The Value of Current Ante Mortem Meat Inspection and Food Chain Information of Dairy Cows in Relation to Post Mortem Findings and the Protection of Public Health: A Case for a More Risk-Based Meat Inspection.

In this study, the contribution of the ante mortem (AM) inspection and the food chain information (FCI) to ensuring meat safety and public health was investigated, by evaluating the slaughterhouse findings of 223,600 slaughtered dairy cows in the Netherlands. The outcome of this study was that the ante mortem (AM) and post mortem (PM) inspections have a substantial overlap, and that with regard to food safety and public health in over 99% of cases the PM could even be omitted on the basis of the AM. In this study, the data provided by the dairy farmers on the current FCI forms contributed little to nothing with regard to the outcomes of AM and PM inspection. It is concluded that current meat inspection procedures need an update and a more risk-based approach needs to be adopted. Regarding this, the AM inspection of dairy cattle should remain, because it plays an important role in ensuring food safety (e.g., by preventing contamination of the slaughter line by excessively dirty animals, or animals with abscesses), monitoring animal welfare and in detecting some important notifiable diseases. The PM inspection, however, could in many cases be omitted, provided there is a strict AM inspection complemented by a vastly improved (automated) way of obtaining reliable FCI.

Modulation of transmembrane pressure in manufacturing scale tangential flow filtration N-1 perfusion seed culture.

Mammalian cells were grown to high density in a 3,000 L culture using perfusion with hollow fibers operated in a tangential flow filtration mode. The high-density culture was used to inoculate the production stage of a biomanufacturing process. At constant permeate flux operation, increased transmembrane pressures (TMPs) were observed on the final day of the manufacturing batches. Small scale studies suggested that the filters were not irreversibly fouled, but rather exposed to membrane concentration polarization that could be relieved by tangential sweeping of the hollow fibers. Studies were undertaken to analyze parameters that influence the hydrodynamic profile within hollow fibers; including filter area, cell density, recirculation flow rate, and permeate flow rate. Results indicated that permeate flow rate had the greatest influence on modulating TMP. Further evaluation showed a significant decrease in TMP when permeate flow was reduced, and this occurred without any negative effect on cell growth or viability. Hence, a 30% reduction of permeate flow rate was implemented at manufacturing scale. A stable operation was achieved as TMP was successfully reduced by 75% while preserving all critical factors for performance in the perfusion bioreactor.

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manß-1,2-Manß-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

Impact of Wheat Bran Hydration Properties As Affected by Toasting and Degree of Milling on Optimal Dough Development in Bread Making.

The impact of the hydration capacity and hydration rate of wheat bran on optimal bread dough development and loaf volume was investigated using coarse bran, both native as well as after toasting, milling, presoaking, and combinations of the latter. It was found that toasting reduces bran's hydration rate, which, during mixing, results in a temporary excess of water in which dough development takes place inefficiently and hence requires additional time. This mechanism was further substantiated by the observation that delayed dough development can be counteracted by the presoaking of bran. Milling of bran increases its hydration rate and results in faster optimal dough development. Presoaking of nonmilled bran, however, did not result in faster dough development. Smaller bran particles do lead to faster dough development, probably due to increased proper contacts between flour particles. Optimal loaf volumes did not change upon milling and toasting.

Dry heat treatment affects wheat bran surface properties and hydration kinetics.

Heat stabilization of wheat bran aims at inactivation of enzymes which may cause rancidity and processability issues. Such treatments may however cause additional unanticipated phenomena which may affect wheat bran technological properties. In this work, the impact of toasting on wheat bran hydration capacity and hydration kinetics was studied. Hydration properties were assessed using the Enslin-Neff and drainage centrifugation water retention capacity methods, thermogravimetric analysis and contact angle goniometry, next to more traditional methods. While equilibrium hydration properties of bran were not affected by the heat treatment, the rate at which the heat treated bran hydrated was, however, very significantly reduced compared to the untreated bran. This phenomenon was found to originate from the formation of a lipid coating during the treatment rendering the bran surface hydrophobic. These insights help to understand and partially account for the modified processability of heat treated bran in food applications.

Wheat (Triticum aestivum L.) Bran in Bread Making: A Critical Review.

Wheat bran, a by-product of the industrial roller milling of wheat, is increasingly added to food products because of its nutritional profile and physiological effects. Epidemiological data and scientific studies have demonstrated the health benefits of consuming bran-rich or whole-grain food products. However, incorporation of wheat bran in cereal-based products negatively affects their production process. Furthermore, the organoleptic quality of the obtained products is mostly perceived as inferior to that of products based on refined wheat flour. This review summarizes the current knowledge on the impact of wheat bran on bread making, provides a comprehensive overview of the bran properties possibly involved, and discusses different strategies that have been evaluated up till now to counteract the detrimental effects of wheat bran on bread making.

