Feeding of wheat bran and sugar beet pulp as sole supplements in high-forage diets emphasizes the potential of dairy cattle for human food supply.

Besides the widely discussed negative environmental effects of dairy production, such as greenhouse gas emissions, the feeding of large amounts of potentially human-edible feedstuffs to dairy cows is another important sustainability concern. The aim of this study was therefore to investigate the effects of a complete substitution of common cereal grains and pulses with a mixture of wheat bran and sugar beet pulp in a high-forage diet on cow performance, production efficiency, feed intake, and ruminating behavior, as well as on net food production potential. Thirteen multiparous and 7 primiparous mid-lactation Holstein dairy cows were randomly assigned to 1 of 2 treatments in a change-over design with 7-wk periods. Cows were fed a high-forage diet (grass silage and hay accounted for 75% of the dry matter intake), supplemented with either a cereal grain-based concentrate mixture (CON), or a mixture of wheat bran and dried sugar beet pulp (WBBP). Human-edible inputs were calculated for 2 different scenarios based on minimum and maximum potential recovery rates of human-edible energy and protein from the respective feedstuffs. Dietary starch and neutral detergent fiber contents were 3.0 and 44.1% for WBBP, compared with 10.8 and 38.2% in CON, respectively. Dietary treatment did not affect milk production, milk composition, feed intake, or total chewing activity. However, chewing index expressed in minutes per kilogram of neutral detergent fiber ingested was 12% lower in WBBP compared with CON. In comparison to CON, the human-edible feed conversion efficiencies for energy and protein, defined as human-edible output per human-edible input, were 6.8 and 5.3 times higher, respectively, in WBBP under the maximum scenario. For the maximum scenario, the daily net food production (human-edible output minus human-edible input) increased from 5.4 MJ and 250 g of crude protein per cow in CON to 61.5 MJ and 630 g of crude protein in the WBBP diet. In conclusion, our data suggest that in forage-based dairy production systems, wheat bran and sugar beet pulp could replace common cereal grains in mid-lactation dairy cows without impairing performance, while strongly increasing human-edible feed conversion efficiency and net food production index.

Feeding of by-products completely replaced cereals and pulses in dairy cows and enhanced edible feed conversion ratio.

When fed human-edible feeds, such as grains and pulses, dairy cows are very inefficient in transforming them into animal products. Therefore, strategies to reduce human-edible inputs in dairy cow feeding are needed to improve food efficiency. The aim of this feeding trial was to analyze the effect of the full substitution of a common concentrate mixture with a by-product concentrate mixture on milk production, feed intake, blood values, and the edible feed conversion ratio (eFCR), defined as human-edible output per human edible input. The experiment was conducted as a change-over design, with each experimental period lasting for 7wk. Thirteen multiparous and 5 primiparous Holstein cows were randomly assigned to 1 of 2 treatments. Treatments consisted of a grass silage-based forage diet supplemented with either conventional ingredients or solely by-products from the food processing industry (BP). The BP mixture had higher contents of fiber and ether extract, whereas starch content was reduced compared with the conventional mixture. Milk yield and milk solids were not affected by treatment. The eFCR in the BP group were about 4 and 2.7 times higher for energy and protein, respectively. Blood values did not indicate negative effects on cows' metabolic health status. Results of this feeding trial suggest that by-products could replace common concentrate supplements in dairy cow feeding, resulting in an increased eFCR for energy and protein which emphasizes the unique role of dairy cows as net food producers.

Substitution of common concentrates with by-products modulated ruminal fermentation, nutrient degradation, and microbial community composition in vitro.

