Orally delivered Bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens.

We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Ensemble learning and personalized training for the improvement of unsupervised deep learning-based synthetic CT reconstruction.

BACKGROUND: The growing adoption of magnetic resonance imaging (MRI)-guided radiation therapy (RT) platforms and a focus on MRI-only RT workflows have brought the technical challenge of synthetic computed tomography (sCT) reconstruction to the forefront. Unpaired-data deep learning-based approaches to the problem offer the attractive characteristic of not requiring paired training data, but the gap between paired- and unpaired-data results can be limiting. PURPOSE: We present two distinct approaches aimed at improving unpaired-data sCT reconstruction results: a cascade ensemble that combines multiple models and a personalized training strategy originally designed for the paired-data setting. METHODS: Comparisons are made between the following models: (1) the paired-data fully convolutional DenseNet (FCDN), (2) the FCDN with the Intentional Deep Overfit Learning (IDOL) personalized training strategy, (3) the unpaired-data CycleGAN, (4) the CycleGAN with the IDOL training strategy, and (5) the CycleGAN as an intermediate model in a cascade ensemble approach. Evaluation of the various models over 25 total patients is carried out using a five-fold cross-validation scheme, with the patient-specific IDOL models being trained for the five patients of fold 3, chosen at random. RESULTS: In both the paired- and unpaired-data settings, adopting the IDOL training strategy led to improvements in the mean absolute error (MAE) between true CT images and sCT outputs within the body contour (mean improvement, paired- and unpaired-data approaches, respectively: 38%, 9%) and in regions of bone (52%, 5%), the peak signal-to-noise ratio (PSNR; 15%, 7%), and the structural similarity index (SSIM; 6%, <1%). The ensemble approach offered additional benefits over the IDOL approach in all three metrics (mean improvement over unpaired-data approach in fold 3; MAE: 20%; bone MAE: 16%; PSNR: 10%; SSIM: 2%), and differences in body MAE between the ensemble approach and the paired-data approach are statistically insignificant. CONCLUSIONS: We have demonstrated that both a cascade ensemble approach and a personalized training strategy designed initially for the paired-data setting offer significant improvements in image quality metrics for the unpaired-data sCT reconstruction task. Closing the gap between paired- and unpaired-data approaches is a step toward fully enabling these powerful and attractive unpaired-data frameworks.

Generation and Evaluation of Synthetic Computed Tomography (CT) from Cone-Beam CT (CBCT) by Incorporating Feature-Driven Loss into Intensity-Based Loss Functions in Deep Convolutional Neural Network.

Deep convolutional neural network (CNN) helped enhance image quality of cone-beam computed tomography (CBCT) by generating synthetic CT. Most of the previous works, however, trained network by intensity-based loss functions, possibly undermining to promote image feature similarity. The verifications were not sufficient to demonstrate clinical applicability, either. This work investigated the effect of variable loss functions combining feature- and intensity-driven losses in synthetic CT generation, followed by strengthening the verification of generated images in both image similarity and dosimetry accuracy. The proposed strategy highlighted the feature-driven quantification in (1) training the network by perceptual loss, besides L1 and structural similarity (SSIM) losses regarding anatomical similarity, and (2) evaluating image similarity by feature mapping ratio (FMR), besides conventional metrics. In addition, the synthetic CT images were assessed in terms of dose calculating accuracy by a commercial Monte-Carlo algorithm. The network was trained with 50 paired CBCT-CT scans acquired at the same CT simulator and treatment unit to constrain environmental factors any other than loss functions. For 10 independent cases, incorporating perceptual loss into L1 and SSIM losses outperformed the other combinations, which enhanced FMR of image similarity by 10%, and the dose calculating accuracy by 1-2% of gamma passing rate in 1%/1mm criterion.

Effect of dietary sophorolipids on growth performance and gastrointestinal functionality of broiler chickens infected with Eimeria maxima.