Wheat milling by-products and their impact on bread making.

This study investigates the relationship between the properties of dietary fiber (DF) rich wheat milling by-products and their impact on bread making. From coarse bran over coarse and fine weatings to low grade flour, the content of starch and lipids increased, while that of DF and ash decreased. Enzyme activity levels differed strongly and were not related to other by-product properties. Average particle size of the by-products was positively correlated with DF and ash contents and their hydration properties. When meals from flour and by-products were composed on the same overall starch level to compensate for differences in endosperm contamination in the by-products, bread specific volume was more strongly depressed with fine weatings and low grade flour than with coarse bran and weatings. This suggests that the properties of the former were intrinsically more detrimental to bread making than those of the latter.

Study of hydration properties of wheat bran as a function of particle size.

New insights in the hydration properties of wheat bran as function of particle size were gained based on a novel water retention capacity test. Upon milling coarse bran with an average particle size of 1687 µm down to 77 µm, the specific surface increases by twofold, structural integrity was lost and water extractable arabinoxylan and damaged starch content were practically unaffected. A standard centrifugation-based water retention capacity, swelling capacity and Enslin-Neff absorption test showed up to threefold higher water absorption for large particles. During these hydration tests, bran is not (continuously) subjected to external forces which allows larger particles to hold more water in between bran particles and probably in micropores. In contrast, the water retention capacity as determined by a novel drainage centrifugation method, and Farinograph absorption were not affected by particle size. In these methods, continuous exposure of bran to external forces causes bran to retain only strongly bound water which is most likely bound in cell wall nanopores and through hydrogen bonding. These insights reconcile contradicting observations in literature with regard to this matter.

Harvesting yeast (Saccharomyces cerevisiae) at different physiological phases significantly affects its functionality in bread dough fermentation.

Fermentation of sugars into CO2, ethanol and secondary metabolites by baker's yeast (Saccharomyces cerevisiae) during bread making leads to leavening of dough and changes in dough rheology. The aim of this study was to increase our understanding of the impact of yeast on dough related aspects by investigating the effect of harvesting yeast at seven different points of the growth profile on its fermentation performance, metabolite production, and the effect on critical dough fermentation parameters, such as gas retention potential. The yeast cells harvested during the diauxic shift and post-diauxic growth phase showed a higher fermentation rate and, consequently, higher maximum dough height than yeast cells harvested in the exponential or stationary growth phase. The results further demonstrate that the onset of CO2 loss from fermenting dough is correlated with the fermentation rate of yeast, but not with the amount of CO2 that accumulated up to the onset point. Analysis of the yeast metabolites produced in dough yielded a possible explanation for this observation, as they are produced in different levels depending on physiological phase and in concentrations that can influence dough matrix properties. Together, our results demonstrate a strong effect of yeast physiology at the time of harvest on subsequent dough fermentation performance, and hint at an important role of yeast metabolites on the subsequent gas holding capacity.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect.

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.

Torsional injuries of the lower limb: an analysis of the frictional torque between different types of football turf and the shoe outsole.

BACKGROUND: Football turf is increasingly used in European soccer competition. Little is known on the rotational torque that players experience on these fields. High rotational torques between the shoe outsole and the sports surface has been correlated with torsional injuries of the lower limb and knee. PURPOSE: To evaluate the effect of six parameters that could influence the rotational torque between the shoe outsole and the latest generation football turf. STUDY DESIGN: Controlled laboratory study. METHODS: A testing apparatus was constructed to measure the peak torque generated during a controlled rotation of the foot. Six parameters that could potentially influence the frictional forces, were considered: (1) the sports surface, (2) the shoe outsole cleat design, (3) the weather conditions, (4) the weight, (5) the presence of an impact and (6) the direction of rotation. RESULTS: The football turf without infill showed significantly lower frictional torques than natural grass whereas a football turf with sand/rubber infill had significantly higher torques. Blades were associated with significantly higher torques than studs on natural grass and on one football turf with sand/rubber infill. Dry weather was associated with higher torques only for the football turf without infill. The torque increased linearly and significantly with an increasing vertical load. The rotational torque increased significantly following an impact. Torques on external rotational movements were significantly higher with blades. CONCLUSIONS: Important differences in rotational torques are found and could be seen as potential risk factors for torsional injuries of the lower limb.