A rumen simulation technique was used to evaluate the effects of the complete substitution of a common concentrate mixture (CON) with a mixture consisting solely of by-products from the food industry (BP) at 2 different forage-to-concentrate ratios on ruminal fermentation profile, nutrient degradation, and abundance of rumen microbiota. The experiment was a 2×2 factorial arrangement with 2 concentrate types (CON and BP) and 2 concentrate levels (25 and 50% of diet dry matter). The experiment consisted of 2 experimental runs with 12 fermentation vessels each (n=6 per treatment). Each run lasted for 10d, with data collection on the last 5d. The BP diets had lower starch, but higher neutral detergent fiber (NDF) and fat contents compared with CON. Degradation of crude protein was decreased, but NDF and nonfiber carbohydrate degradation were higher for the BP diets. At the 50% concentrate level, organic matter degradation tended to be lower for BP and CH4 formation per unit of NDF degraded was also lower for BP. The BP mixture led to a higher concentration of propionate and a lower acetate-to-propionate ratio, whereas concentrations of butyrate and caproate decreased. Concentrate type did not affect microbial community composition, except that the abundance of bacteria of the genus Prevotella was higher for BP. Increasing the concentrate level resulted in higher degradation of organic matter and crude protein. At the higher concentrate level, total short-chain fatty acid formation increased and concentrations of isobutyrate and valerate decreased. In addition, at the 50% concentrate level, numbers of protozoa increased, whereas numbers of methanogens, anaerobic fungi, and fibrolytic bacteria decreased. No interaction was noted between the 2 dietary factors on most variables, except that at the higher concentrate level the effects of BP on CH4 and CO2 formation per unit of NDF degraded, crude protein degradation, and the abundance of Prevotella were more prominent. In conclusion, the results of this study suggest that BP in the diet can adequately substitute CON with regard to ruminal fermentation profile and microbiota, showing even favorable fermentation patterns when fed at 50% inclusion rate.

A compression transmission device for the evaluation of bonding strength of biocompatible microfluidic and biochip materials and systems.

Bonding of a variety of inorganic and organic polymers as multi-layered structures is one of the main challenges for biochip production even to date, since the chemical nature of these materials often does not allow easy and straight forward bonding and proper sealing. After selection of an appropriate method to bond the chosen materials to form a complex biochip, function and stability of bonding either requires qualitative burst tests or expensive mechanical multi-test stations, that often do not have the right adaptors to clamp biochip slides without destruction. Therefore, we have developed a simple and inexpensive bonding test based on 3D printed transmission elements that translate compressive forces via manual compression, hand press or hydraulic press compression into shear and tensile force. Mechanical stress simulations showed that design of the bonding geometry and size must be considered for bonding tests since the stress distribution thus bonding strength heavily varies with size but also with geometry. We demonstrate the broad applicability of our 3D printed bonding test system by testing the most frequent bonding strategies in combination with the respective most frequently used biochip material in a force-to-failure study. All evaluated materials are biocompatible and used in cell-based biochip devices. This study is evaluating state-of-the-art bonding approaches used for sealing of microfluidic biochips including adhesive bonding, plasma bonding, solvent bonding as well as bonding mediated by amino-silane monolayers or even functional thiol-ene epoxy biochip materials that obviate intermediate adhesive layers.

Characterization of four functional biocompatible pressure-sensitive adhesives for rapid prototyping of cell-based lab-on-a-chip and organ-on-a-chip systems.

In the advent of affordable photo- and soft-lithography using polydimethylsiloxane (PDMS), low cost multi-step microfabrication methods have become available to a broad scientific community today. Although these methods are frequently applied for microfluidic prototype production in academic and industrial settings, fast design iterations and rapid prototyping within a few minutes with a high degree of flexibility are nearly impossible. To reduce microfluidic concept-to-chip time and costs, a number of alternative rapid prototyping techniques have recently been introduced including CNC micromachining, 3D printing and plotting out of numeric CAD designs as well as micro-structuring of thin PDMS sheets and pressure sensitive adhesives. Although micro-structuring of pressure sensitive adhesives promises high design flexibility, rapid fabrication and simple biochip assembly, most adhesives are toxic for living biological systems. Since an appropriate bio-interface and proper biology-material interaction is key for any cell chip and organ-on-a-chip system, only a limited number of medical-grade materials are available for microfluidic prototyping. In this study, we have characterized four functional biomedical-grade pressure sensitive adhesives for rapid prototyping (e.g. less than 1 hour) applications including structuring precision, physical and optical properties as well as biocompatibilities. While similar biocompatibility was found for all four adhesives, significant differences in cutting behavior, bonding strength to glass and polymers as well as gas permeability was observed. Practical applications included stability testing of multilayered, membrane-integrated organ-on-a-chip devices under standard cell culture conditions (e.g. 2-3 weeks at 37 °C and 100% humidity) and a shear-impact up to 5 dynes/cm2. Additionally, time- and shear-dependent uptake of non-toxic fluorescently labelled nanoparticles on human endothelial cells are demonstrated using micro-structured adhesive-bonded devices. Our results show that (a) both simple and complex microdevices can be designed, fabricated and tested in less than 1 hour, (b) these microdevices are stable for weeks even under physiological shear force conditions and (c) can be used to maintain cell monolayers as well as 3D cell culture systems.