Two experiments were conducted to evaluate the effects of dietary sophorolipids (SLs) supplementation as antibiotic alternatives on growth performance and gut health of chickens infected with Eimeria maxima. In experiment 1, 336 (zero-day-old) male broilers were used. The chickens were weighed and randomly allocated to the following 6 treatments groups with 7 chickens/cage and 8 cages/treatment: control group that received a basal diet (NC), positive control group that received a basal diet and was challenged with E. maxima (PC), PC+C18:1 lactonic diacetyled SL (SL1), PC+C18:1 deacetyled SL (SL2), PC+C18:1 monoacetyled SL (SL3), and PC+C18:1 diacetyled SL (SL4). Each SL (200 mg/kg feed) was added to the corresponding treatment group. In experiment 2, 588 (zero-day-old) male broilers were used. The chickens were randomly allocated to the following experimental groups with 10 or 11 chickens/cage and 8 cages/treatment: NC, PC, PC+ monensin at 90 mg/kg feed (MO), PC+SL1 at 200 mg/kg feed (SL1 200), PC+SL1 at 500 mg/kg feed (SL1 500), PC+SL4 at 200 mg/kg feed (SL4 200), and PC+SL4 at 500 mg/kg of feed (SL4 500). The chickens and feed were weighed at 0, 7, 14, 20, and 22 d to determine growth performance. In both experiments, all chickens except the NC group were orally infected with E. maxima (10,000 oocysts/chicken) at d 14. One chicken per cage was euthanized at d 20 to sample jejunal tissue to measure lesion scores, cytokines, and tight junction (TJ) proteins. Excreta samples were collected daily between d 20 and 22 to measure oocyst numbers. Data were analyzed using Mixed Model (PROC MIXED) in SAS. In experiment 1, SLs did not affect the growth of broiler chickens, but SL4 decreased (P < 0.05) the lesion score and oocyst number compared to PC chickens. In terms of cytokines and TJ protein gene expression, SLs increased (P < 0.05) IL-1ß, IL-6, IL-17F, IL-4, IL-13, occludin, and ZO1 levels compared to PC chickens. In experiment 2, monensin increased (P < 0.05) body weight, and decreased (P < 0.05) the lesion score and oocyst number compared to the PC group. SL4 500 increased (P < 0.05) average daily gain and feed conversion ratio but decreased (P < 0.05) lesion score and fecal oocyst number. SL4 decreased (P < 0.05) IL-6, IL-17F, TNFSF-15, IL-2, and IL-10 levels but increased (P < 0.05) occludin and ZO-1 levels. Overall, dietary SL supplementation, especially SL4, improved growth and gastrointestinal functionality of young broiler chickens, demonstrating significant potential as an antibiotic alternative.

Synthetic contrast-enhanced computed tomography generation using a deep convolutional neural network for cardiac substructure delineation in breast cancer radiation therapy: a feasibility study.

BACKGROUND: Adjuvant radiation therapy improves the overall survival and loco-regional control in patients with breast cancer. However, radiation-induced heart disease, which occurs after treatment from incidental radiation exposure to the cardiac organ, is an emerging challenge. This study aimed to generate synthetic contrast-enhanced computed tomography (SCECT) from non-contrast CT (NCT) using deep learning (DL) and investigate its role in contouring cardiac substructures. We also aimed to determine its applicability for a retrospective study on the substructure volume-dose relationship for predicting radiation-induced heart disease. METHODS: We prepared NCT-CECT cardiac scan pairs of 59 patients. Of these, 35, 4, and 20 pairs were used for training, validation, and testing, respectively. We adopted conditional generative adversarial network as a framework to generate SCECT. SCECT was validated in the following three stages: (1) The similarity between SCECT and CECT was evaluated; (2) Manual contouring was performed on SCECT and CECT with sufficient intervals and based on this, the geometric similarity of cardiac substructures was measured between them; (3) The treatment plan was quantitatively analyzed based on the contours of SCECT and CECT. RESULTS: While the mean values (± standard deviation) of the mean absolute error, peak signal-to-noise ratio, and structural similarity index measure between SCECT and CECT were 20.66 ± 5.29, 21.57 ± 1.85, and 0.77 ± 0.06, those were 23.95 ± 6.98, 20.67 ± 2.34, and 0.76 ± 0.07 between NCT and CECT, respectively. The Dice similarity coefficients and mean surface distance between the contours of SCECT and CECT were 0.81 ± 0.06 and 2.44 ± 0.72, respectively. The dosimetry analysis displayed error rates of 0.13 ± 0.27 Gy and 0.71 ± 1.34% for the mean heart dose and V5Gy, respectively. CONCLUSION: Our findings displayed the feasibility of SCECT generation from NCT and its potential for cardiac substructure delineation in patients who underwent breast radiation therapy.

Deep-Learning-Based Automatic Segmentation of Head and Neck Organs for Radiation Therapy in Dogs.

