Efficiency of energy utilisation and voluntary feed intake in ruminants

Energy requirements of animals are most readily expressed in terms of net energy (NE), while the energy yield of feed is, at least initially, expressed in terms of metabolisable energy (ME). Energy evaluation systems 'translate' NE requirements into ME requirements (ME systems) or assign NE values to feeds (NE systems). Efficiency of ME utilisation is higher for maintenance than for production and the NE yield of a feed varies, therefore, with ME intake. In addition, energetic efficiency for maintenance and production is thought to be different for lactating and non-lactating animals and to be affected by diet quality. As a result, there are currently many national energy evaluation systems that are complex, differ in their approach and are, as a result, difficult to compare. As ruminants in most production systems are fed ad libitum, this is also the most appropriate intake level at which to estimate energetic efficiency. Analyses of older as well as more recent data suggest that ad libitum feeding (i) abolishes the effects of diet quality on energetic efficiency (almost) completely, (ii) abolishes the differences between lactating and non-lactating animals (almost) entirely and (iii) results in overall energetic efficiencies that are always close to 0.6. The paper argues that there is now sufficient information to develop an international energy evaluation system for ad libitum fed ruminants. Such a system should (i) unify ME and NE systems, (ii) avoid the systematic bias and large errors that can be associated with current systems (iii) be simpler than current systems and (iv) have as a starting point a constant efficiency of ME utilisation, with a value of around 0.6. The remarkably constant efficiency of ME utilisation in ad libitum fed ruminants could be the result of energetic efficiency as well as feed intake regulation being affected by the same variables or of a direct role of energetic efficiency in feed intake regulation. Models to predict intake on the basis of the latter hypothesis are already available for non-reproducing ruminants but remain to be developed for reproducing animals.     

Energy intake and milk production in mink (MUSTELA vison)‐effect of litter size

Energy intake and milk production were measured in 12 mink dams raising litters of 3, 6 and 9 kits one to four weeks post partum by means of balance experiments and measurements of milk intake of the kits by the water isotope dilution technique. The dams were fed ad libitum on a conventional wet mink diet (DM: 323 g/kg; CP: 173 g/kg; ME: 4.4 MJ/kg). Milk samples collected from dams with corresponding litter sizes and lactation weeks, and body composition of kits nursed by these dams, were analysed for content of DM, ash, N and fat. The ME and drinking water consumption were higher in dams nursing 9 kits than in dams nursing 3 kits. The N and water balances as well as the live weight of dams were not affected by litter size. Daily milk production was higher in dams nursing 9 kits than in dams nursing 3 kits. The DM, N and fat content of the milk increased during lactation, but were not affected by litter size. Individual kit live weight was higher in litters of 3 than in litters of 6 and 9 kits four weeks post partum. The DM and fat content of the kits were lowest in kits from litters of 9 kits, whereas these kits had the highest protein content. Daily ME for maintenance of kits and the efficiency of utilisation of ME in milk for body gain were estimated to 356 kJ/kg^0.75, k_p ≈0.53 and k_f ≈0.71, respectively. In conclusion, daily milk production increased with increasing litter size, but not in proportion to the number of kits, indicating that milk production limits the growth rate of the young. In the fourth week of lactation, milk production was not different between dams nursing 6 or 9 kits, indicating a maximum capacity.     

Photobioreactors: production systems for phototrophic microorganisms

Microalgae have a large biotechnological potential for producing valuable substances for the feed, food, cosmetics and pharmacy industries as well as for biotechnological processes. The design of the technical and technological basis for photobioreactors is the most important issue for economic success in the field of phototrophic biotechnology. For future applications, open pond systems for large-scale production seem to have a lower innovative potential than closed systems. For high-value products in particular, closed systems of photobioreactors seem to be the more promising field for technical developments despite very different approaches in design.     

