ratio (aOR): 11.66, 95% CI 2.56-53.18), pre-operative hemoglobin ≤10 mg/dl (aOR 2.70, 95% CI 1.02-7.14) and presence of 3+ comorbidities (aOR: 2.76, 95% CI 1.07-7.10)., Conclusions: Major complications after exenteration are common. Surgical complexity and patient selection play a considerable role in predicting complications. These data can be used to better risk stratify patients undergoing PE., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to assess the role of frozen section (FS) in identifying an absence of lymph nodes during sentinel lymph node (SLN) biopsy for apparent early-stage endometrial cancer (EC)., Methods: Consecutive apparent early-stage EC patients who had SLNs removed after cervical injection with indocyanine green (ICG) from 1 June 2014 to 30 June 2016 were analyzed. An empty node (EN) was defined as an SLN specimen without evidence of lymph node(s). The association of tumor and patient characteristics with an EN was evaluated, and trend analysis to compare the rate of ENs over calendar quarters was performed. A decision-tree model was then created to compare the use of FS versus no FS for SLN evaluation in the hypothetical cohort affected by early-stage EC in the US each year., Results: Over the study period, 300 patients met the inclusion criteria. FS revealed ENs in 24 (8%) patients. No association between patient demographic characteristics (age, body mass index, prior abdominopelvic surgery, international federation of gynecology and obstetrics (FIGO) stage, histology, myometrial invasion, cervical stromal invasion) and presence of ENs was observed. The rate of ENs at FS did not change over time (p = 0.68). The hypothetical analysis showed a 4.3% decrease of inappropriately staged patients with the use of FS on the SLN (95% confidence interval 4.1-4.5)., Conclusions: ENs during SLN biopsy for EC staging is not a rare event and can be easily detected with FS. The implementation of FS of SLN might reduce inadequate staging of EC. Individual institutions may want to examine their own EN rates and determine if this would assist them in their SLN practices for EC.

Enhanced recovery protocols consist of a bundle of concepts including early feeding, opioid-sparing multimodal pain management, and euvolemia, with the overarching goal of hastening postoperative recovery. Enhanced recovery after surgery has been shown to reduce hospital length of stay, reduce costs, and decrease perioperative opioid requirements in benign and oncologic gynecologic surgery. Interventions without supporting evidence of benefit, such as the use of mechanical bowel preparation, routine use of nasogastric tubes and surgical drains, caloric restriction, routine use of intravenous opioid analgesics, and over-vigorous intravenous hydration should be discouraged to improve broader endpoints such as patient satisfaction and overall recovery. Successful implementation requires engagement from a multidisciplinary team including surgeons, anesthesiologists, nurses, and pharmacists., (© 2018 The Authors. International Journal of Gynecology & Obstetrics published by John Wiley & Sons Ltd on behalf of International Federation of Gynecology and Obstetrics.)