Purpose: This study was conducted to develop a deep learning-based automatic segmentation (DLBAS) model of head and neck organs for radiotherapy (RT) in dogs, and to evaluate the feasibility for delineating the RT planning. Materials and Methods: The segmentation indicated that there were potentially 15 organs at risk (OARs) in the head and neck of dogs. Post-contrast computed tomography (CT) was performed in 90 dogs. The training and validation sets comprised 80 CT data sets, including 20 test sets. The accuracy of the segmentation was assessed using both the Dice similarity coefficient (DSC) and the Hausdorff distance (HD), and by referencing the expert contours as the ground truth. An additional 10 clinical test sets with relatively large displacement or deformation of organs were selected for verification in cancer patients. To evaluate the applicability in cancer patients, and the impact of expert intervention, three methods-HA, DLBAS, and the readjustment of the predicted data obtained via the DLBAS of the clinical test sets (HA_DLBAS)-were compared. Results: The DLBAS model (in the 20 test sets) showed reliable DSC and HD values; it also had a short contouring time of ~3 s. The average (mean ± standard deviation) DSC (0.83 ± 0.04) and HD (2.71 ± 1.01 mm) values were similar to those of previous human studies. The DLBAS was highly accurate and had no large displacement of head and neck organs. However, the DLBAS in the 10 clinical test sets showed lower DSC (0.78 ± 0.11) and higher HD (4.30 ± 3.69 mm) values than those of the test sets. The HA_DLBAS was comparable to both the HA (DSC: 0.85 ± 0.06 and HD: 2.74 ± 1.18 mm) and DLBAS presented better comparison metrics and decreased statistical deviations (DSC: 0.94 ± 0.03 and HD: 2.30 ± 0.41 mm). In addition, the contouring time of HA_DLBAS (30 min) was less than that of HA (80 min). Conclusion: In conclusion, HA_DLBAS method and the proposed DLBAS was highly consistent and robust in its performance. Thus, DLBAS has great potential as a single or supportive tool to the key process in RT planning.

Abdominal synthetic CT reconstruction with intensity projection prior for MRI-only adaptive radiotherapy.

Objective. Owing to the superior soft tissue contrast of MRI, MRI-guided adaptive radiotherapy (ART) is well-suited to managing interfractional changes in anatomy. An MRI-only workflow is desirable, but producing synthetic CT (sCT) data through paired data-driven deep learning (DL) for abdominal dose calculations remains a challenge due to the highly variable presence of intestinal gas. We present the preliminary dosimetric evaluation of our novel approach to sCT reconstruction that is well suited to handling intestinal gas in abdominal MRI-only ART.Approach. We utilize a paired data DL approach enabled by the intensity projection prior, in which well-matching training pairs are created by propagating air from MRI to corresponding CT scans. Evaluations focus on two classes: patients with (1) little involvement of intestinal gas, and (2) notable differences in intestinal gas presence between corresponding scans. Comparisons between sCT-based plans and CT-based clinical plans for both classes are made at the first treatment fraction to highlight the dosimetric impact of the variable presence of intestinal gas.Main results. Class 1 patients (n= 13) demonstrate differences in prescribed dose coverage of the PTV of 1.3 ± 2.1% between clinical plans and sCT-based plans. Mean DVH differences in all structures for Class 1 patients are found to be statistically insignificant. In Class 2 (n= 20), target coverage is 13.3 ± 11.0% higher in the clinical plans and mean DVH differences are found to be statistically significant.Significance. Significant deviations in calculated doses arising from the variable presence of intestinal gas in corresponding CT and MRI scans result in uncertainty in high-dose regions that may limit the effectiveness of adaptive dose escalation efforts. We have proposed a paired data-driven DL approach to sCT reconstruction for accurate dose calculations in abdominal ART enabled by the creation of a clinically unavailable training data set with well-matching representations of intestinal gas.

Impacts of energy feeds and supplemental protease on growth performance, nutrient digestibility, and gut health of pigs from 18 to 45 kg body weight.

A total of 144 pigs with 18.4 ± 2.3 kg initial body weight (BW) at 6 wk of age were used in a 40-d trial to evaluate effects of protease (300,000 U/kg feed, BioResource International Inc., Durham, NC, USA) on growth performance, apparent ileal digestibility (AID) of nutrients, and gut health of pigs fed diets with sorghum. Pigs were randomly allotted to 4 treatments (12 pens per treatment, 3 pigs per pen) in a 2 × 2 factorial arrangement (corn or sorghum basal diets, and 0 or 0.05% protease as 2 factors) with sex and initial BW as blocks. Experimental period had phase 1 (d 1 to 21) and phase 2 (d 22 to 40). About 65% (phase 1) and 72% (phase 2) of cereal grains were used in corn or sorghum based diets. Both grains were ground to 400 µm. Body weight and feed intake were recorded weekly. On d 35, serum was collected to quantify tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA). Titanium dioxide (0.3%) was added as an indigestible marker for an additional 4 d feeding. On d 40, 32 pigs (8 pigs per treatment) were euthanized to collect digesta from jejunum and ileum (for viscosity and AID), tissues (for morphology) and mucosa samples (for TNF-α and MDA) from duodenum, jejunum, and ileum. Replacing corn with sorghum in the diet increased (P < 0.05) overall average daily gain (from 756 to 787 g/day) and average daily feed intake (from 1,374 to 1,473 g/day), reduced (P < 0.05) overall gain:feed ratio (from 0.553 to 0.537), and did not affect AID. Pigs fed diets with sorghum had lower (P < 0.05) MDA content in serum (from 14.61 to 6.48 µmol/L) and jejunum (from 1.42 to 0.91 µmol/g protein), and reduced (P < 0.05) villus height (from 492 to 396 µm) and crypt depth (from 310 to 257 µm) in jejunum. Dietary protease improved (P < 0.05) AID of crude protein (from 81.8% to 86.0%), decreased MDA level (from 1.20 to 0.98 µmol/g protein) in duodenum, and increased (P < 0.05) the ratio of villus height to crypt depth (from 1.08 to 1.21) in duodenum. Overall, use of sorghum fully replacing corn in diets could benefit pigs with enhanced growth and feed intake potentially by reducing oxidative stress, whereas feed efficiency was compromised. Supplementation of protease improved protein digestion and maintained gut health, irrespective of sorghum or corn based diets.