CD44 and HCELL: preventing hematogenous metastasis at step 1.

Despite great strides in our knowledge of the genetic and epigenetic changes underlying malignancy, we have limited information on the molecular basis of metastasis. Over 90% of cancer deaths are caused by spread of tumor cells from a primary site to distant organs and tissues, highlighting the pressing need to define the molecular effectors of cancer metastasis. Mounting evidence suggests that circulating tumor cells (CTCs) home to specific tissues by hijacking the normal leukocyte trafficking mechanisms. Cancer cells characteristically express CD44, and there is increasing evidence that hematopoietic cell E-/L-selectin ligand (HCELL), a sialofucosylated glycoform of CD44, serves as the major selectin ligand on cancer cells, allowing interaction of tumor cells with endothelium, leukocytes, and platelets. Here, we review the structural biology of CD44 and of HCELL, and present current data on the function of these molecules in mediating organ-specific homing/metastasis of CTCs.

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate.

BACKGROUND: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce. RESULTS: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man5GlcNAc2 N-glycosylation of the secreted proteins is achieved at all specific growth rates tested. CONCLUSIONS: Together, these data illustrate that the GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins.

The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins.

BACKGROUND: The unfolded protein response (UPR) in eukaryotes upregulates factors that restore ER homeostasis upon protein folding stress and in yeast is activated by a non-conventional splicing of the HAC1 mRNA. The spliced HAC1 mRNA encodes an active transcription factor that binds to UPR-responsive elements in the promoter of UPR target genes. Overexpression of the HAC1 gene of S. cerevisiae can reportedly lead to increased production of heterologous proteins. To further such studies in the biotechnology favored yeast Pichia pastoris, we cloned and characterized the P. pastoris HAC1 gene and the splice event. RESULTS: We identified the HAC1 homologue of P. pastoris and its splice sites. Surprisingly, we could not find evidence for the non-spliced HAC1 mRNA when P. pastoris was cultivated in a standard growth medium without any endoplasmic reticulum stress inducers, indicating that the UPR is constitutively active to some extent in this organism. After identification of the sequence encoding active Hac1p we evaluated the effect of its overexpression in Pichia. The KAR2 UPR-responsive gene was strongly upregulated. Electron microscopy revealed an expansion of the intracellular membranes in Hac1p-overexpressing strains. We then evaluated the effect of inducible and constitutive UPR induction on the production of secreted, surface displayed and membrane proteins. Wherever Hac1p overexpression affected heterologous protein expression levels, this effect was always stronger when Hac1p expression was inducible rather than constitutive. Depending on the heterologous protein, co-expression of Hac1p increased, decreased or had no effect on expression level. Moreover, alpha-mating factor prepro signal processing of a G-protein coupled receptor was more efficient with Hac1p overexpression; resulting in a significantly improved homogeneity. CONCLUSIONS: Overexpression of P. pastoris Hac1p can be used to increase the production of heterologous proteins but needs to be evaluated on a case by case basis. Inducible Hac1p expression is more effective than constitutive expression. Correct processing and thus homogeneity of proteins that are difficult to express, such as GPCRs, can be increased by co-expression with Hac1p.

Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology.

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes approximately 3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal(2)GlcNAc(2)Man(3)GlcNAc(2)N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains.

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications.

Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study.

The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates.

Toluene removal from waste air using a flat composite membrane bioreactor.

In this report, gaseous toluene biodegradation results in a flat composite membrane reactor inoculated with Pseudomonas putida TVA8 are presented. Preliminary abiotic experiments showed that transport of toluene through the membrane was linearly and negatively correlated with the gas residence time (tau). During a 339-day biofiltration experiment, the influence of gas residence time (2-24 sec) and mass loading rate (B(v); 10-483 g x m(-3) h(-1)) on the toluene elimination capacity was investigated. A maximum elimination capacity (EC(max)) of 397 g x m(-3) h(-1) was achieved at tau = 24 sec and B(v) = 473 g x m(-3) h(-1). Expressed per unit membrane area, the EC(m,max) was 0.793 g x m(-2) h(-1), which is five times higher than results obtained with other membrane bioreactor experiments in the same range of loading rates. At low gas residence times, reactor performance was limited by mass transfer. Toluene concentration profiles along the membrane were measured for several biotic and abiotic conditions. For inlet concentrations (C(in)) up to 1 g x m(-3), more than 90% was eliminated at 15 cm from the reactor inlet. For C(in) > 1.65 g x m(-3), longer membranes are necessary to obtain these high removal efficiencies.