Enhanced resolution of molecular recognition to distinguish structurally similar molecules by different conformational responses of a protein upon ligand binding

MutT distinguishes substrate 8-oxo-dGTP from dGTP and also 8-oxo-dGMP from dGMP despite small differences of chemical structures between them. In this paper we show by the method of molecular dynamics simulation that the transition between conformational substates of MutT is a key mechanism for a high-resolution molecular recognition of the differences between the very similar chemical compounds. (1) The native state MutT has two conformational substates with similar free energies, each characterized by either open or closed of two loops surrounding the substrate binding active site. Between the two substates, the open substate is more stable in free MutT and in dGMP-MutT complex, and the closed substate is more stable in 8-oxo-dGMP-MutT complex. (2) Conformational fluctuation of the open substate is much larger than that of the closed substate. An estimate of associated entropy difference was found to be consistent with the experimentally found difference of entropy contribution to the binding free energies of the two molecules. (3) A hydrogen bond between H7 atom of 8-oxo-dGMP and the sidechain of Asn119 plays a crucial role for maintaining the closed substate in 8-oxo-dGMP-MutT complex. When this hydrogen bond is absent in the H7-deficient dGMP-MutT complex, the closed substate is no more maintained and transition to the more entropically-favored open substate is induced. (4) Thus, this mechanism of the hydrogen bond controlling the relative stabilities of the drastically different two conformational substates enhances the resolution to recognize the small difference of the chemical structures between the two molecules, dGMP and 8-oxo-dGMP.     

Comparison of energy evaluation systems and a mechanistic model for milk production by dairy cattle offered fresh grass-based diets

Grass-based diets are of increasing social-economic importance in dairy cattle farming, but their low supply of glucogenic nutrients may limit the production of milk. Current evaluation systems that assess the energy supply and requirements are based on metabolisable energy (ME) or net energy (NE). These systems do not consider the characteristics of the energy delivering nutrients. In contrast, mechanistic models take into account the site of digestion, the type of nutrient absorbed and the type of nutrient required for production of milk constituents, and may therefore give a better prediction of supply and requirement of nutrients. The objective of the present study is to compare the ability of three energy evaluation systems, viz. the Dutch NE system, the agricultural and food research council (AFRC) ME system, and the feed into milk (FIM) ME system, and of a mechanistic model based on Dijkstra et al. [Simulation of digestion in cattle fed sugar cane: prediction of nutrient supply for milk production with locally available supplements. J. Agric. Sci., Cambridge 127, 247–60] and Mills et al. [A mechanistic model of whole-tract digestion and methanogenesis in the lactating dairy cow: model development, evaluation and application. J. Anim. Sci. 79, 1584–97] to predict the feed value of grass-based diets for milk production. The dataset for evaluation consists of 41 treatments of grass-based diets (at least 0.75 g ryegrass/g diet on DM basis). For each model, the predicted energy or nutrient supply, based on observed intake, was compared with predicted requirement based on observed performance. Assessment of the error of energy or nutrient supply relative to requirement is made by calculation of mean square prediction error (MSPE) and by concordance correlation coefficient (CCC). All energy evaluation systems predicted energy requirement to be lower (6–11%) than energy supply. The root MSPE (expressed as a proportion of the supply) was lowest for the mechanistic model (0.061), followed by the Dutch NE system (0.082), FIM ME system (0.097) and AFRC ME system (0.118). For the energy evaluation systems, the error due to overall bias of prediction dominated the MSPE, whereas for the mechanistic model, proportionally 0.76 of MSPE was due to random variation. CCC analysis confirmed the higher accuracy and precision of the mechanistic model compared with energy evaluation systems. The error of prediction was positively related to grass protein content for the Dutch NE system, and was also positively related to grass DMI level for all models. In conclusion, current energy evaluation systems overestimate energy supply relative to energy requirement on grass-based diets for dairy cattle. The mechanistic model predicted glucogenic nutrients to limit performance of dairy cattle on grass-based diets, and proved to be more accurate and precise than the energy systems. The mechanistic model could be improved by allowing glucose maintenance and utilization requirements parameters to be variable.     