The use of lung ultrasonography to confirm lung isolation in an infant who underwent emergent video-assisted thoracoscopic surgery: a case report.

Video-assisted thoracoscopic surgery for pediatric patients has gained popularity due to better outcomes than open surgery. For this procedure, one-lung ventilation may be necessary to provide an adequate surgical field. Confirming lung isolation is crucial when one-lung ventilation is required. Recently, we experienced a case in which one-lung ventilation was confirmed by ultrasonography using the lung sliding sign and the lung pulse in an infant. Since lung ultrasonography can be performed easily and quickly, it may be a useful method to confirm lung isolation, particularly in emergency surgeries with limited time, devices, and experienced anesthesiologists.

Impacts of low level aflatoxin in feed and the use of modified yeast cell wall extract on growth and health of nursery pigs.

This study was to investigate the effect of corn naturally contaminated with aflatoxins (AF) under the regulatory level on the growth performance and health of nursery pigs, and the efficiency of yeast cell wall based feed additive (YC) mainly composed of ß-glucans and mannan oligosaccharide (MOS) (Integral A+, Alltech, Lexington, KY) in prevention of mycotoxicosis. Pigs (60 barrows and 60 gilts at 6.02 ± 0.83 kg BW) were randomly allotted to 4 treatments in a randomized complete block design based on a 2 × 2 factorial arrangement with 10 pens (5 barrow and 5 gilt pens) per treatment and 3 pigs per pen. Pigs were fed experimental diets for 5 wk. First factor was AF (0 or 20 µg/kg in feed) and the second factor was YC (0 or 2 g/kg in feed). Feed intake and body weight were measured weekly, and blood samples were used to measure blood cell counts, immunoglobulin G (IgG), tumor necrosis factor-a (TNF-a), oxidative damage status, and serological evaluation related to liver health. Aflatoxin decreased (P < 0.05) the number of platelet count (247.4 to 193.5 × 103/µL), and it also tended to increase the level of albumin (P = 0.055, 3.46 to 3.63 g/dL), albumin:globulin ratio (P = 0.050, 2.09 to 2.37), and Ca (P = 0.080, 10.79 to 10.97 mg/dL). Yeast cell wall based feed additive increased (P < 0.05) ADG (493 to 524 g/d), and ADFI (796 to 846 g/d) of pigs whereas G:F was not affected, and it also tended to increase (P = 0.055) albumin level (3.46 to 3.63 g/dL). Interactions (P < 0.05) on hemoglobin, hematocrit, and platelet count indicated that YC further increased their levels when pigs were eating AF contaminated feed. Interactions (P < 0.05) on urea nitrogen and blood urea N to creatinine ratio indicated that YC further decreased their levels when feed were contaminated with AF. In conclusion, low level of 20 µg AF/kg under the regulatory level had minor effects on hematology without affecting growth performance, however the supplementation of 2 g/kg YC as a source of ß-glucans and MOS in feed can improve feed intake and therefore the growth of pigs.