Microfluidic nutrient gradient-based three-dimensional chondrocyte culture-on-a-chip as an in vitro equine arthritis model.

In this work, we describe a microfluidic three-dimensional (3D) chondrocyte culture mimicking in vivo articular chondrocyte morphology, cell distribution, metabolism, and gene expression. This has been accomplished by establishing a physiologic nutrient diffusion gradient across the simulated matrix, while geometric design constraints of the microchambers drive native-like cellular behavior. Primary equine chondrocytes remained viable for the extended culture time of 3 weeks and maintained the low metabolic activity and high Sox9, aggrecan, and Col2 expression typical of articular chondrocytes. Our microfluidic 3D chondrocyte microtissues were further exposed to inflammatory cytokines to establish an animal-free, in vitro osteoarthritis model. Results of our study indicate that our microtissue model emulates the basic characteristics of native cartilage and responds to biochemical injury, thus providing a new foundation for exploration of osteoarthritis pathophysiology in both human and veterinary patients.

An approach to including protein quality when assessing the net contribution of livestock to human food supply.

The production of protein from animal sources is often criticized because of the low efficiency of converting plant protein from feeds into protein in the animal products. However, this critique does not consider the fact that large portions of the plant-based proteins fed to animals may be human-inedible and that the quality of animal proteins is usually superior as compared with plant proteins. The aim of the present study was therefore to assess changes in protein quality in the course of the transformation of potentially human-edible plant proteins into animal products via livestock production; data from 30 Austrian dairy farms were used as a case study. A second aim was to develop an approach for combining these changes with quantitative aspects (e.g. with the human-edible feed conversion efficiency (heFCE), defined as kilogram protein in the animal product divided by kilogram potentially human-edible protein in the feeds). Protein quality of potentially human-edible inputs and outputs was assessed using the protein digestibility-corrected amino acid score and the digestible indispensable amino acid score, two methods proposed by the Food and Agriculture Organization of the United Nations to describe the nutritional value of proteins for humans. Depending on the method used, protein scores were between 1.40 and 1.87 times higher for the animal products than for the potentially human-edible plant protein input on a barn-gate level (=protein quality ratio (PQR)). Combining the PQR of 1.87 with the heFCE for the same farms resulted in heFCE×PQR of 2.15. Thus, considering both quantity and quality, the value of the proteins in the animal products for human consumption (in this case in milk and beef) is 2.15 times higher than that of proteins in the potentially human-edible plant protein inputs. The results of this study emphasize the necessity of including protein quality changes resulting from the transformation of plant proteins to animal proteins when evaluating the net contribution of livestock to the human food supply. Furthermore, these differences in protein quality might also need to be considered when choosing a functional unit for the assessment of environmental impacts of the production of different proteins.

Structural studies of the acquired immunodeficiency syndrome virus reverse transcriptase.

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized. Crystallographic studies currently underway are aimed at elucidating the three-dimensional structure of HIV RT. The availability of a bacterial expression system has enabled structural/functional studies of the RT by site-directed mutagenesis. These studies have identified amino acid residues that are essential for activity of the enzyme and might be involved in substrate binding. It is hoped that structural information of this nature will allow the rational design of HIV RT inhibitors.

Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties.