Purpose: To determine the relationship between chemotherapy dose modification (dose adjustment or treatment delay), overall survival (OS) and progression-free survival (PFS) for women with advanced-stage epithelial ovarian carcinoma (EOC) and primary peritoneal carcinoma (PPC) who receive carboplatin and paclitaxel., Methods: Women with stages III and IV EOC and PPC treated on the Gynecologic Oncology Group phase III trial, protocol 182, who completed eight cycles of carboplatin with paclitaxel were evaluated in this study. The patients were grouped per dose modification and use of granulocyte colony stimulating factor (G-CSF). The primary end point was OS; Hazard ratios (HR) for PFS and OS were calculated for patients who completed eight cycles of chemotherapy. Patients without dose modification were the referent group. All statistical analyses were performed using the R programming language and environment., Results: A total of 738 patients were included in this study; 229 (31%) required dose modification, 509 did not. The two groups were well-balanced for demographic and prognostic factors. The adjusted hazard ratios (HR) for disease progression and death among dose-modified patients were: 1.43 (95% CI, 1.19-1.72, P < 0.001) and 1.26 (95% CI, 1.04-1.54, P = 0.021), respectively. Use of G-CSF was more frequent in dose-modified patients with an odds ratio (OR) of 3.63 (95% CI: 2.51-5.26, P < 0.001) compared to dose-unmodified patients., Conclusion: Dose-modified patients were at a higher risk of disease progression and death. The need for chemotherapy dose modification may identify patients at greater risk for adverse outcomes in advanced stage EOC and PPC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: We hypothesized that mutations in homologous recombination repair (HRR) genes beyond BRCA1 and BRCA2 improve outcomes for ovarian carcinoma patients treated with platinum therapy and would impact the relative benefit of adding prolonged bevacizumab. Experimental Design: We sequenced DNA from blood and/or neoplasm from 1,195 women enrolled in GOG-0218, a randomized phase III trial in advanced ovarian carcinoma of bevacizumab added to carboplatin and paclitaxel. Defects in HRR were defined as damaging mutations in 16 genes. Proportional hazards models were used to estimate relative hazards for progression-free survival (PFS) and overall survival (OS). Results: Of 1,195 women with ovarian carcinoma, HRR mutations were identified in 307 (25.7%). Adjusted hazards for progression and death compared with those without mutations were lower for women with non- BRCA HRR mutations [HR = 0.73; 95% confidence interval (CI), 0.57-0.94; P = 0.01 for PFS; HR = 0.67; 95% CI, 0.50-0.90; P = 0.007 for OS] and BRCA1 mutations (HR = 0.80; 95% CI, 0.66-0.97; P = 0.02 for PFS; HR = 0.74; 95% CI, 0.59-0.94; P = 0.01 for OS) and were lowest for BRCA2 mutations (HR = 0.52; 95% CI, 0.40-0.67; P < 0.0001 for PFS; HR = 0.36; 95% CI, 0.25-0.53; P < 0.0001 for OS). A test of interaction showed no difference in the effect of bevacizumab on PFS between cases with and without mutations. Conclusions: HRR mutations, including non- BRCA genes, significantly prolong PFS and OS in ovarian carcinoma and should be stratified for in clinical trials. The benefit of adding bevacizumab was not significantly modified by mutation status. Clin Cancer Res; 24(4); 777-83. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Objectives: To construct a tool, using computed tomography (CT) imaging and preoperative clinical variables, to estimate successful primary cytoreduction for advanced epithelial ovarian cancer (EOC)., Methods: Women who underwent primary cytoreductive surgery for stage IIIC/IV EOC at Mayo Clinic between 1/2/2003 and 12/30/2011 and had preoperative CT images of the abdomen and pelvis within 90days prior to their surgery available for review were included. CT images were reviewed for large-volume ascites, diffuse peritoneal thickening (DPT), omental cake, lymphadenopathy (LP), and spleen or liver involvement. Preoperative factors included age, body mass index (BMI), Eastern Cooperative Oncology Group performance status (ECOG PS), American Society of Anesthesiologists (ASA) score, albumin, CA-125, and thrombocytosis. Two prediction models were developed to estimate the probability of (i) complete and (ii) suboptimal cytoreduction (residual disease (RD) >1cm) using multivariable logistic analysis with backward and stepwise variable selection methods. Internal validation was assessed using bootstrap resampling to derive an optimism-corrected estimate of the c-index., Results: 279 patients met inclusion criteria: 143 had complete cytoreduction, 26 had suboptimal cytoreduction (RD>1cm), and 110 had measurable RD ≤1cm. On multivariable analysis, age, absence of ascites, omental cake, and DPT on CT imaging independently predicted complete cytoreduction (c-index=0.748). Conversely, predictors of suboptimal cytoreduction were ECOG PS, DPT, and LP on preoperative CT imaging (c-index=0.685)., Conclusions: The generated models serve as preoperative evaluation tools that may improve counseling and selection for primary surgery, but need to be externally validated., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ovarian carcinosarcoma is a rare subtype of ovarian cancer with poor clinical outcomes. The low incidence of this disease makes accrual to large clinical trials challenging. However, studies have shown that treatment responses in patient-derived xenograft (PDX) models correlate with matched-patient responses in the clinic, supporting their use for preclinical testing of standard and novel therapies. An ovarian carcinosarcoma PDX is presented herein and showed resistance to carboplatin and paclitaxel (similar to the patient) but exhibited significant sensitivity to ifosfamide and paclitaxel. The PDX demonstrated overexpression of EGFR mRNA and gene amplification by array comparative genomic hybridization (log2 ratio 0.399). EGFR phosphorylation was also detected. Angiogensis and insulin-like growth factor pathways were also implicated by overexpression of VEGFC and IRS1. In order to improve response to chemotherapy, the PDX was treated with carboplatin/paclitaxel with or without a pan-HER and VEGF inhibitor (BMS-690514) but there was no tumor growth inhibition or improved animal survival, which may be explained by a KRAS mutation. Resistance was also observed when the IGF-1R inhibitor BMS-754807 was combined with carboplatin/paclitaxel. Because poly (ADP-ribose) polymerase inhibitors have activity in ovarian cancer patients, with and without BRCA mutations, ABT-888 was also tested but found to have no activity. Pathogenic mutations were also detected in TP53 and PIK3CA. In conclusion, ifosfamide/paclitaxel was superior to carboplatin/paclitaxel in this ovarian carcinosarcoma PDX and gene overexpression or amplification alone was not sufficient to predict response to targeted therapy. Better predictive markers of response are needed.