Production of extracellular α-galactosidase by Bacillus sp. LX-1 in solid state fermentation for application as a potential feed additive / Producción de α-galactosidasa por Bacillus sp. LX-1 en fermentación en estado sólido para su aplicación como potencial aditivo alimenticio / Produção de α-galactosidase por Bacillus sp. LX-1 em fermentação em estado sólido para o uso como um potencial aditivo na alimentação

Background: α-galacto-oligosaccharides, including raffinose and stachyose, are present in soybean meal and used widely as a protein source in poultry diets. These compounds have anti-nutritive effects that ultimately reduce performance and value of birds. Thus, the addition of exogenous α-galactosidase to poultry diets -which can initiate the digestion of these non-digestible sugars- can be an effective strategy to solve the nutritional disorders associated with consumption of these oligosacharides. Solid state fermentation (SSF) has drawn attention for the production of microbial enzymes, due to the possibility of using cheap and abundant agro-industrial residues as substrates. Objective: to present information on α-galactosidase production under SSF conditions by an Antarctic bacterial isolate, Bacillus LX-1. Methods: initially, wheat bran, soybean meal, corn flour and the combinations of these individual substrates with nutritive supplements containing 1% galactose, 0.5% yeast extract, 1% tryptone, and 0.001% MnSO(4)4H2O were evaluated to select an optimal medium in SSF to produce extracellular α-galactosidase. Certain fermentation parameters involving incubation time, moisture content, and initial pH were investigated separately. Additional studies were conducted to evaluate the influence on enzyme production of different carbon sources (glucose, sucrose, galactose, lactose, and maltose) and nitrogen sources (peptone, tryptone, sodium nitrate and ammonium sulfate). Results: a medium containing soybean meal resulted in best α-galactosidase synthesis and was used for further SSF explorations with Bacillus sp. LX-1. Maximum enzyme production was observed at a growth period of 72 h, 75% moisture content and pH 8.0. Enzyme activity was enhanced in the presence of galactose or lactose as the carbon source, and tryptone or peptone as the nitrogen source. Conclusion: this SSF technique could be potentially used to produce α-galactosidase for poultry feed.

Antecedentes: los α-galacto-oligosacáridos, incluyendo rafinosa y estaquiosa, están presentes en la harina de soja, la cual es utilizada ampliamente como fuente de proteína en dietas de aves. Estos compuestos tienen efectos anti-nutricionales que reducen el rendimiento de las aves. La adición de α-galactosidasa exógena a dietas de aves puede iniciar la digestión de esos azúcares, resultando en una estrategia eficaz para resolver los desórdenes nutricionales asociados con el consumo de dichos oligosacáridos. La fermentación en estado sólido (SSF) se puede usar para producir enzimas microbianas, debido a la posibilidad de utilizar residuos agroindustriales abundantes y baratos como sustrato. Objetivo: informar sobre la producción de α-galactosidasas por una bacteria antártica (Bacillus LX-1) bajo condiciones de SSF. Métodos: se evaluaron salvado de trigo, harina de soja, harina de maíz y las combinaciones individuales de estos sustratos con suplementos nutritivos conteniendo 1% de galactosa, 0,5% de extracto de levadura, 1% de triptona y 0,001% de MnSO(4)4H2O para seleccionar un medio óptimo de SSF para producir α-galactosidasa extracelular. Ciertos parámetros de fermentación incluyendo tiempo de incubación, contenido de humedad, y pH inicial se evaluaron por separado. Se realizaron estudios adicionales para evaluar la influencia de diferentes fuentes de carbono (glucosa, sucrosa, galactosa, lactosa y maltosa) y de nitrógeno (peptona, triptona, nitrato de sodio y sulfato de amonio) sobre la producción enzimatica. Resultados: un medio con harina de soja resultó ser el mejor para la síntesis de α-galactosidasa y se utilizó para nuevas exploraciones de SSF con Bacillus sp. LX-1. La producción máxima de la enzima se observó en un periodo de crecimiento de 72 h, a 75% de humedad y pH 8,0. La actividad enzimática mejoró en presencia de galactosa o lactosa como fuente de carbono, y triptona o peptona como fuente de nitrógeno. Conclusión: la técnica de SSF podría ser utilizada para producir α-galactosidasa destinada a la alimentación de aves de corral.

Antecedentes: os α-galacto-oligossacarídeos, incluindo rafinose e estaquiose, estão presentes na farinha de soja, que é amplamente utilizado como uma fonte de proteína em dietas de aves domésticas. Estes compostos têm efeitos anti-nutricionais que reduzem o desempenho das aves. A adição de α-galactosidase exógena em dietas de aves pode começar a digerir estes açúcares, resultando em uma estratégia eficaz para resolver os problemas nutricionais associados com o consumo desses oligossacarídeos. A fermentação em estado sólido (SSF) pode ser usada para produzir enzimas microbianas, devido à possibilidade de utilização de resíduos agroindustriais como substrato abundante e barato. Objetivo: relatar a produção de α-galactosidase por uma bactéria da Antártida (Bacillus LX- 1) em SSF. Métodos: foram avaliados o farelo de trigo, farelo de soja, farelo de milho e combinações específicas destes substratos com suplementos nutricionais contendo 1% de galactose, 0,5% de extrato de levedura, 1% de triptona e 0,001% de MnSO(4)4H2O para selecionar um meio ideal de SSF para produzir extracelular α-galactosidase. Certos parâmetros de fermentação, incluindo tempo de incubação, teor de umidade e pH inicial foram avaliados separadamente. Estudos adicionais foram conduzidos para avaliar a influência de diferentes fontes de carbono (glucose, sacarose, galactose, lactose e maltose) e de azoto (peptona, triptona, nitrato de sódio e sulfato de amónio) na produção da enzima. Resultados: um meio com farelo de soja acabou por ser o melhor para a síntese de α-galactosidase e foi usado para uma maior exploração de SSF com Bacillus sp. LX - 1. A produção máxima da enzima foi observada em um período de crescimento de 72 h, 75% de humidade e pH 8,0. Melhoria da atividade enzimática na presença de galactose ou lactose como fonte de carbono e de triptona e peptona como fonte de azoto. Conclusão: A técnica da SSF poderia ser usada para produzir α-galactosidase para a alimentação de aves domésticas.

