A case of an inverted appendiceal stump with dysplastic mucosa mimicking cecal polyp managed by a combined endoscopic laparoscopic approach / Un caso de muñón apendicular invertido con mucosa displásica que imita Pólipo cecal tratado mediante un abordaje laparoscópico endoscópico combinado

Endoscopic finding of invaginated appendiceal stump mimicking polyp is very rare. We present a case of patient with appendiceal stump mimicking cecal polyp covered with dysplastic mucosa which makes it even more rare. Polypoid lesions involving the appendiceal area represent a specific diagnostic-therapeutic dilemma. In these situations simple colonoscopic polypectomy poses increased risk for perforation. In our case biopsy of the polypoid cecal lesion revealed dysplastic mucosa. Due to the non-lifting sign and increased risk of perforation from simple polypectomy, polyp was removed by cecal wedge resection using combined endoscopic laparoscopic approach. Histopathology of the specimen confirmed medium dysplastic epithelium covering lymphoid follicles specific for appendix. In our case, decision on the need to remove the polyp, based on biopsy histology, was quite clear; however, the question is what should we have done if the biopsy finding was negative? In such a situation, conservative approach with endoscopic follow-up of the polyp and regular biopsy specimen analysis, or a more active approach of polyp removal should be considered. This issue can be discussed, however, we do believe that decision on approaching such patients should be made individually, based on the patient's age, comorbidities, general condition and operative risk.(AU)

Anatomy of the mastoid triangle and morphometric sex differences.

OBJECTIVE OF THE STUDY: This study aimed to quantify the area of the mastoid triangle (MT) and assess potential morphometric differences between males and females. PATIENTS: The sample consisted of 244 dry human skulls, with biological sex known based on genetic analysis, collected from a medicolegal osteological database from Central-Western Brazil. MATERIALS AND METHODS: The study was observational, analytical, and cross-sectional. The skulls were analyzed using Heron's equation to calculate the area of the MT. The landmarks connecting each of the sides of the triangle were: Porion (Po)>Mastoidale (Ma)>Asterion (Ast). Morphometric references were calculated and compared based on sex. RESULTS: The area of the MT was nearly 14% larger in males compared to females (p<0.05). The mean MT area for the right and left sides of males were 684.11±93.25mm2 and 668.94±111.95mm2, respectively. In females, the mean MT for the right and left sides were 588.93±91.09mm2 and 582.88±102.98mm2, respectively. Right and left side measurements were significantly different (p<0.05), except for Po-Ast (p=0.232). CONCLUSION: Morphometric features regarding the MT were slightly different between males and females. Application of the MT as a dimorphic tool should be adjuvant. Moreover, this tool should be considered carefully, especially because the sex-based differences were statistically significant, but discrete between males and females.

Biotechnological production of γ-decalactone, a peach like aroma, by Yarrowia lipolytica.

The request for new flavourings increases every year. Consumer perception that everything natural is better is causing an increase demand for natural aroma additives. Biotechnology has become a way to get natural products. γ-Decalactone is a peach-like aroma widely used in dairy products, beverages and others food industries. In more recent years, more and more studies and industrial processes were endorsed to cost-effect this compound production. One of the best-known methods to produce γ-decalactone is from ricinoleic acid catalyzed by Yarrowia lipolytica, a generally regarded as safe status yeast. As yet, several factors affecting γ-decalactone production remain to be fully understood and optimized. In this review, we focus on the aromatic compound γ-decalactone and its production by Y. lipolytica. The metabolic pathway of lactone production and degradation are addressed. Critical analysis of novel strategies of bioprocess engineering, metabolic and genetic engineering and other strategies for the enhancement of the aroma productivity are presented.

Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media.

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.

Optimization of xylanase production by filamentous fungi in solid-state fermentation and scale-up to horizontal tube bioreactor.

Five microorganisms, namely Aspergillus niger CECT 2700, A. niger CECT 2915, A. niger CECT 2088, Aspergillus terreus CECT 2808, and Rhizopus stolonifer CECT 2344, were grown on corncob to produce cell wall polysaccharide-degrading enzymes, mainly xylanases, by solid-state fermentation (SSF). A. niger CECT 2700 produced the highest amount of xylanases of 504 ± 7 U/g dry corncob (dcc) after 3 days of fermentation. The optimization of the culture broth (5.0 g/L NaNO3, 1.3 g/L (NH4)2SO4, 4.5 g/L KH2PO4, and 3 g/L yeast extract) and operational conditions (5 g of bed loading, using an initial substrate to moistening medium of 1:3.6 (w/v)) allowed increasing the predicted maximal xylanase activity up to 2,452.7 U/g dcc. However, different pretreatments of materials, including destarching, autoclaving, microwave, and alkaline treatments, were detrimental. Finally, the process was successfully established in a laboratory-scale horizontal tube bioreactor, achieving the highest xylanase activity (2,926 U/g dcc) at a flow rate of 0.2 L/min. The result showed an overall 5.8-fold increase in xylanase activity after optimization of culture media, operational conditions, and scale-up.