Background: High tumor dissemination (HTD) is a major risk factor for serious morbidity after primary ovarian cancer (OC) surgery, particularly in medically compromised patients. We performed a pilot study of whether CT findings could predict extent of disease and surgical complexity necessary in advanced OC., Methods: Preoperative CT images for patients with advanced OC from 1997-2003 were evaluated for rigorously defined disease-related findings and compared to both the findings at exploration and the required surgical procedures. Associations were assessed by the chi-square test., Results: Forty-six cases met inclusion criteria. Mean age was 66.4y, and 76% had residual disease (RD) 1cm or less. CT and surgical findings correlated (sensitivity/specificity) as follows: diaphragm disease (48%/100%); surface liver (100%/93%); omental cake (72%/65%); any sigmoid involvement (54%/100%); ascites (44%/100%); extra-pelvic large bowel involvement (29%/91%). When diaphragm disease and omental cake were present, HTD was found in all cases (positive predictive value and specificity=100%, sensitivity 48%). For CT findings of liver, large bowel and spleen involvement there was a strong trend toward resection (P=0.001, P=0.06 and P=0.06, respectively)., Conclusions: The findings of diaphragm disease and omental cake on CT scan are highly predictive for high tumor dissemination (HTD) and thus likelihood of extensive surgery required to achieve low residual disease. In addition, multiple CT findings correlate strongly with the need for higher surgical complexity which should facilitate preoperative planning and/or triage to specialized centers. These preliminary data suggest specific CT findings can be used to optimize treatment planning., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a deeper insight on the SAR for a better understanding of their potential interactions and inhibitory activity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our previously reported Relacin., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Bacterial persistence has been shown to be an underlying factor in the failure of antibiotic treatments. Although many pathways, among them the stringent response and toxin-antitoxin modules, have been linked to antibiotic persistence, a clear molecular mechanism for the growth arrest that characterizes persistent bacteria remained elusive. Here, we screened an expression library for putative targets of HipA, the first toxin linked to persistence, and a serine/threonine kinase. We found that the expression of GltX, the glutamyl-tRNA-synthetase, reverses the toxicity of HipA and prevents persister formation. We show that upon HipA expression, GltX undergoes phosphorylation at Ser239, its ATP-binding site. This phosphorylation leads to accumulation of uncharged tRNA(Glu) in the cell, which results in the activation of the stringent response. Our findings demonstrate a mechanism for persister formation by the hipBA toxin-antitoxin module and provide an explanation for the long-observed connection between persistence and the stringent response.

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.