Splanchnic oxygen saturation immediately after weaning from cardiopulmonary bypass can predict early postoperative outcomes in children undergoing congenital heart surgery.

This study compared the abilities of cerebral, renal, and splanchnic regional oxygen saturation (rSO2) immediately after weaning from cardiopulmonary bypass (CPB) to predict early postoperative outcomes for children undergoing congenital heart surgery. The study enrolled 73 children (ages 0.1-72 months) undergoing corrective or palliative cardiac surgery requiring CPB. Laboratory and hemodynamic variables were analyzed at the time of successful weaning from CPB. Using near-infrared spectroscopy, cerebral, renal, and splanchnic rSO2 values were obtained simultaneously. Early postoperative outcome measures included the maximum vasoactive inotropic score (VIS(max)) during the first 36 postoperative hours, the duration of mechanical ventilation, and the postoperative hospital length of stay. In the univariate analysis, cerebral, renal, and splanchnic rSO2 values correlated significantly with early postoperative outcomes. However, splanchnic rSO2 was the only independent factor predicting VIS(max) (ß = -0.302, P = 0.021), duration of mechanical ventilation (ß = -0.390, P = 0.002), and postoperative hospital length of stay (ß = -0.340, P = 0.001) by multivariate analyses. Splanchnic rSO2 had a larger receiver operating characteristic area under the curve (AUC) for determining high VIS(max), prolonged mechanical ventilation, and longer postoperative hospital stay (AUC 0.775, 0.792, and 0.776, respectively) than cerebral (AUC 0.630, 0.638, and 0.632, respectively) and renal (AUC 0.703, 0.716, and 0.715, respectively) rSO2. After weaning from CPB, splanchnic rSO2 may be superior to rSO2 measured from brain and kidney in predicting an increased requirement for vasoactive inotropic support, a prolonged mechanical ventilation, and a longer postoperative hospital stay for children.

Bacillus strains as feed additives: In vitro evaluation of its potential probiotic properties / Bacilos utilizados como aditivos para piensos: Evaluación in vitro de sus potenciales propiedades probióticas / Bacilos utilizados como aditivos para concentrados: Avaliação in vitro de suas propriedades probióticas

Background: much recent attention has been devoted to the genuine value of Bacillus species as multifunctional probiotic products, which produce various extracellular enzymes that enhance feed digestibility as well as many antimicrobial compounds for the purpose of improving animal performance. Objective: to describe novel, in vitro potential probiotic properties such as acid tolerance, bile tolerance, safety, and antimicrobial activity of mesophilic and psychrophilic Bacillus strains in conjunction with their extracellular enzymatic activities. Methods: four Bacillus strains (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isolated from different sources were used. Strains were identified according to 16S rDNA sequences. Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus were used as indicator bacteria for the antimicrobial activity trial. Strains were activated and cultured in tryptic soy broth (pH 7.0) or broth solidified with 1.5% agar. Results: B. sp. JSP1 was fully resistant to both pH 2 and 3, whereas B. sp. SM2 showed relatively good viability at pH 3. All strains tolerated oxgall (0.3%) bile salt and were not cytotoxic to the HEK 293 human embryonic kidney cells. Three strains, except B. sp. T3, displayed differential production of extracellular enzymes including amylase, xylanase, cellulase, protease, phytase, and α-galactosidase. In particular, B. sp. SM2 inhibited six indicator pathogens: Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus. Conclusion: the single use of B. sp. SM2 or the mixed use of the strain combined with acid or bile tolerant Bacillus strains secreting extracellular enzymes may be an alternative to antibiotics as a feed additive in farm animal production.

