Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner-Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner-Doudoroff and Embden-Meyerhof-Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. RESULTS: Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner-Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden-Meyerhof-Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. CONCLUSIONS: Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.

Insights into metabolic osmoadaptation of the ectoines-producer bacterium Chromohalobacter salexigens through a high-quality genome scale metabolic model.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. RESULTS: We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. CONCLUSIONS: Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.

Understanding the interplay of carbon and nitrogen supply for ectoines production and metabolic overflow in high density cultures of Chromohalobacter salexigens.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. RESULTS: Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect - mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L-1) and volumetric ectoine yields (up to 4.21 g L-1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes. CONCLUSIONS: Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.

Investigating vaginal bleeding in postmenopausal women found to have an endometrial thickness of equal to or greater than 10 mm on ultrasonography.

PURPOSE: This aim of this study is to determine the risk of endometrial cancer in symptomatic postmenopausal women, when endometrial thickness on transvaginal ultrasonography is equal to or greater than 10 mm, and subsequent office-based endometrial sampling histology is negative. METHODS: This is a prospective cross-sectional study, performed in a gynaecological oncology centre in the United Kingdom between February 2008 and July 2012. All postmenopausal women presenting with vaginal bleeding were investigated using transvaginal ultrasonography. Women with endometrial thickness measurements equal to or greater than 10 mm and negative office-based endometrial biopsy underwent hysteroscopy and endometrial biopsies. RESULTS: Over a 52-month period, 4148 women were investigated for postmenopausal vaginal bleeding. 588 (14.2%) women were found to have endometrial thickness measurements of equal to or greater than 10 mm on transvaginal ultrasonography. 170 (28.9%) cases of endometrial cancer were diagnosed in this group: 149 (87.6%) of the cancer cases were diagnosed in the outpatient setting with a Pipelle® endometrial sampler, whilst 21 (12.4%) had a negative Pipelle® sample and were diagnosed with hysteroscopy. The group diagnosed with hysteroscopy had lower BMI (32.7 kg/m2 versus 39.7 kg/m2, p < 0.001) whilst the group diagnosed with Pipelle was more likely to have a history of hypertension and diabetes mellitus (p = 0.019 for both). The sensitivity of Pipelle was 87.65%. CONCLUSION: For women presenting with postmenopausal bleeding and where the endometrial thickness is equal to or greater than 10 mm and Pipelle sampling is negative, hysteroscopic evaluation with directed biopsy is strongly recommended.

The impact of ultra-radical surgery in the management of patients with stage IIIC and IV epithelial ovarian, fallopian tube, and peritoneal cancer.

OBJECTIVE: The aim of this study is to estimate the percentage of patients with metastatic ovarian, fallopian tube, and primary peritoneal cancer requiring ultra-radical surgery to achieve cytoreduction to less than 1 cm (optimal) or no macroscopic residual disease (complete). METHODS: Perioperative data were collected prospectively on consecutive patients undergoing elective cytoreductive surgery for metastatic epithelial ovarian, fallopian tube, or primary peritoneal cancer at the Norfolk and Norwich University Hospital, a tertiary referral cancer centre in the United Kingdom from November 2012 to June 2016. RESULTS: Over a 42-month period, 135 consecutive patients underwent cytoreductive surgery for stage IIIC and IV ovarian, fallopian tube, or primary peritoneal cancer. The median age of the patients was 69 years. 47.4% of the patients underwent diaphragmatic peritonectomy and/or resection, 20% underwent splenectomy, 14.1% had excision of disease from porta hepatis and celiac axis, and 5.2% of the patients had gastrectomy. Cytoreduction to no macroscopic visible disease (complete) and to disease with greater tumour diameter of less than 1 cm (optimal) was achieved in 54.1 and 34.1% of the cases, respectively. Without incorporating surgical procedures in the upper abdomen ('ultra-radical'), the combined rate of complete and optimal cytoreduction would be only 33.3%. CONCLUSIONS: Up to 50.4% of the patients in this study required at least one surgical procedure classified as ultra-radical, emphasizing the importance of cytoreductive surgery in the upper abdomen in management of women with stage IIIC and IV ovarian, fallopian tube, and primary peritoneal cancer.

Role of central metabolism in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens.

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-(13)C]-, [2-(13)C]-, [6-(13)C]-, and [U-(13)C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.

Temperature- and salinity-decoupled overproduction of hydroxyectoine by Chromohalobacter salexigens.

