Metabolic Engineering of Saccharomyces cerevisiae for Rosmarinic Acid Production.

Rosmarinic acid is a hydroxycinnamic acid ester commonly found in the Boraginaceae and Lamiaceae plant families. It exhibits various biological activities, including antioxidant, anti-inflammatory, antibacterial, antiallergic, and antiviral properties. Rosmarinic acid is used as a food and cosmetic ingredient, and several pharmaceutical applications have been suggested as well. Rosmarinic acid is currently produced by extraction from plants or chemical synthesis; however, due to limited availability of the plant sources and the complexity of the chemical synthesis method, there is an increasing interest in producing this compound by microbial fermentation. In this study, we aimed to produce rosmarinic acid by engineered baker's yeast Saccharomyces cerevisiae. Multiple biosynthetic pathway variants, carrying only plant genes or a combination of plant and Escherichia coli genes, were implemented using a full factorial design of experiment. Through analysis of variances, the effect of each enzyme variant (factors), together with possible interactions between these factors, was assessed. The best pathway variant produced 2.95 ± 0.08 mg/L rosmarinic acid in mineral medium with glucose as the sole carbon source. Increasing the copy number of rosmarinic acid biosynthetic genes increased the titer to 5.93 ± 0.06 mg/L. The study shows the feasibility of producing rosmarinic acid by yeast fermentation.

Metabolic engineering and transcriptomic analysis of Saccharomyces cerevisiae producing p-coumaric acid from xylose.

BACKGROUND: Aromatic amino acids and their derivatives are valuable chemicals and are precursors for different industrially compounds. p-Coumaric acid is the main building block for complex secondary metabolites in commercial demand, such as flavonoids and polyphenols. Industrial scale production of this compound from yeast however remains challenging. RESULTS: Using metabolic engineering and a systems biology approach, we developed a Saccharomyces cerevisiae platform strain able to produce 242 mg/L of p-coumaric acid from xylose. The same strain produced only 5.35 mg/L when cultivated with glucose as carbon source. To characterise this platform strain further, transcriptomic analysis was performed, comparing this strain's growth on xylose and glucose, revealing a strong up-regulation of the glyoxylate pathway alongside increased cell wall biosynthesis and unexpectedly a decrease in aromatic amino acid gene expression when xylose was used as carbon source. CONCLUSIONS: The resulting S. cerevisiae strain represents a promising platform host for future production of p-coumaric using xylose as a carbon source.

Use of a case-control study and control bank to investigate an outbreak of locally acquired cyclosporiasis in Canada, 2016.

BACKGROUND: Cyclospora is an intestinal parasite that is not endemic in Canada. However, national outbreaks of locally acquired cases have been reported since 2013. These outbreaks were suspected to be associated with consumption of produce imported from countries where Cyclospora is endemic. Identification of the source can be challenging because of reporting delays and limited traceability of produce. OBJECTIVE: To report on a national outbreak of locally acquired cyclosporiasis, highlight the challenges of investigating these outbreaks and document the first time use of a control bank to recruit controls for a national outbreak case-control study in Canada. METHODS: Cases of cyclosporiasis were identified through provincial laboratory testing and reported through provinces to the national level. Cases were interviewed about food exposures using a questionnaire and food exposures reported by cases were compared to Foodbook reference values. To narrow down the food items of interest, a matched case-control study was conducted. Controls for the study were recruited primarily from a control bank, that is, a list of individuals who had previously agreed to participate in public health-related surveys. RESULTS: In total, 87 cases of locally acquired cyclosporiasis with onset or report dates between May 19, 2016 and August 10, 2016 were reported by four provinces. Comparing case exposures to Foodbook reference values identified several food items of interest, including blackberries, other berries, herbs and leafy greens. The case-control study identified only blackberries and mesclun greens as significantly more frequently consumed by cases than controls. Due to lack of product details for blackberries and mesclun greens, the source of the outbreak was not conclusively identified. CONCLUSION: Blackberries were the primary food item of interest, but could not be identified as the conclusive source due to lack of traceability. The control bank was found to be a useful tool for control recruitment.

[We are living longer, but in good health? Inequality in quality-adjusted life expectancy]. / We leven langer, maar ook in goede gezondheid?

OBJECTIVE: Quantifying trends in quality-adjusted life expectancy (QALE) by level of education in the Netherlands 2001-2011. DESIGN: Retrospective study. METHOD: For this study we used data from several sources. Using regression models we estimated mortality rates and quality of life as functions of age, gender, calendar year and educational level. Quality of life was measured using the SF-6D questionnaire. In order to calculate QALE we combined estimates of mortality rates and quality of life into Sullivan's life tables. RESULTS: Over the period 2001-2011 quality of life and survival increased at all educational levels. This resulted in an increase of QALE varying from 1.5 to 3 years depending on gender and education. QALE increased less strongly in people with lower education than in those with higher education, which to a large extent was due to widening inequalities in mortality. CONCLUSION: The Dutch are living longer and have a better quality of life but inequalities in QALE have increased.

EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.

Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway.

Giant Esophageal GIST: Diagnostic and Therapeutic Challenge - Case Report.

GIST are rare mesenchymal tumors of the digestive tract (less than 1% of the digestive tract neoplasia). Of these, less than 1% are found in the esophagus. Surgery is the main treatment of GIST and is supported by targeted therapy with tyrosine kinase inhibitors like imatinibmesylate. We present the case of a female patient of 51 years, admitted in our clinic for a bulky tumor in the posterior mediastinum, diagnosed after investigations performed for fatigue for the great efforts. Clinical examination was unspecific. Chest X-ray and thoraco-abdominal CT identified a widening of the mediastinum through a posterior mediastinal tumor mass, determining a deviation to the left of the thoracic esophagus without causing not abledysphagia or respiratory symptoms. It was decided surgery by thoracotomy in V right intercostal space and total excision of the tumor was performed. Histopathology examination confirmed the preoperative suspicion of esophageal GIST. Prognosis is reserved, the risk of relapse is very high given the fact that the tumor was extracted fragmented. Currently the patient is under treatment with imatinib mesylate and entered into clinical and imaging follow-up program, according to clinical guidelines.

Integration testing of heterotic systems.

Computational theory and practice generally focus on single-paradigm systems, but relatively little is known about how best to combine components based on radically different approaches (e.g. silicon chips and wetware) into a single coherent system. In particular, while testing strategies for single-technology artefacts are generally well developed, it is unclear at present how to perform integration testing on heterotic systems: can we develop a test-set generation strategy for checking whether specified behaviours emerge (and unwanted behaviours do not) when components based on radically different technologies are combined within a single system? In this paper, we describe an approach to modelling multi-technology heterotic systems using a general-purpose formal specification strategy based on Eilenberg's X-machine model of computation. We show how this approach can be used to represent disparate technologies within a single framework, and propose a strategy for using these formal models for automatic heterotic test-set generation. We illustrate our approach by showing how to derive a test set for a heterotic system combining an X-machine-based device with a cell-based P system (membrane system).

Clinical and therapeutic considerations of GIST.

Gastrointestinal stromal tumors (GIST) are rare tumors of the digestive tract, with an incidence of about 1.5 per 100,000/year. Clinical features may vary depending on location, size and aggressiveness. The diagnosis is confirmed by immunohistochemistry tests that identify CD 117 or DOG1 (typical receptors/markers for most GISTs) at the level of biopsy specimen. The treatment of localized GIST is based primarily on the surgery, while for metastatic GIST the targeted therapy with tyrosine kinase inhibitors represents the current standard. The neoadjuvant and adjuvant therapy indications guided and depending on genetic analysis included in the diagnostic and treatment algorithm as well as the strategy for cases surveillance are listed in the journal. All these data obtained from the literature have been integrated in a practical experience of 19 cases of GIST, operated in the clinic in the last 10 years for which we have proposed an adapted diagnostic algorithm.

The vascularization pattern of the colon and surgical decision in esophageal reconstruction with colon. A selective SMA and IMA arteriographic study.

INTRODUCTION: No matter the reconstructive technique, the fundamental concepts in visceral reconstruction have as main grounds the mandatory vascular support for the graft replacement. Individual vascular particularities can influence or even oblige the surgeon to choose a certain procedure. This is why the vascularization is beyond doubt the dominant factor in mobilizing the colon for reconstruction. MATERIAL AND METHOD: Our arteriographic study entails an investigation upon the vascularization pattern of the two main sources that participate in the arterial irrigation of the colon via the emerging vessels: superior mesenteric artery (SMA) and inferior mesenteric artery (IMA). We did not consider certain patients upon a specific criterion; also, we did not exclude any patients due to various reasons. We took into account 49 patients as study group, all of them having registered into the clinic for a reconstructive technique, throughout the years from 2000 to 2010. From 1981 to 2012 there have been 187 reconstructive techniques performed due to post caustic pathology. From a total of 49 patients, 11 had suffered major abdominal surgeries, 5 of which had had unsuccessful reconstructive attempts. RESULTS: Out of the 49 patients on whom we have performed the exploration, arteriography showed a favorable situation for reconstruction in 31 of them. In the other 18 patients anomalies or atypical distributions were identified, in 5 of the SMA and in 13 of the IMA, respectively. Operative decision was modified in 22 patients. One important thing to note from the point of view of the segment to be moved: we had no graft necrosis in patients with preoperative arteriographic examination. CONCLUSIONS: Due to the need for good mobilization, arterial ligations should be adjusted and modified depending on the particular vascular distribution, to maintain a sufficient blood flow in the marginal artery, in order to reach the colic sections and the straight arteries near them.