Antecedentes: recientemente el valor de las especies de Bacillus como productos probióticos multifuncionales ha recibido bastante atención, debido a que estos producen varias enzimas extracelulares que potencian la digestibilidad de los alimentos, así como también compuestos antimicrobianos que mejoran el desempeño del animal. Objetivo: describir y evaluar potenciales propiedades probióticas ''in vitro'' -tales como acidez, tolerancia a la bilis, seguridad y actividad antimicrobiana- de cuatro cepas de Bacilos (B. sp. T3, B. sp. T4, B. sp. SM2 y B. sp. JSP1) aisladas de diferentes fuentes, en conjunción con sus actividades enzimáticas extracelulares. Métodos: se usaron cuatro cepas de Bacillus (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) aisladas de diferentes fuentes. Las cepas se identificaron de acuerdo a secuencias 16S rDNA. Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, y Staphylococcus aureus fueron empleadas como bacterias indicadoras para el ensayo de actividad antimicrobiana. Las cepas fueron activadas y cultivadas en caldo soya tripticasa (PH 7.0) o caldo solidificado con 1.5% de agar. Resultados: el B. sp. JSP1 resultó totalmente resistente tanto a pH 2 como a pH 3, mientras que el B. sp. SM2 mostró viabilidad relativamente alta a pH 3. Todas las cepas toleraron oxgall (0.3%) de sales biliares y no resultaron citotóxicas para las células humanas HEK 293 de riñón embrionario. Tres cepas, con excepción de B. sp. T3, presentaron producción diferencial de enzimas extracelulares -incluyendo amilasa, xilanasa, celulasa, proteasa, fitasa y α-galactosidasa. En particular, el B. sp. SM2 inhibió seis indicadores patógenos (Escherichia coli K88, E. coli O157: H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes y Staphylococcus aureus). Conclusiones: el uso específico de B. sp. SM2, o el uso combinado de esta cepa junto con cepas secretoras de enzimas extracelulares y tolerantes a ácidos o bilis puede ser una alternativa para reemplazar los antibióticos frecuentemente usados como aditivos en alimentación animal.

Antecedentes: o valor das espécies de Bacillus como produtos probióticos multifuncionais tem recebido bastante atenção recentemente, devido a que produzem várias enzimas extracelulares que potenciam a digestibilidade dos alimentos, como também compostos antimicrobianos que melhoram o desempenho do animal. Objetivo: descrever e avaliar as propriedades potenciais ''in vitro'' - como acidez, tolerância à bile, segurança e atividade antimicrobial- de quatro cepas de Bacilos (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isoladas de diferentes fontes, em conjunto com as suas atividades enzimáticas extracelulares. Métodos: foram usadas quatro cepas de Bacillus (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isoladas de diferentes fontes. As cepas foram identificadas de acordo às sequências 16S rDNA. As seguintes bactérias foram empregadas como indicadoras no teste de atividade antimicrobiana: Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, y Staphylococcus aureus. As cepas foram ativadas e cultivadas em caldo soja tripticase (pH 7.0) ou caldo solidificado com 1.5 de Agar. Resultados: o B. sp. JSP1 foi totalmente resistente tanto no pH 2.0 como no pH 3.0, enquanto que o B. sp. SM2 mostrou viabilidade relativamente alta no pH 3.0. Todas as cepas toleraram oxgall (0.3%) de sais biliares e não foram citotóxicas para as células humanas HEK 293 de rim embrionário. Tres cepas, com exceção de B. sp. T3, apresentaram produção diferenciada de enzimas extracelulares -incluindo amilase, xilanase, celulase, protease, fitase e α-galactosidase. Particularmente, o B. sp, SM2 inibiu seis indicadores patógenos (Escherichia coli K88, E. coli O157: H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes y Staphylococcus aureus). Conclusões: O uso específico de B. sp. SM2 ou o uso combinado desta cepa junto com as cepas secretoras de enzimas extracelulares e tolerantes a ácidos ou bile, pode ser uma alternativa para substituir os antibióticos frequentemente usados como aditivos na alimentação animal.

Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors.

The Wnt/ß-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

Production of an extracellular protease by an Antarctic bacterial isolate (Bacillus sp. JSP1)as a potential feed additive / Proteasa extracelular de un aislado bacteriano Antartico (Bacillus sp. JSP1) con uso potencial como aditivo alimenticio para animales / Produção de proteasa extracelular por bactérias antárcticas isoladas, Bacillus sp. JSP1 como um aditivo potencial em concentrado

Extracellular proteolytic activity was found in JSP1, an Antarctic bacterial isolate. The strain was related to Bacillus sp, based on 16S rRNA gene sequence analysis. The JSP1 protease was partially purified by ammonium sulfate precipitation. Optimal enzyme activity occurred at 40 C and pH 7.4. Enzyme activity was significantly enhanced in the presence of Mg2+ and Ca2+ and was completely inactivated in presence of Cu2+, Zn2+, Hg2+, EDTA and SDS. The enzyme hydrolyzed casein the most effectively among the protein substrates tested. The enzyme also exhibited relatively high activity on keratin and gluten, and was active against peptidyl conjugates such asL-Leu-p-Nitroanilide and N-Succinyl-L-Phe-p-Nitroanilide. This study suggests that JSP1 protease could be utilized as a potential environmentally-friendly feed additive in animal production.