Hydroxyectoine overproduction by the natural producer Chromohalobacter salexigens is presented in this study. Genetically engineered strains were constructed that at low salinity coexpressed, in a vector derived from a native plasmid, the ectoine (ectABC) and hydroxyectoine (ectD) genes under the control of the ectA promoter, in a temperature-independent manner. Hydroxyectoine production was further improved by increasing the copies of ectD and using a C. salexigens genetic background unable to synthesize ectoines.

Interval Cytoreductive Surgery and Heated Intraperitoneal Chemotherapy for Ovarian Cancer: Report of a Single-center Experience.

BACKGROUND/AIM: Emerging data suggest that addition of hyperthermic intraperitoneal chemotherapy (HIPEC) at the time of interval cytoreduction for patients with metastatic ovarian cancer is associated with a survival benefit. However, the implementation of this treatment is affected by concerns related to its potential morbidity. We present data from the first centre in the UK implementing HIPEC as part of treatment for patients with advanced ovarian cancer undergoing interval cytoreductive surgery. PATIENTS AND METHODS: This is a prospective study of patients planned to undergo cytoreductive surgery and HIPEC for advanced ovarian cancer over a 30-month period. All patients had undergone neoadjuvant chemotherapy prior to surgery. Patients with stage III/IV ovarian cancer who underwent complete or near complete cytoreduction (<2.5 mm residual disease) received HIPEC using a closed technique. RESULTS: A total of 31 patients were included in the study, of which 30 had complete cytoreduction and 1 patient had residual disease <2.5 mm. The mean age of the patients was 63.7±2.8 years. Median peritoneal cancer index score was 9 (range=3-31). The mean operating time was 515.4±55.1 min. The mean length of hospital stay was 7.6±0.8 days. In total, 24 complications were observed in 18 patients (58.1%), while 6.5% of the patients experienced grade 3/4 complications. There were no deaths within 30-days from the surgery. Age was found to be an independent predictor of both postoperative complications of any grade and prolonged hospital stay. CONCLUSION: Interval cytoreductive surgery and HIPEC for patients with advanced ovarian cancer is associated with low perioperative morbidity.

Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli.

BACKGROUND: The compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions. RESULTS: Besides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage. CONCLUSIONS: Our findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.

Outcome of investigations for postmenopausal vaginal bleeding in women under the age of 50 years.

OBJECTIVE: The objective of this study is to determine the incidence of endometrial cancer in young postmenopausal women presenting with vaginal bleeding. METHODS: Cross-sectional study of postmenopausal women presenting with vaginal bleeding in a gynaecological oncology centre in the United Kingdom. All women underwent transvaginal ultrasound scanning (TVS) as the initial investigation tool to evaluate the endometrium. Endometrial biopsy was performed only in cases where endometrial thickness measured equal to or greater than 5mm. The patients were divided into two groups based on their age: less than 50 years (Group A) and 50 years or older (Group B). RESULTS: Over a 57-month period, 4454 women were investigated for postmenopausal vaginal bleeding. Of these, 259 (5.8%) women were diagnosed with endometrial carcinoma. 260 (5.8%) women were younger than 50 years. Endometrial biopsy was not performed in 130 women in Group A that had an endometrial thickness measurement of less than 5mm on ultrasonography. With a median follow-up period of 3 (1-5) years, we found no cases of endometrial cancer in women under the age of 50 that did not undergo endometrial biopsy at the time of initial evaluation. Overall, no cases of endometrial cancer were diagnosed in postmenopausal women under the age of 50 years. CONCLUSIONS: We found no cases of endometrial cancer amongst 260 women presenting with postmenopausal vaginal bleeding under the age of 50 years. These women could be investigated on a less urgent basis depending on the available resources.

Management of postmenopausal women with vaginal bleeding when the endometrium can not be visualized.

OBJECTIVE: To determine the risk of endometrial cancer when endometrial thickness is not visualized using ultrasonography. DESIGN: Cross-sectional study. SETTING: Gynecological oncology center in the United Kingdom. POPULATION: All postmenopausal women referred with vaginal bleeding. METHODS: All women were investigated using gray-scale transvaginal ultrasonography. Women were arbitrarily stratified into four groups according to the endometrial thickness measurement. Women with endometrial thickness that was not adequately visualized on ultrasonography were included in a separate group. MAIN OUTCOME MEASURES: Endometrial cancer diagnosis. RESULTS: Over a 50-month period, 4454 women were investigated for postmenopausal vaginal bleeding. A total of 259 (6%) of women were diagnosed with endometrial carcinoma. Endometrial thickness measured 5-9.9 mm in 1201 (27%), 10-14.9 mm in 468 (11%), 15-19.9 mm in 209 (5%), and equal to or greater than 20mm in 197 (4%) of women. In 174 (4%) of women, the endometrial thickness was not visualized on transvaginal ultrasonography. For women where the endometrial thickness was not adequately visualized, the final histology included benign endometrium (124), endometrial cancer (26), endometrial polyps (11), endometritis (7), and other pathology (7). The odds of endometrial cancer in women where the endometrial thickness was not visualized were found to be significantly higher than the odds of cancer for women with an endometrial thickness of 5-9.9 mm (OR = 5.23, 95%CI 3.10-8.85, p-value <0.0001). CONCLUSIONS: For women presenting with postmenopausal bleeding and where the endometrial thickness cannot be adequately visualized on ultrasonography, hysteroscopic evaluation is recommended.