Molecular factors and criteria for predicting the response to neoadjuvant treatment in patients with esophageal squamous cell carcinoma (ESCC) -- responder/non-responder.

UNLABELLED: Regarding the mortality causes, esophageal squamous cell carcinoma (ESCC) is on the 7th place in the US and the 6th place in the world, with 5 year overall survival rate of 14%, which is still modest in comparison to other digestive neoplasia. Multiples strategies were involved to improve this percentage, associating surgical procedures and chemo- and radio- oncologic therapies. The distant results showed sensitive improvement, after introduction of multimodality neoadjuvant therapies. MATERIAL AND METHOD: Fifty-seven patients diagnosed with ESCC were evaluated between 2006 and 2010, male preponderance (77%), average age of 55. A multimodality therapeutic protocol was used: first - radio-chemotherapy (RCT), second - surgery and/or third - chemo- or radiotherapy. Four weeks post RCT all patients were evaluated to determine the response to neoadjuvant treatment followed by surgery - esophageal resection. Histopathological (HP) and immunohistochemical (IHC) analysis of the pathological specimens were performed in order to identify the molecular predictors with responsive or non responsive character; the studied markers were p53 (Dako 1:50), Ki-67 (Biogenex, 1:20), c-erbB-2 (Dako, 1:250). Based on these results, the working model used to determine the response to neoadjuvant therapy was tumor regression grade (TRG). RESULTS: After HP and IHC examination, the patients were included in two groups: responders and non responders (tumor cells > 10%). Complete neoplasia sterilization was achieved in 5 of the patients. CONCLUSIONS: We are able to state that the identification of potential predictive markers along with HP and IHC results represents a great perspective alternative in the ESCC therapy outcome. The detection of molecular type aggressiveness of the neoplastic process allows therapeutic orientation or guidance to certain therapy sequences or even to specific molecular targeted treatments.

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode.

BACKGROUND: Plasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC) using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in E. coli (strain VH33) allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the recA, deoR, nupG and endA genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions. RESULTS: The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X). The triple mutant VH33 Δ (recA deoR nupG) accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g), whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation. CONCLUSION: The pDNA concentration obtained with the engineered strain VH33 Δ (recA deoR nupG) is, to the best of our knowledge, the highest reported for a batch cultivation, and its supercoiled fraction remained close to 80%. Strain VH33 Δ (recA deoR nupG) and its cultivation using elevated glucose concentrations represent an attractive technology for fast and efficient pDNA production and a valuable alternative to fed-batch cultivations of commercial strains.

Entorhinal verrucae geometry is coincident and correlates with Alzheimer's lesions: a combined neuropathology and high-resolution ex vivo MRI analysis.

Entorhinal cortex displays a distinctive organization in layer II and forms small elevations on its surface called entorhinal verrucae. In Alzheimer's disease, the verrucae disappear due to neurofibrillary tangle formation and neuronal death. Isosurface models were reconstructed from high-resolution ex vivo MRI volumes scanned at 7.0 T and individual verruca were measured quantitatively for height, width, volume, and surface area on control and mild Alzheimer's cases. Mean verruca height was 0.13 ± 0.04 mm for our cognitively normal (controls) sample set whereas for mild AD samples mean height was 0.11 mm ± 0.05 mm (p < 0.001) in entorhinal cortex (n = 10 cases). These quantitative methods were validated by a significant correlation of verrucae height and volume with qualitative verrucae ratings (n = 36 cases). Entorhinal surfaces were significantly different from other cortical heights such as, cingulate, frontal, occipital, parietal and temporal cortices. Colocalization of verrucae with entorhinal islands was confirmed in ex vivo MRI and, moreover, verrucae ratings were negatively correlated to Braak and Braak pathological stage. This study characterizes novel methods to measure individual entorhinal verruca size, and shows that verrucae size correlates to Alzheimer's pathology. Taken together, these results suggest that verrucae may have the potential to serve as an early and specific morphological marker for mild cognitive impairment and Alzheimer's disease.

[Surgical treatment of esophageal diverticula]. / Tratamentul chirurgical al diverticulilor esofagieni.

The aim of this paper is to evaluate the methods and therapeutic principles of esophageal diverticula pathology. We analyze the main pathological mechanisms which establish the therapeutic attitude linked with a complex pretherapeutic evaluation. In our study we enrolled 12 patients operated between 2001-2009 for esophageal diverticula with different topography. In this period of time there were much more patients diagnosed with this pathology, but the need for surgery was establish very tight regarding the actual practical guide which impose the identification and interception of physiological mechanisms by the surgical procedure. We highlight the particular technical details, as well as the important differences of postoperatory complications according to the topography of the diverticula pouch.