From laboratory to commercial production: a case study of a Spirulina (Arthrospira) facility in Musina, South Africa

Until recently, most large commercial scale microalgal production systems employed open systems. However, several large-scale closed systems have now been built and, for the first time, actual comparisons can be made. There are major operational differences between open and closed photobioreactors and, consequently, the growth physiology of the microalgae is different between the two systems. Several of the factors governing growth can, within certain boundaries, be manipulated while others are specific to the cultivation system. Crucial factors are the optical depth, turbulence, light acclimated state of the organism, nutrient availability and metabolite accumulation. In the final analyses, systems are used for specific purposes and each will determine which system is the most suitable, since there is no universal all-purpose photobioreactor.     

Factors governing algal growth in photobioreactors: the “open” versus “closed” debate

Until recently, most large commercial scale microalgal production systems employed open systems. However, several large-scale closed systems have now been built and, for the first time, actual comparisons can be made. There are major operational differences between open and closed photobioreactors and, consequently, the growth physiology of the microalgae is different between the two systems. Several of the factors governing growth can, within certain boundaries, be manipulated while others are specific to the cultivation system. Crucial factors are the optical depth, turbulence, light acclimated state of the organism, nutrient availability and metabolite accumulation. In the final analyses, systems are used for specific purposes and each will determine which system is the most suitable, since there is no universal all-purpose photobioreactor.     

Two-dimensional kinetic analysis suggests nonsequential gating of mechanosensitive channels in Xenopus oocytes

Xenopus oocytes express mechanosensitive (MS(XO)) channels that can be studied in excised patches of membrane with the patch-clamp technique. This study examines the steady-state kinetic gating properties of MS(XO) channels using detailed single-channel analysis. The open and closed one-dimensional dwell-time distributions were described by the sums of 2-3 open and 5-7 closed exponential components, respectively, indicating that the channels enter at least 2-3 open and 5-7 closed kinetic states during gating. Dependency plots revealed that the durations of adjacent open and closed intervals were correlated, indicating two or more gateway states in the gating mechanism for MS channels. Maximum likelihood fitting of two-dimensional dwell-time distributions to both generic and specific models was used to examine gating mechanism and rank models. A kinetic scheme with five closed and five open states, in which each closed state could make a direct transition to an open state (two-tiered model) could account for the major features of the single-channel data. Two-tiered models that allowed direct transitions to subconductance open states in addition to the fully open state were also consistent with multiple gateway states. Thus, the gating mechanism of MS(XO) channels differs from the sequential (linear) gating mechanisms considered for MS channels in bacteria, chick skeletal muscle, and Necturus proximal tubule.     

Superposition properties of interacting ion channels.

Quantitative analysis of patch clamp data is widely based on stochastic models of single-channel kinetics. Membrane patches often contain more than one active channel of a given type, and it is usually assumed that these behave independently in order to interpret the record and infer individual channel properties. However, recent studies suggest there are significant channel interactions in some systems. We examine a model of dependence in a system of two identical channels, each modeled by a continuous-time Markov chain in which specified transition rates are dependent on the conductance state of the other channel, changing instantaneously when the other channel opens or closes. Each channel then has, e.g., a closed time density that is conditional on the other channel being open or closed, these being identical under independence. We relate the two densities by a convolution function that embodies information about, and serves to quantify, dependence in the closed class. Distributions of observable (superposition) sojourn times are given in terms of these conditional densities. The behavior of two channel systems based on two- and three-state Markov models is examined by simulation. Optimized fitting of simulated data using reasonable parameters values and sample size indicates that both positive and negative cooperativity can be distinguished from independence.     