Objective: To evaluate the efficacy and biological effects of the gemcitabine/tanespimycin combination in patients with advanced ovarian and peritoneal cancer. To assess the effect of tanespimycin on tumor cells, levels of the chaperone proteins HSP90 and HSP70 were examined in peripheral blood mononuclear cells (PBMC) and paired tumor biopsy lysates., Methods: Two-cohort phase II clinical trial. Patients were grouped according to prior gemcitabine therapy. All participants received tanespimycin 154 mg/m(2) on days 1 and 9 of cycle 1 and days 2 and 9 of subsequent cycles. Patients also received gemcitabine 750 mg/m(2) on day 8 of the first treatment cycle and days 1 and 8 of subsequent cycles., Results: The tanespimycin/gemcitabine combination induced a partial response in 1 gemcitabine naïve patient and no partial responses in gemcitabine resistant patients. Stable disease was seen in 6 patients (2 gemcitabine naïve and 4 gemcitabine resistant). The most common toxicities were hematologic (anemia and neutropenia) as well as nausea and vomiting. Immunoblotting demonstrated limited upregulation of HSP70 but little or no change in levels of most client proteins in PBMC and paired tumor samples., Conclusions: Although well tolerated, the tanespimycin/gemcitabine combination exhibited limited anticancer activity in patients with advanced epithelial ovarian and primary peritoneal carcinoma, perhaps because of failure to significantly downregulate the client proteins at clinically achievable exposures., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A prominent feature of the stringent response is the accumulation of two unusual phosphorylated derivatives of GTP and GDP (pppGpp: 5'-triphosphate-3'-diphosphate, and ppGpp: 5'-3'-bis-diphosphate), collectively called (p)ppGpp, within a few seconds after the onset of amino-acid starvation. The synthesis of these 'alarmone' compounds is catalyzed by RelA homologues. Other features of the stringent response include inhibition of stable RNA synthesis and modulation of transcription, replication, and translation. (p)ppGpp accumulation is important for virulence induction, differentiation and antibiotic resistance. We have synthesized a group of (p)ppGpp analogues and tested them as competitive inhibitors of Rel proteins in vitro. 2'-Deoxyguanosine-3'-5'-di(methylene bisphosphonate) [compound (10)] was found as an inhibitor that reduces ppGpp formation in both Gram-negative and Gram-positive bacteria. In silico docking together with competitive inhibition analysis suggests that compound (10) inhibits activity of Rel proteins by competing with GTP/GDP for its binding site. As Rel proteins are completely absent in mammalians, this appears to be a very attractive approach for the development of novel antibacterial agents., (Copyright 2010 Elsevier Ltd. All rights reserved.)

MazG is a homodimeric alpha-helical protein that belongs to the superfamily of all-alpha NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-alpha NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual alpha-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH <6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.

Immediately after sensing the inception of amino acid starvation, bacteria respond pleiotropically with the stringent response via RelA, mainly resulting in the accumulation of the signal molecule (p)ppGpp. A series of analogues of ppGpp that inhibit RelA activity was prepared in order to control the ability of bacteria cells to react to the changes in their environment. Some of those compounds presented very clear inhibitory effect on both Gram positive and negative bacteria in vitro.

Objective: To report the first placement of an abdominal cervicoisthmic cerclage using the da Vinci robot., Design: Case report., Setting: Tertiary-care hospital., Patient(s): A 39-year-old female with a history of cervical insufficiency who required a cerclage and was not a candidate for transvaginal cerclage placement., Intervention(s): Abdominal cervicoisthmic cerclage placement using the da Vinci robot., Main Outcome Measure(s): Ability to safely and successfully place an abdominal cerclage using the da Vinci robot., Result(s): Abdominal cerclage was successfully placed using the da Vinci robot. The patient had minimal blood loss and was discharged to home on the same day as surgery., Conclusion(s): Da Vinci robot-assisted abdominal cerclage placement is an innovative application of robotic surgery and may alter the standard of care for women who require this surgery.

Background: Butterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occurring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-beta signaling pathways as putative morphogens for eyespot development., Results: Saturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development., Conclusion: Wingless and TGF-beta ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution.