Yarrowia lipolytica lipase production enhanced by increased air pressure.

AIMS: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. METHODS AND RESULTS: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96.6 U l(-1) at 1 bar to 533.5 U l(-1) at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. CONCLUSIONS: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. SIGNIFICANCE AND IMPACT OF THE STUDY: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high-density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.

Decalactone production by Yarrowia lipolytica under increased O2 transfer rates.

Yarrowia lipolytica converts methyl ricinoleate to gamma-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-gamma-decalactone produced by the yeast (263 mg l(-1)) occurred by increasing the k(L)a up to 120 h(-1) at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O(2) solubility, but, although growth was accelerated, gamma-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec-2-en-4-olide and dec-3-en-4-olide (70 mg l(-1)).

Morphological and physiological changes in Saccharomyces cerevisiae by oxidative stress from hyperbaric air.

Increase in air or oxygen pressure in microbial cell cultures can cause oxidative stress and consequently affect cell physiology and morphology. The behaviour of Saccharomyces cerevisiae grown under hyperbaric atmospheres of air and pure oxygen was studied. A limit of 1.0 MPa for the air pressure increase (i.e. 0.21 MPa of oxygen partial pressure) in a fed-batch culture of S. cerevisiae was established. Values of 1.5 MPa air pressure and 0.32 MPa pure oxygen pressure strongly inhibited the metabolic activity and the viability of the cells. Also, morphological changes were observed, especially cell-size distribution and the genealogical age profile. Pressure caused cell compression and an increase in number of aged cells. These effects were attributed to oxygen toxicity since similar results were obtained using air or oxygen, if oxygen partial pressure was equal to or higher than 0.32 MPa. The activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD) (cytosolic and mitochondrial isoformes) indicated that the enzymes have different roles in oxidative stress cell protection, depending on other factors that affect the cell physiological state.

Effect of hyperbaric stress on yeast morphology: study by automated image analysis.

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO2.

Growth and beta-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure.

AIMS: To investigate the effect of total air pressure raise on cell growth and intracellular beta-galactosidase activity in batch cultures of Kluyveromyces marxianus CBS 7894. METHODS AND RESULTS: A pressurized bioreactor was used for K. marxianus batch cultivation under increased air pressure from 1.2 to 6 bar. Under these conditions no inhibition of cell growth was observed. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization led to an enhancement of the cell growth rate obtained at atmospheric pressure without aeration. The specific beta-galactosidase productivity increased from 5.8 to 17.0 U gCD-1 h-1 using a 6-bar air pressure instead of air at atmospheric pressure. The antioxidant enzyme superoxide dismutase (SOD) was slightly induced by the air pressure raise, which indicates that the defensive mechanisms of the cells can cope with an air pressure up to 6 bar. CONCLUSIONS: These experiments showed that the increase of air pressure up to 6 bar is an alternative to other methods of preventing the oxygen limitation and can be applied in the beta-galactosidase production by K. marxianus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results here reported proved that, in what biological aspects are concerned, it is possible to use the air pressure increase as an optimization parameter of beta-galactosidase production in high-density cell cultures of K. marxianus strains.

Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor.

Fed-batch is the dominating mode of operation in high-cell-density cultures of Saccharomyces cerevisae in processes such as the production of baker's yeast and recombinant proteins, where the high oxygen demand of these cultures makes its supply an important and difficult task. The aim of this work was to study the use of hyperbaric air for oxygen mass transfer improvement on S. cerevisiae fed-batch cultivation. The effects of increased air pressure up to 1.5 MPa on cell behavior were investigated. The effects of oxygen and carbon dioxide were dissociated from the effects of total pressure by the use of pure oxygen and gas mixtures enriched with CO(2). Fed-batch experiments were performed in a stirred tank reactor with a 600 mL stainless steel vessel. An exponential feeding profile at dilution rates up to 0.1 h(-)(1) was used in order to ensure a subcritical flux of substrate and, consequently, to prevent ethanol formation due to glucose excess. The ethanol production observed at atmospheric pressure was reduced by the bioreactor pressurization up to 1.0 MPa. The maximum biomass yield, 0.5 g g(-)(1) (cell mass produced per mass of glucose consumed) was attained whenever pressure was increased gradually through time. This demonstrates the adaptive behavior of the cells to the hyperbaric conditions. This work proved that hyperbaric air up to 1.0 MPa (0.2 MPa of oxygen partial pressure) could be applied to S. cerevisiae cultivation under low glucose flux. Above that critical oxygen partial pressure value, i.e., for oxygen pressures of 0.32 and 0.5 MPa, a drastic cell growth inhibition and viability loss were observed. The increase of carbon dioxide partial pressure in the gas mixture up to 48 kPa slightly decreased the overall cell mass yield but had negligible effects on cell viability.

Oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide, paraquat and pressure.