Molecular polar surface area (PSA), i.e., surface belonging to polar atoms, is a descriptor that was shown to correlate well with passive molecular transport through membranes and, therefore, allows prediction of transport properties of drugs. The calculation of PSA, however, is rather time-consuming because of the necessity to generate a reasonable 3D molecular geometry and the calculation of the surface itself. A new approach for the calculation of the PSA is presented here, based on the summation of tabulated surface contributions of polar fragments. The method, termed topological PSA (TPSA), provides results which are practically identical with the 3D PSA (the correlation coefficient between 3D PSA and fragment-based TPSA for 34 810 molecules from the World Drug Index is 0.99), while the computation speed is 2-3 orders of magnitude faster. The new methodology may, therefore, be used for fast bioavailability screening of virtual libraries having millions of molecules. This article describes the new methodology and shows the results of validation studies based on sets of published absorption data, including intestinal absorption, Caco-2 monolayer penetration, and blood-brain barrier penetration.

World Wide Web-based system for the calculation of substituent parameters and substituent similarity searches.

Easy to use, interactive, and platform-independent WWW-based tools are ideal for development of chemical applications. By using the newly emerging Web technologies such as Java applets and sophisticated scripting, it is possible to deliver powerful molecular processing capabilities directly to the desk of synthetic organic chemists. In Novartis Crop Protection in Basel, a Web-based molecular modelling system has been in use since 1995. In this article two new modules of this system are presented: a program for interactive calculation of important hydrophobic, electronic, and steric properties of organic substituents, and a module for substituent similarity searches enabling the identification of bioisosteric functional groups. Various possible applications of calculated substituent parameters are also discussed, including automatic design of molecules with the desired properties and creation of targeted virtual combinatorial libraries.

Herpesvirus protease assays.

Herpesviruses encode a serine protease that is essential for the maturation of viral capsids (1,2). The protease is expressed as part of a polyprotein. The catalytic domain is contained within the N-terminal third of the protein, and the remainder comprises a structural "scaffold" protein. The scaffold protein is independently expressed in excess to the polyprotein from an internal initiation codon. The protease cleaves the polyprotein at two sites: one at the c-terminus of the protease catalytic domain, the release or R-site, and the other close to the c-terminus of the scaffold protein, the maturation or M-site (Fig. 1). Cleavage of the M-site follows assembly of the viral procapsids and precedes packaging of the viral DNA. The M-site sequence is conserved among the herpesviruses and has a consensus sequence (V/L)-X-A-S, with cleavage between A-S (3). Structural studies have shown that the herpesvirus proteases have a novel structure, and their essential role in capsid maturation makes them a potential target for antiviral intervention. Fig. 1. Cartoon illustration of the structure of the herpesvirus protease/scaffold polyprotein showing the position of the protease catalytic domain, the scafold protein, and the release and maturation cleavage sites.

Regio- and stereo-selective synthesis of carbohydrate isoxazolidines by 1,3-dipolar cycloaddition of nitrones to 5,6-dideoxy-1,2-O-isopropylidene- alpha-D-xylo-hex-5-enofuranose.

The synthesis of 2-phenyl-3-aryl and 2-phenyl-3-aroyl derivatives 5-(1,2-O-isopropylidene-alpha-D-xylo-tetrofuranos-4-yl)isoxazolidi ne (3) from nitrones and 5,6-dideoxy-1,2-O-isopropylidene-alpha-D-xylo-hex-5- enofuranose (1) is described. The 1,3-dipolar cycloaddition reactions given mainly anti adducts 3 and 4 (greater than or equal to 95% pi-facial stereoselectivity). The cycloadducts 3 with H-3,5 cis are formed either exclusively or preponderate over the trans diastereoisomers 4.

Web-based cheminformatics and molecular property prediction tools supporting drug design and development at Novartis.

Web-based tools offer many advantages for processing chemical information, most notably ease of use and high interactivity. Therefore more and more pharmaceutical companies are using web technology to deliver sophisticated molecular processing tools directly to the desks of their chemists, to assist them in the process of designing and developing new drugs. In this paper, the web-based cheminformatics system developed at Novartis and currently used by more than thousand users is described. The system allows various molecular modeling and molecular processing tasks, including the calculation of molecular and substituent properties, property-based virtual screening, visualization of molecules, bioisosteric design, diversity analysis, and support of combinatorial chemistry. The methodology to calculate various molecular properties relevant to drug design is described, including the prediction of intestinal absorption, blood-brain barrier penetration, efflux, and water solubility. Information about the web technology used is also provided.