In the central nervous system, Connexin32 and Connexin47 are confined to oligodendrocytes where they contribute to myelin formation and maintenance, and are essential for establishing a functional glial syncytium that ensures ionic homeostasis. Despite their importance, not much is known about the regulation of connexin gene expression in oligodendrocytes. Here, we identify group E Sox proteins, in particular Sox10, as essential transcriptional regulators of both connexins. Not only was expression of Connexin32 and Connexin47 severely compromised in spinal cords of mouse mutants with reduced amounts of group E Sox proteins. Sox10 also stimulated in transient transfections the Connexin32 promoter as well as Connexin47 promoter 1b which is the main Connexin47 promoter active in the postnatal spinal cord. Detailed characterization of Connexin47 promoter 1b identified a single monomer binding site that mediated Sox10-dependent promoter activation. The region containing this binding site was also occupied by endogenous Sox10 in 33B oligodendroglioma cells. These results add Connexin47 and Connexin32 to the list of Sox10 target genes and argue that Sox10 may influence transcription of many terminal differentiation and myelination genes in oligodendrocytes as an essential regulatory component of the myelination program.

We have previously reported that mazEF, the first regulatable chromosomal 'addiction module' located on the Escherichia coli chromosome, downstream from the relA gene, plays a crucial role in the programmed cell death in bacteria under stressful conditions. It consists of a pair of genes encoding a stable toxin, MazF, and MazE, a labile antitoxin interacting with MazF to form a complex. The cellular target of MazF toxin was recently described to be cellular mRNA, which is degraded by this toxin. On the same operon, downstream to the mazEF genes, we found another open reading frame, which was called mazG. Recently, it was shown that the MazG protein has a nucleotide pyrophosphohydrolase activity. Here we show that mazG is being transcribed in the same polycistronic mRNA with mazEF. We also show that the enzymatic activity of MazG is inhibited by MazEF proteins. When the complex MazEF was added, the enzymatic activity of MazG was about 70% inhibited. We demonstrate that the enzymatic activity of MazG in vivo causes depletion of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation conditions. Based on our results, we propose a model in which this third gene, which is unique for chromosomal addiction systems, has a function of limiting the deleterious activity of MazF toxin. In addition, MazG solves a frequently encountered biological problem: how to avoid the persistence of a toxic product beyond the time when its toxicity is useful to the survival of the population.

The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions. It codes for the toxin MazF and the antitoxin MazE, which interferes with the lethal action of the toxin. To better understand the role of various conformations of MazE in bacterial life, its order-disorder transitions were monitored by differential scanning calorimetry, spectropolarimetry, and fluorimetry. The changes in spectral and thermodynamic properties accompanying MazE dimer denaturation can be described in terms of a compensating reversible process of the partial folding of the unstructured C-terminal half (high mean net charge, low mean hydrophobicity) and monomerization coupled with the partial unfolding of the structured N-terminal half (low mean net charge, high mean hydrophobicity). At pH

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

Sox8 belongs to a family of transcription regulators characterized by a unique DNA-binding domain known as the high mobility group box. Many Sox proteins play fundamental roles in vertebrate development and differentiation processes. Expression of Sox8 is strong during embryonic muscle development and gradually declines postnatally. In this study, we report that in adult skeletal muscle Sox8 is confined to satellite cells. Down-regulation during myogenic differentiation was also detected in cell culture systems and occurred in parallel with down-regulation of the related Sox9. Overexpression of Sox8 or Sox9 on the other hand disrupted myoblasts in their ability to form myotubes. Concomitantly, expression of MyoD and myogenin decreased and basal as well as MyoD-induced activities of the myogenin promoter were strongly reduced in a Sox8-dependent manner. Our data suggest that Sox8 acts as a specific negative regulator of skeletal muscle differentiation, possibly by interfering with the function of myogenic basic helix-loop-helix proteins.

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.

Sox10 belongs to a family of transcription regulators characterized by a DNA-binding domain known as the HMG box. It plays fundamental roles in neural crest development, peripheral gliogenesis, and terminal differentiation of oligodendrocytes. In accord with its function as transcription factor, Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus. In this study, we report that Sox10 is an active nucleocytoplasmic shuttle protein, competent of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export signal within the DNA-binding domain of Sox10. Mutational inactivation of this nuclear export signal or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited nuclear export and consequently nucleocytoplasmic shuttling of Sox10. Importantly, the inhibition of the nuclear export of Sox10 led to decreased transactivation of transfected reporters and endogenous target genes, arguing that continuous nucleocytoplasmic shuttling is essential for the function of Sox10. To our knowledge this is the first time that nuclear export has been reported and shown to be functionally relevant for any Sox protein.