The aim of this work was to study the oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide (50 mM), paraquat (1 mM), an increase in air pressure (120 kPa, 600 kPa) and pure oxygen pressure (120-600 kPa) in a pressurized bioreactor. The effect of these oxidants on metabolism and on the induction of antioxidant enzymes was investigated. The exposure for 1 h of K. marxianus at exponential growth phase with either H(2)O(2) or paraquat, under air pressure of 120 kPa or 600 kPa, induced an increase in both superoxide dismutase (SOD) and glutathione reductase (GR) content. SOD induction by the chemical oxidants was independent of the air pressure values used. A 2-fold increase in SOD activity was observed after 1 h of exposure to H(2)O(2) and a 3-fold increase was obtained by the presence of paraquat, with both air pressures studied. In contrast, GR activity was raised 1.7-fold by the exposure to both chemicals with 120 kPa, but a 2.4-fold GR induction was obtained with 600 kPa. As opposed to Saccharomyces cerevisiae, catalase was not induced and was even lower than the normal basal levels. This antioxidant enzyme seemed to be inhibited under increasing oxygen partial pressure. The cells showed a significant increase in SOD and GR activity levels, 4.7-fold and 4.4-fold, when exposed for 24 h to 120 kPa pure oxygen pressure. This behaviour was even more patent with 400 kPa. However, whenever cells were previously exposed to low air pressures, low enzymatic activity levels were measured after subsequent exposure to pure oxygen pressure.

Air pressure effects on biomass yield of two different Kluyveromyces strains.

The use of air pressure as a way of improving oxygen transfer in aerobic bioreactors was investigated. To compare the air pressure effects with traditional air bubbled cultures, experiments using a pressure reactor and a stirred flask, with the same oxygen transfer rate, were made. Kluyveromyces marxianus is an important industrial yeast and some of it show a "Kluyver effect" for lactose: even under oxygen limited growth conditions, certain disaccharides that support aerobic, respiratory growth, are not fermented. This study deals with the effect of increased pressure on the physiological behavior of two Kluyveromyces strains: K. marxianus ATCC10022 is a lactose-fermenting strain, whereas K. marxianus CBS 7894 has a Kluyver-effect for lactose. For K. marxianus ATCC10022 an air pressure increase of 2 bar led to a 3-fold increase in biomass yield. When air pressure increased an enhancement of ethanol oxidation of cell yeasts was also observed. Batch cultures of K. marxianus CBS 7894 exhibited different growth behaviour. Its metabolism was always oxidative and ethanol was never produced. With the increase in air pressure, it was possible to increase the productivity in biomass of K. marxianus CBS 7894. As a response to high oxygen concentrations, due to the increase in oxygen partial pressure, oxidative stress in the cells was also studied. Antioxidant defences, such as superoxide dismutase, catalase, and glutathione reductase, were at high activity levels, suggesting that these yeast strains could tolerate the increased pressures applied.

Response of the thermophile Thermus sp. RQ-1 to hyperbaric air in batch and fed-batch cultivation.

The effects of increased air pressure in a culture of the thermophilic microorganism Thermus sp. RQ-1 were investigated. Cell growth dependence on oxygen supply was investigated in a fermenter at atmospheric pressure. Total oxygen depletion from the medium for low values of kLa was observed during the exponential growth phase. It was possible with this strain to enhance the oxygen transfer rate by increasing the air pressure. Cell productivity was improved by pressurisation up to 0.56 MPa for batch cultivation; and an induction of the antioxidant enzymes, superoxide dismutase and catalase, was observed with the rise in pressure. Cell pre-cultivation under pressurised conditions conferred to the cells more resistance to an exposure to hydrogen peroxide and more sensitivity to paraquat (methyl viologen). The usefulness of bioreactor pressurisation on the cultivation of Thermus sp. RQ-1 was demonstrated for fed-batch operation, with the attainment of higher cell densities. A two-fold increase in cell mass productivity was obtained by the use of hyperbaric air (0.5 MPa). With the pressurisation of the head-space in the reactor, it was also possible to eliminate the loss of liquid by evaporation, which amounted to more than 10% at 70 degrees C and atmospheric pressure.

Freeze/thawing and sonication of Escherichia coli TB1 cells for cytochrome b5 recovery.

The influence of sonication power, suspension volume and cell concentration on the kinectics of cytochrome b5 and intracellular protein release by sonication of Escherichia coli TB1 cells was studied. The influence of freezing and thawing of the cell suspension was also evaluated. Freezing and thawing increased the recovery yield of cytochrome b5. The sonication efficiency increased with the increase of sonication power and with the decrease of the suspension volume and cell concentration.

Optimization study of Escherichia coli TB1 cell disruption for cytochrome b5 recovery in a small-scale bead mill.

The recovery of a recombinant intracellular protein, cytochrome b5, from Escherichia coli TB1 cells was carried out by bead mill disintegration in a discontinuous small-scale instrument. This process was optimized by the use of experimental factorial design. Several parameters were studied: operating time, amount and size of beads, cellular suspension concentration, and presence of toluene and lysozyme. For the experimental conditions used, only the time of treatment and bead load had significant effects. The optimal values of these variables were found by applying the response surface methodology.