A novel approach to postoperative bladder care in women after radical hysterectomy.

PURPOSE: Currently, controversy exists with regard to the duration of bladder drainage and choice of catheter used in women who undergo radical hysterectomy. In this manuscript, we propose a novel approach to improving postoperative bladder care in women who undergo radical hysterectomy. METHODS: This is a retrospective study of women who underwent Type 3 Piver radical hysterectomy in a gynaecological oncology centre in the United Kingdom from January 2009 to September 2011. We report the outcomes of removal of urinary catheter 48-72 h following radical hysterectomy. RESULTS: Over a 32-month period, 30 women underwent radical hysterectomy. 19 (63.3 %) women underwent surgery for treatment of cervical cancer, 5 (16.7 %) women for management of endometrial cancer, 6 (20 %) women for other conditions. One patient underwent partial cystectomy at the time of radical hysterectomy and was not included in the analysis. Of the 29 patients, only five (17.2 %) were found to have urinary residuals greater than 100 ml following the removal of the indwelling catheter on the second postoperative day and required recatheterisation. 82.8 % of the patients had the catheter removed within 48-72 h postoperatively. None of these patients required re-admission with urinary retention. CONCLUSION: Removal of urinary catheter on the second postoperative day following radical hysterectomy is feasible and not associated with increased morbidity. This approach may be particularly useful to complement the introduction of laparoscopic and robotic surgical approaches for surgical management of cervical cancer.

How to improve training in bowel surgery for gynecological oncologists-experience from a single center in the United kingdom.

INTRODUCTION: There is now a growing realization of the lack of experience of gynecological oncology trainees in gastrointestinal surgery. Advanced fellowship programs in gastrointestinal surgery have been suggested as a potential solution to this problem. PATIENTS AND METHODS: We present data relating to gastrointestinal procedures performed by the gynecological oncology trainee during a fellowship program over a 3-year period in a single gynecological oncology center in the United Kingdom. RESULTS: Over a 36-month period, 369 cases of invasive ovarian cancer were diagnosed in our institute, of which 278 (75.3%) were stage III/IV disease. Bowel surgery was performed in 86 patients (30.9%) with stage III/IV ovarian cancer. A total of 121 gastrointestinal procedures were performed during the study period, as some patients had more than one procedure. We present the procedures the gynecological oncology fellow performed and assisted during this period. DISCUSSION: To improve competencies in performing bowel surgery among gynecological oncology trainees, we suggest sustained exposure in bowel surgery over the entire duration of the training program.

Estimating the risk of endometrial cancer in symptomatic postmenopausal women: a novel clinical prediction model based on patients' characteristics.

INTRODUCTION: The aim of this study was to develop a multivariable model to predict the risk of endometrial carcinoma in postmenopausal women with vaginal bleeding using individuals' clinical characteristics. PATIENTS AND METHODS: This prospective study of consecutive postmenopausal women presenting with vaginal bleeding was conducted at a gynecological oncology center in the United Kingdom for a 46-month period. All women underwent transvaginal ultrasound scanning as the initial investigation tool to evaluate the endometrium. Women found to have an endometrial thickness 5 mm or more had endometrial sampling performed. RESULTS: Of a total of 3548 women presenting with vaginal bleeding during the study period, 201 (6%) women had a diagnosis of endometrial carcinoma. An investigator-led best model selection approach used to select the best predictors of cancer in the multiple logistic regression model showed that patient's age (odds ratio [OR], 1.06), body mass index (OR, 1.07), recurrent episodes of bleeding (OR, 3.64), and a history of diabetes (OR, 1.48) increased the risk of endometrial malignancy when corrected for other characteristics. The mentioned clinical variables satisfied the criteria for inclusion in our predictive model called FAD 31 (F for the frequency of bleeding episodes, A for the age of the patient, D for diabetes, and the number 31 represents the BMI cut-off value). The total score for the model varies from 0 to 8. The area under the receiver operating characteristics curve for the developed model was 0.73 (95% confidence interval, 0.70-0.77). DISCUSSION: We have developed a simple model based on patients' clinical characteristics in estimating the risk of endometrial cancer for postmenopausal women presenting with vaginal bleeding. The model shows reasonable discriminatory ability for women with cancer and without, with an area under the receiver operating characteristics curve of 0.73. This will allow clinicians to individualize the diagnostic pathway for women with postmenopausal vaginal bleeding.