Structural models for the KCNQ1 voltage-gated potassium channel

Mutations in the human voltage-gated potassium channel KCNQ1 are associated with predisposition to deafness and various cardiac arrhythmia syndromes including congenital long QT syndrome, familial atrial fibrillation, and sudden infant death syndrome. In this work 3-D structural models were developed for both the open and closed states of human KCNQ1 to facilitate structurally based hypotheses regarding mutation-phenotype relationships. The KCNQ1 open state was modeled using Rosetta in conjunction with Molecular Operating Environment software, and is based primarily on the recently determined open state structure of rat Kv1.2 (Long, S. B., et al. (2005) Science 309, 897-903). The closed state model for KCNQ1 was developed based on the crystal structures of bacterial potassium channels and the closed state model for Kv1.2 of Yarov-Yarovoy et al. ((2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7292-7207). Using the new models for KCNQ1, we generated a database for the location and predicted residue-residue interactions for more than 85 disease-linked sites in both open and closed states. These data can be used to generate structure-based hypotheses for disease phenotypes associated with each mutation. The potential utility of these models and the database is exemplified by the surprising observation that four of the five known mutations in KCNQ1 that are associated with gain-of-function KCNQ1 defects are predicted to share a common interface in the open state structure between the S1 segment of the voltage sensor in one subunit and both the S5 segment and top of the pore helix from another subunit. This interface evidently plays an important role in channel gating.     

Fractal model of ion-channel kinetics

Markov models with discrete states, such as closed in equilibrium with closed in equilibrium with open have been widely used to model the kinetics of ion channels in the cell membrane. In these models the transition probabilities per unit time (the kinetic rate constants) are independent of the time scale on which they are measured. However, in many physical systems, a property, L, depends on the scale, epsilon, at which it is measured such that L(epsilon) alpha epsilon 1-D where D is the fractal dimension. Such systems are said to be 'fractal'. Based on the assumption that the kinetic rates are given by k(t) alpha t1-D we derive a fractal model of ion-channel kinetics. This fractal model has fewer adjustable parameters, is more consistent with the dynamics of protein conformations, and fits the single-channel recordings from the corneal endothelium better than the discrete-state Markov model.     

Kinetics and thermodynamics of the rate-limiting conformational change in the actomyosin V mechanochemical cycle

We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain-sensitive step in many muscle and non-muscle myosins. We also explored essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time-resolved FRET analysis demonstrates a temperature-dependent reversible conformational change in the nucleotide-binding pocket (NBP). Our kinetic results demonstrate that the NBP goes from a closed to an open conformation prior to the release of ADP, while the actin-binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating that this is the rate-limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates that the transition from the open to closed NBP conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the NBP and actin-binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open NBP conformation may be altered by lever arm position.     

Mild cycles open closed communities to ecological restoration

Species loss is a global issue. With up to a million species at risk and insufficient protected area to maintain the world's biodiversity, humanity will increasingly need to rely on species re‐introductions to locally restore diversity and function. However, such restoration attempts are bound to fail when ecological communities get locked in a closed state that is resistant to recovery. It is presently unknown how to repair these closed systems. We use mathematical models to identify ways out of this problem. We first show how ecological communities may enter a closed state, to then explain how to open them up again for restoration of their original diversity. We find that restoration is often still possible shortly after initial species loss, as (1) the secondary extinctions that produce closure have not happened yet and (2) mild population fluctuations still allow successful repair during a transient postdisturbance phase. However, after this typically short window of opportunity for restoration, the system enters a new equilibrium, which may be a closed state. Our analysis shows how to take ecological communities out of the closed state: Appropriate management of carrying capacities produces a regime of mild population fluctuations that opens a window for successful species re‐introductions. These windows can be perpetually recurring or permanently open. Such opportunities for repair can be absent under regimes of wild cycles or perfect stability. We conclude that mild cycles may open windows of opportunity for the repair of communities that have become resistant to recovery.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Good design practices for an integrated containment and production system for advanced therapies

Advances in molecular biology and the possibility of differentiating stem cells have opened up new scenarios in therapies that use progenitor or variously differentiated cells. Regardless of the choice of the system, designing a plant for producing advanced therapies requires a clear understanding of the final objective (the product), taking into account all the regulatory, environment, process, risk assessment, asepsis, and validation aspects involved until its implementation. Good Manufacturing Practice (GMP) compliant procedures are a prerequisite for cell production in clinical application, and clean rooms are zones for producing cell therapies. Clean rooms for clinical application require high running and maintenance costs and need trained operators and strict procedures to prepare the rooms and the people involved in the processes. While today production mainly occurs in open systems (clean rooms), there is evidence of processes in closed systems (isolators). The isolator is a Grade A aseptic closed system that requires a controlled environment and at least a Grade D environment in the case of sterile productions (A in D closed system). The use of isolators can ensure a very high level of protection against the risk of product contamination and, at the same time, provide the operators with a very safe working environment. Furthermore, working with closed systems can optimize and facilitate the production of Advanced Therapy Medical Products in GMP environments, by providing an easily reproducible working tool even for large-scale production, with generally lower costs compared to a classical clean room approach. In conclusion, the isolator workstation as a possible alternative to the classic clean room, due to its small size and the simplification of the working and maintenance operational procedures, may represent an interesting solution in the perspective of the increasingly more stringent requests for cost reductions of GMP in clinical application.     