Binding of the Escherichia coli global transcription factor FIS to the upstream activating sequence (UAS) of stable RNA promoters activates transcription on the outgrowth of cells from stationary phase. Paradoxically, while these promoters require negative supercoiling of DNA for optimal activity, FIS counteracts the increase of negative superhelical density by DNA gyrase. We demonstrate that binding of FIS at the UAS protects the rrnA P1 promoter from inactivation at suboptimal superhelical densities. This effect is correlated with FIS-dependent constraint of writhe and facilitated untwisting of promoter DNA. We infer that FIS maintains stable RNA transcription by stabilizing local writhe in the UAS. These results suggest a novel mechanism of transcriptional regulation by a transcription factor acting as a local topological homeostat.

The discovery of toxin-antitoxin gene pairs (also called addiction modules) on extrachromosomal elements of Escherichia coli, and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures. We have reported on the E. coli mazEF system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and MazE is a labile antitoxin. Here we show that cell death mediated by the E. coli mazEF module can be triggered by several antibiotics (rifampicin, chloramphenicol, and spectinomycin) that are general inhibitors of transcription and/or translation. These antibiotics inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes cell death. Our results have implications for the possible mode(s) of action of this group of antibiotics.

The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death. This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE. Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF. We show that the expression of this module is positively regulated by the factor for inversion stimulation. What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c". When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results. We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions.

The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA(+) cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752-5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

In bacteria, programmed cell death is mediated through "addiction modules" consisting of two genes. The product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. Here we extensively review what is known about those modules that are borne by one of a number of Escherichia coli extrachromosomal elements and are responsible for the postsegregational killing effect. We focus on a recently discovered chromosomally borne regulatable addiction module in E. coli that responds to nutritional stress and also on an antideath gene of the E. coli bacteriophage lambda. We consider the relation of these two to programmed cell death and antideath in bacterial cultures. Finally, we discuss the similarities between basic features of programmed cell death and antideath in both prokaryotes and eukaryotes and the possibility that they share a common evolutionary origin.

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.

The immediate early BRLF1 and BZLF1 promoters of Epstein-Barr virus are crucial for triggering the replicative cycle of the virus. To better understand the cell type dependence of the lytic cycle we conducted an analysis of the BRLF1-promoter in the epithelial cell line HeLa and the lymphoid cell line IM9. To analyze promoter activities, transient transfections with 5'-deletions of the BRLF1-promoter in front of luciferase as reporter gene were conducted. Besides the already known cis-acting elements of the promoter close to the TATA-box, more distal elements were located and functionally tested. A nuclear factor 1 consensus site was found to act positively in HeLa cells, but did not in lymphoid IM9 cells. The NF1 site was shown to bind protein by electrophoretic mobility shift assays, antibody-supershifts and in vitro footprinting. Thus, a protein belonging to the nuclear factor 1 family of proteins was identified as additional cellular trans-acting factor for the BRLF1-promoter besides the already described factors Sp1, Zta and Zif268.

Most bacterial RNA consists of stable RNA which is composed of rRNA and tRNA. We have followed by primer extension analysis the level of ribosomal RNA synthesis along the growth phases of a cell culture. A sharp drop in rRNA synthesis was observed upon the transition from the exponential to the stationary phase of growth. Our results demonstrate that an effective shut-off of rRNA synthesis occurs also in the absence of ppGpp. Mutations in the host factors Fis and H-NS, which are known to regulate rrn P1 promoters, did not affect the shut-off process of ribosomal RNA promoters. We also tested the effect of RpoS, the sigma factor known to induce a number of genes in the stationary phase. It was shown that the host factors Fis, H-NS and RpoS do not play a major role in the regulation of the shut-off process of rRNA synthesis. The results presented demonstrate that the rate of rRNA synthesis provides a sensitive measure of the growth phase of the bacterial culture.

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.