Se reporta haber encontrado actividad proteolítica extracelular en una bacteria antártica denominada JSP1. Con base en el análisis de secuencia genética 16S ARNr, la cepa fué relacionada con Bacillus sp. La proteasa JSP1 fué parcialmente purificada por precipitación con sulfato de amonio. La actividad óptima de la enzima se produjo a 40 ºC y pH 7,4. La actividad enzimática fue significativamente mayor en presencia de Mg2+ y Ca2+ y se inactivó completamente en presencia de Cu2+, Zn2+ , Hg2+ , EDTA y SDS. Entre todos los sustratos ensayados, el más eficientemente hidrolizado por la enzima fue la caseína. La enzima tuvo actividad relativamente alta sobre la queratina y el gluten, y participó activamente contra conjugados peptídicos como la L-Leu-p-nitroanilida y la N-succinil-L-fenilalanina-p-nitroanilida. La enzima podría ofrecer potencial para su uso como aditivo alimenticio ecológico en producción animal.

A actividade proteolítica extracelular foi encontrada a partir de uma bactéria antárctica denominada JSP1, mediante uma análises de sequencia do gene 16S rRNA, a cepa foi relacionada para Bacillus sp. A proteasa JSP1 foi parcialmente purificada por precipitação com sulfato de amônia. Uma óptima actividade enzimática ocorreu em 40 ºC e pH 7,4. A actividade da enzima foi significativamente maior na presença de Mg2 + e Ca2 +, e foi completamente inactivada em presença de Cu2 + Zn2 +, Hg2 +, EDTA e SDS. A enzima hidrolisada de caseína foi mais eficaz entre os substratos de proteína testados, apresentando maior actividade na queratina e no glúten e foi activo contra os peptidil conjugados como L-Leu-p Nitroanilida e N-succinil-L-Phe-p-Nitroanilida. A enzima pode ser utilizado como aditivo ambiental na alimentação animal.

DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency.

Previous studies have shown that blocking DLL4 signaling reduced tumor growth by disrupting productive angiogenesis. We developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. We found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes and reduced proliferation of tumor cells. Furthermore, we found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.

Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy.

BACKGROUND: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. METHODS AND FINDINGS: Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3), at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. CONCLUSIONS: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/- multipotent progenitors.

Haematopoiesis is maintained by a hierarchical system where haematopoietic stem cells (HSCs) give rise to multipotent progenitors, which in turn differentiate into all types of mature blood cells. HSCs maintain themselves for the lifetime of the organism because of their ability to self-renew. However, multipotent progenitors lack the ability to self-renew, therefore their mitotic capacity and expansion potential are limited and they are destined to eventually stop proliferating after a finite number of cell divisions. The molecular mechanisms that limit the proliferation capacity of multipotent progenitors and other more mature progenitors are not fully understood. Here we show that bone marrow cells from mice deficient in three genes genetically downstream of Bmi1--p16Ink4a, p19Arf and Trp53 (triple mutant mice; p16Ink4a and p19Arf are alternative reading frames of the same gene (also called Cdkn2a) that encode different proteins)--have an approximately 10-fold increase in cells able to reconstitute the blood long term. This increase is associated with the acquisition of long-term reconstitution capacity by cells of the phenotype c-kit+Sca-1+Flt3+CD150-CD48-Lin-, which defines multipotent progenitors in wild-type mice. The pattern of triple mutant multipotent progenitor response to growth factors resembles that of wild-type multipotent progenitors but not wild-type HSCs. These results demonstrate that p16Ink4a/p19Arf and Trp53 have a central role in limiting the expansion potential of multipotent progenitors. These pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self-renew and become malignant with further oncogenic mutations.

Phenotypic characterization of human colorectal cancer stem cells.

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the "cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM)(high)/CD44+ epithelial cells. Tumors originated from EpCAM(high)/CD44+ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM(high)/CD44+ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.

Bmi1, stem cells, and senescence regulation.

Stem cells generate the differentiated cell types within many organs throughout the lifespan of an organism and are thus ultimately responsible for the longevity of multicellular organisms. Therefore, senescence of stem cells must be prevented. Bmi1 is required for the maintenance of adult stem cells in some tissues partly because it represses genes that induce cellular senescence and cell death.