[Gastrointestinal stromal tumor (GIST)--medical rarities?]. / Tumora stromala gastrointestinala (GIST) o raritate medicala?

Although their overall incidence is low, GISTs are distinctive subgroup of gastrointestinal mesenchymal tumors which express CD117 or platelet derived growth factor receptor alpha (PDGFRA). Considered as rare digestive cancers, tumors like schwannomas, neurofibromas, gastrointestinal leiomiomas are now reclassified as GIST based on immunohistochemistry studies. GIST are more frequent in stomach (40-70%), small bowel (20-40%), colon (5-15%), meanwhile locations such as mesentery, omentum, retro peritoneum in less of 5%. 10 GIST patients were surgically managed during 2004-2009. 5 gastric and 5 small bowel GIST. Most with symptomatic disease: palpable tumor, abdominal pain, anemia, fatigue, superior digestive hemorrhage or occlusion. Imagistic diagnosis consisted of: barium swallow, abdominal sonography, CT and PET-CT. Confirmation was made by hystopathological exam and immunohistochemistry. All patients had more or less wide surgical resections. For some patients there was also a specific adjuvant treatment. All patients survived after surgery. The principle of surgery for GIST is RO resection of the tumor. Tumor rupture or R1 resection of the primary tumor has a negative impact on disease free survival. Some patients (great volume tumors, R1 or R2 resection) had adjuvant treatment. Imatinib mesylate and derivates showed a significant improvement of recurrence free survival with one condition: permanent treatment. Surgery remains the mainstay of treatment in patients with localized, resectable GIST. Recurrence rate of 17-21% and 5 years survival rate of 48-70%, even in resectable GIST, impose an adjuvant treatment.

Exogenous platelet-activating factor improves the motility of human spermatozoa evaluated with C.A.S.A.: optimal concentration and incubation time.

The objective of this study is to determine the optimal conditions for human semen incubation treated with exogenous platelet activating factor (ePAF) for intra-uterine insemination (IUI). This prospective study was carried out on 32 infertile men and each semen sample was processed with the ISolate Sperm Separation Medium, washed with sperm washing medium (SWM) and resuspended either in SWM alone (control samples), or with ePAF 0.1, 0.5, and 1.0 µM. Each concentration was subsequently incubated and evaluated at 5, 15, 30, and 60 min. The motility parameters were evaluated by the computer-aided sperm analysis (C.A.S.A.) system. Curvilinear velocity, straight line velocity, average path velocity, rapid and progressive motility significantly increased compared to control samples at an ePAF concentration of 0.1 µM (with at least 15 min of incubation). The best results were obtained with ePAF concentrations of: 0.1 µM (60 min of incubation) and 0.5 µM (30-60 min of incubation). In conclusion, results are enhanced when ePAF is added to standard semen preparation for IUI. An ePAF concentration of 0.1 µM, with an incubation time of 15 min, can be used for semen samples with normal motility. Whilst, for semen samples with poor motility, the ePAF concentration is best increased to 0.5 µM and/or the incubation time prolonged to 60 min.

Clinical anatomy and surgical repair of prepubic hernia in dogs and cats.

Prepubic hernia is a traumatic hernia in small animals, most often associated with severe trauma to the caudal abdomen. Common causes include vehicular trauma, dog fights, and kicks by large animals. Rupture of the prepubic tendon in dogs and of its equivalent in cats results in a ventral abdominal hernia. Due to the traumatic nature of the injury, concurrent injuries are frequently seen. Clinical signs of herniation are often nonspecific, and the resultant hernia may not be readily apparent during routine physical examination. This being so, diagnosis is often based on a thorough physical examination in conjunction with abdominal radiography and, possibly, abdominal ultrasonography. Multiple methods of repair of prepubic hernia have been reported, and survival rates are quite good if concurrent injuries are not severe.

[Vitamin intake and pregnancy]. / Apporto vitaminico ed evoluzione della gravidanza.

A suitable intake of vitamins and minerals both during preconceptional period and pregnancy is essential in reducing the incidence of adverse maternal and perinatal outcomes. This is more evident in developing countries, particularly during periods of famine, when women suffer from an inadequate intake of vitamins and minerals. Even in developed countries, however, most women's diet does not meet their increased needs for micronutrients. The association of different micronutrients in a single multivitamin preparation is consequently a useful, easy to take solution and with a good cost/benefit ratio, and can prevent some important obstetrics pathologies, as preterm delivery, fetal growth restriction and preeclampsia.