Biotechnology of algal biomass production: a review of systems for outdoor mass culture

Microalgae are very efficient solar energy converters and they can produce a great variety of metabolites. Man has always tried to take advantage of these proporties through algal mass culture. Despite the fact that many applications for microalgae have been described in the literature, these micro-organisms are still of minor economic importance. Industrial reactors for algal culture are at present, all designed as open race-ways (shallow open ponds where culture is circulated by a paddle-wheel). Technical and biological limitations of these open systems have given rise to the development of enclosed photoreactors (made of transparent tubes, sleeves or containers and where light source may be natural or artificial). The present review surveys advances in these two technologies for cultivation of microalgae. Starting from published results, the advantages and disadvantages of open systems and closed photobioreactors are discussed. A few open systems are presented for which particularly reliable results are available. Emphasis is then put on closed systems, which have been considered as capital intensive and are justified only when a fine chemical is to be produced.     

Structural and functional alterations of myoglobin by glucose-protein interactions

Quorum sensing (QS) is a process of bacterial communication and cooperation mediated by the release of jointly exploited signals and “public goods” into the environment. There are conflicting reports on the behavior of mutants deficient in the release of these materials. Namely, mutants that appear perfectly viable and capable of outgrowing wild type cells in a closed model system such as a culture flask, may not be viable or invasive on open surfaces such as agar plates. Here we show via agent-based computational simulations that this apparent discrepancy is due to the difference between open and closed systems. We suggest that the experimental difference is due to the fact that wild type cells can easily saturate a well-mixed culture flask with signals and public goods so QS will be not necessary after a certain time point. As a consequence, QS-deficient mutants can continue to grow even after the wild type population has vanished. This phenomenon is not likely to occur in open environments including open surfaces and agar plate models. In other words, even if QS is required for survival, QS deficient mutants may grow faster initially in short term laboratory experiments or computer simulations, while only WT cells appear stable over longer time scales, especially when adaptation to changing environments is important.     

Are organisms committed to lower their rates of entropy production?: Possible relevance to evolution of the Prigogine theorem and the ergodic hypothesis

The physiology at limiting and stress conditions challenges the current view that the overall reaction of metabolic processes is always far from equilibrium and, therefore, that organisms are not committed to lower their rates of entropy production. Plausibly, critical steps of natural selection takes place at limiting conditions, near equilibrium, in the linear range response of entropy production, and consequently the trend to lower the rate of entropy production could be the fitness arrow of biological evolution. The evolutionary relevance of the Prigogine theorem is discussed in connection with the ergodic hypothesis of Boltzmann. The emergence of metabolic strategies to economise carbon/energy resources, of resource-waste systems like active transport and the irreversible increase in the complexity of organisms during evolution may be consequences of a more general trend of metabolic systems to lower the rates of entropy production.     

A model of the gating of ion channels

The gating of ion channels in biological membranes has usually been described in terms of Markov transitions between a few discrete open or closed states. Such models predict that the distributions of open and closed durations decay as a sum of exponential terms. Recent experimental data have indicated that certain channels are not easily described by these models. We show that distributions of open and closed times similar to those seen experimentally are predicted by a model that involves only one open and closed state but that assumes the activation energy of the gating process to be stochastic. This model involves only a few parameters and these have direct physical interpretations. Measurements of the correlation between the durations of successive open or closed events is shown to provide an experimental method for distinguishing between this and other models.