New insights into hydroxyectoine synthesis and its transcriptional regulation in the broad-salt growing halophilic bacterium Chromohalobacter salexigens.

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyectoine production.

Interplay between iron homeostasis and the osmotic stress response in the halophilic bacterium Chromohalobacter salexigens.

In this study, the connection between iron homeostasis and the osmostress response in the halophile Chromohalobacter salexigens was investigated. A decrease in the requirement for both iron and histidine and a lower level of siderophore synthesis were observed at high salinity, and these findings were correlated with a lower protein content in salt-stressed cells. A six-gene operon (cfuABC-fur-hisI-orf6 operon) located downstream of the ectABC ectoine synthesis genes was characterized. A fur strain (in which the ferric iron uptake regulator Fur was affected) had the Mn resistance phenotype typical of fur mutants, was deregulated for siderophore production, and displayed delayed growth under iron limitation conditions, indicating that fur encodes a functional iron regulator. hisI was essential for histidine synthesis, which in turn was necessary for siderophore production. Fur boxes were found in the promoters of the cfuABC-fur-hisI-orf6 and ectABC operons, suggesting that Fur directly interacts with DNA in these regions. Fur mediated the osmoregulated inhibition of cfuABC-fur-hisI-orf6 operon expression by iron and functioned as a positive regulator of the ectABC genes under high-salinity conditions, linking the salt stress response with iron homeostasis. Excess iron led to a higher cytoplasmic hydroxyectoine content, suggesting that hydroxyectoine protects against the oxidative stress caused by iron better than ectoine. This study provides the first evidence of involvement of the iron homeostasis regulator Fur as part of the complex circuit that controls the response to osmotic stress in halophilic bacteria.

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens.

BACKGROUND: Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions. RESULTS: A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain. CONCLUSIONS: This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields.

BACKGROUND: Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T. RESULTS: The most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the beta-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined. CONCLUSIONS: The soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.

Cholecystectomy as Part of Cytoreductive Surgery for Advanced Ovarian Cancer: Perioperative Outcomes.

BACKGROUND/AIM: To assess the perioperative outcomes of cholecystectomy in cytoreductive procedures for epithelial ovarian cancer (EOC). PATIENTS AND METHODS: Prospectively collected perioperative data of patients that underwent cytoreduction for advanced EOC, between 2014 and 2018, were analysed. Patients were divided in two groups on the basis of whether cholecystectomy was performed. RESULTS: A total of 144 patients with stage IIIC/IV EOC were included. Cholecystectomy was performed in 22 (15.3%) patients. Those who underwent cholecystectomy more likely required diaphragmatic peritonectomy, splenectomy, lesser omentectomy, excision of disease from the porta hepatis and liver's capsule (p<0.001). There was no difference in the cytoreductive outcomes (complete or optimal) and the rate of grade 3-5 complications between the two groups (p=0.10 & p=0.06, respectively). No direct complications related to cholecystectomy were observed. CONCLUSION: A significant percentage of patients with advanced EOC require cholecystectomy. Gynecologic oncologists should embrace the opportunity to develop advanced surgical skills including cholecystectomy.

The Role of Magnetic Resonance Imaging in the Pre-operative Evaluation of Women Diagnosed With Atypical Endometrial Hyperplasia.

BACKGROUND/AIM: To evaluate the role of MRI in patients with atypical endometrial hyperplasia (AEH) and incorporate MRI findings in predictive models estimating the risk of co-existent endometrial cancer (EC). PATIENTS AND METHODS: Data from 189 women diagnosed with AEH and had MRI scan prior to operation, over nine years, were retrospectively collected. RESULTS: Histology showed EC in 51 (27%) cases. Presence of myometrial invasion on MRI was more commonly detected in patients with EC compared to those with benign pathology (37.3% versus 10.9%, p<0.001). The sensitivity and specificity of MRI in identifying cancer were 37% and 89%, respectively. Age, menopausal status and presence of invasion on MRI were the best predictors for the presence of malignancy. CONCLUSION: Myometrial invasion on MRI is associated with increased risk of EC in women with AEH. Its accuracy in detecting malignancy improves when combined with clinical parameters. This could be of value for conservative-management candidates.