The multiple benefits of accurate assessment. Effective management of leg ulcers.

1. To be effective, the treatment of leg ulcers should be directed at the underlying cause. 2. If the underlying cause is chronic venous insufficiency, the most effective treatment is compression therapy. 3. There are a number of effective bandaging systems in use; the choice of any particular regimen is of less importance than the skill of the practitioner in applying it. 4. The prevention of recurrence should form a significant part of the care plan.

Planning a route to treatment. A framework for leg ulcer assessment.

There are three distinct stages to leg ulcer assessment. Basic knowledge of the anatomy of the leg is essential for identifying and recognising predisposing factors. A holistic approach ensures that perpetuating factors, frequently the cause of failure to heal, are eliminated or reduced. Careful observation and measurement of presenting factors assist in identifying predisposing and perpetuating factors and provide the baseline by which to determine the ulcer's progress.

A valuable contribution to care. The role of leg ulcer resource groups.

The survey on which this series has been based suggests district nurses are becoming more competent and confident in treating most leg ulcers. This final article discusses possible future research and the role of leg ulcer resource groups.

Look beyond the ulcer itself: Assessment of leg ulcers.

Accurate assessment of leg ulcers involves identifying their predisposing, precipitating and perpetuating causes. Once these have been established, effective treatment can be instigated or even, in some cases, recurrence avoided.

Allow the ulcer to heal. Treatment of leg ulcers.

A key priority when treating leg ulcers is to identify the underlying cause and reduce any exacerbating factors. A problem-solving approach, which allows the ulcer to heal, will achieve optimum results.

Contemporaneous cell spreading and phagocytosis: magneto-resistive real-time monitoring of membrane competing processes.

Adhesion and spreading of cells strongly depend on the properties of the underlying surface, which has significant consequences in long-term cell behavior adaption. This relationship is important for the understanding of both biological functions and their bioactivity in disease-related applications. Employing our magnetic lab-on-a-chip system, we present magnetoresistive-based real-time and label-free detection of cellular phagocytosis behavior during their spreading process on particle-immobilized sensor surfaces. Cell spreading experiments carried out on particle-free and particle-modified surfaces reveal a delay in spreading rate after an elapsed time of about 2.2h for particle-modified surfaces due to contemporaneous cell membrane loss by particle phagocytosis. Our associated magnetoresistive measurements show a high uptake rate at early stages of cell spreading, which decreases steadily until it reaches saturation after an average elapsed time of about 100 min. The corresponding cellular average uptake rate during the entire cell spreading process accounts for three particles per minute. This result represents a four times higher phagocytosis efficiency compared to uptake experiments carried out for confluently grown cells, in which case cell spreading is already finished and, thus, excluded. Furthermore, other dynamic cell-surface interactions at nano-scale level such as cell migration or the dynamics of cell attachment and detachment are also addressable by our magnetic lab-on-a-chip approach.

Magnetoresistive-based real-time cell phagocytosis monitoring.

The uptake of large particles by cells (phagocytosis) is an important factor in cell biology and also plays a major role in biomedical applications. So far, most methods for determining the phagocytic properties rely on cell-culture incubation and end-point detection schemes. Here, we present a lab-on-a-chip system for real-time monitoring of magnetic particle uptake by human fibroblast (NHDF) cells. It is based on recording the time evolution of the average position and distribution of magnetic particles during phagocytosis by giant-magnetoresistive (GMR) type sensors. We employ particles with a mean diameter of 1.2 µm and characterize their phagocytosis-relevant properties. Our experiments at physiological conditions reveal a cellular uptake rate of 45 particles per hour and show that phagocytosis reaches saturation after an average uptake time of 27.7h. Moreover, reference phagocytosis experiments at 4°C are carried out to mimic environmental or disease related inhibition of the phagocytic behavior, and our measurements clearly show that we are able to distinguish between cell-membrane adherent and phagocytosed magnetic particles. Besides the demonstrated real-time monitoring of phagocytosis mechanisms, additional nano-biointerface studies can be realized, including on-chip cell adhesion/spreading as well as cell migration, attachment and detachment dynamics. This versatility shows the potential of our approach for providing a multifunctional platform for on-chip cell analysis.