Multibody dynamics analysis (MDA) as a numerical modelling tool to reconstruct the function and palaeobiology of extinct organisms

Recent advances in computer technology have substantially changed the field of palaeontology in the last two decades. Palaeontologists now have a whole new arsenal of powerful digital techniques available to study fossil organisms in unprecedented detail and to test hypotheses regarding function and behaviour. Multibody dynamics analysis (MDA) is one of these techniques and although it originated as a tool used in the engineering and automotive industry, it holds great potential to address palaeontological questions as well. MDA allows the simulation of dynamic movements in complex objects consisting of multiple linked components. As such, this technique is ideally suited to model biological structures and to obtain quantifiable results that can be used to test the function of musculoskeletal systems rigorously. However, despite these advantages, MDA has seen a slow uptake by the palaeontological community. The most likely reason for this lies in the steep learning curve and complexity of the method. This paper provides an overview of the underlying principles of MDA and outlines the main steps involved in conducting analyses. A number of recent studies using MDA to reconstruct the palaeobiology of fossil organisms are presented and the potential for future studies is discussed. Similar to other computational techniques, including finite element analysis and computational fluid dynamics, the non-invasive and exploratory power of MDA makes it ideally suited to study the form and function in vertebrates for which no modern analogues exist.     

A portable flow chamber for in situ determination of benthic metabolism

1. Many stream ecologists are interested in determining the metabolic rates of benthic organisms, particularly those of production and respiration. It is often necessary to make these measurements on fresh material in the field at remote sites. Recirculating chambers are commonly used for this purpose.
2. A broad variety of recirculating chambers are described in the literature, but each design has inherent limitations. The most common are inability to control flow in the chamber and match it with external flow rates, and a lack of the power required to do this for extended periods. Alteration of spectral irradiance, temperature rise and elevated internal chamber pressures are additional limitations that have received little attention.
3. We have designed and constructed a flow chamber that eliminates some of these problems. The chamber utilizes a DC motor-driven propeller as an efficient recirculator (axial impeller), minimizing power requirements and it is constructed of UVB transparent acrylic to allow a full spectral complement of solar irradiance in the interior. Modular components allow the chamber to be taken apart quickly for cleaning and replacement of parts, making it more functional than some previous designs.
4. The axial impeller chamber was compared to a similar sized conventional chamber that had a small diameter return line and a high capacity centrifugal pump. The axial impeller chamber had less of a temperature rise during field incubations, lower power consumption and less internal pressure in the return line when producing equivalent water velocities.
5. The reported axial impeller design had relatively homogeneous velocity across the working section relative to other chambers and was capable of water velocities in excess of 1 m s^–1.     

Evolutionary principles and the regulation of engineered bacteria

The introduction of engineered bacteria to the environment is being overregulated, on the basis of several assumptions: (i) the danger from deliberate introduction on a large scale is much greater than that from accidental release; (ii) the more distant the source of the DNA the greater the risk; (iii) novel organisms are likely to cause unexpected ecological damage, like that seen with native organisms transplanted to a novel location; (iv) even if the probability of harm is very small, great care must be taken because the harm might be large; (v) products of recombinant DNA must be treated differently from products of classical genetic manipulation; and (vi) our unlimited power to manipulate DNA implies an unlimited power to refashion organisms. Evolutionary principles contradict all these assumptions. Moreover, our increased power of genetic manipulation must be recognized as an expansion of the biotechnology of domestication; and unlike the physical technologies, the long history of domestication has not adventitiously created harmful by-products. I propose that in dealing with such novel and unpredictable developments it would be better to respond with speed and resilience to problems as they arise, rather than to hamper advances by clumsy regulations based on unsubstantiated guesses.     

The chlorosome: a prototype for efficient light harvesting in photosynthesis

Three phyla of bacteria include phototrophs that contain unique antenna systems, chlorosomes, as the principal light-harvesting apparatus. Chlorosomes are the largest known supramolecular antenna systems and contain hundreds of thousands of BChl c/d/e molecules enclosed by a single membrane leaflet and a baseplate. The BChl pigments are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Their excitation energy flows via a small protein, CsmA embedded in the baseplate to the photosynthetic reaction centres. Chlorosomes allow for photosynthesis at very low light intensities by ultra-rapid transfer of excitations to reaction centres and enable organisms with chlorosomes to live at extraordinarily low light intensities under which no other phototrophic organisms can grow. This article reviews several aspects of chlorosomes: the supramolecular and molecular organizations and the light-harvesting and spectroscopic properties. In addition, it provides some novel information about the organization of the baseplate.     

CONFINE-MAS: a magic-angle spinning NMR probe that confines the sample in case of a rotor explosion

Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called “rotor crash” involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials. 1D and 2D NMR experiments show that such a hazardous material-confining MAS probe (“CONFINE-MAS” probe) has a similar sensitivity compared to a standard probe and performs equally well in terms of spinning stability. We illustrate the CONFINE-MAS probe properties and performance by application to a fungal amyloid.     

A model of the protein–pigment baseplate complex in chlorosomes of photosynthetic green bacteria

In contrast to photosynthetic reaction centers, which share the same structural architecture, more variety is found in the light-harvesting antenna systems of phototrophic organisms. The largest antenna system described, so far, is the chlorosome found in anoxygenic green bacteria, as well as in a recently discovered aerobic phototroph. Chlorosomes are the only antenna system, in which the major light-harvesting pigments are organized in self-assembled supramolecular aggregates rather than on protein scaffolds. This unique feature is believed to explain why some green bacteria are able to carry out photosynthesis at very low light intensities. Encasing the chlorosome pigments is a protein-lipid monolayer including an additional antenna complex: the baseplate, a two-dimensional paracrystalline structure containing the chlorosome protein CsmA and bacteriochlorophyll a (BChl a). In this article, we review current knowledge of the baseplate antenna complex, which physically and functionally connects the chlorosome pigments to the reaction centers via the Fenna–Matthews–Olson protein, with special emphasis on the well-studied green sulfur bacterium Chlorobaculum tepidum (previously Chlorobium tepidum). A possible role for the baseplate in the biogenesis of chlorosomes is discussed. In the final part, we present a structural model of the baseplate through combination of a recent NMR structure of CsmA and simulation of circular dichroism and optical spectra for the CsmA–BChl a complex.     

The role of stress waves in thoracic visceral injury from blast loading: modification of stress transmission by foams and high-density materials

Materials have been applied to the thoracic wall of anaesthetised experimental animals exposed to blast overpressure to investigate the coupling of direct stress waves into the thorax and the relative contribution of compressive stress waves and gross thoracic compression to lung injury. The ultimate purpose of the work is to develop effective personal protection from the primary effects of blast overpressure--efficient protection can only be achieved if the injury mechanism is identified and characterized. Foam materials acted as acoustic couplers and resulted in a significant augmentation of the visceral injury; decoupling and elimination of injury were achieved by application of a high acoustic impedance layer on top of the foam. In vitro experiments studying stress wave transmission from air through various layers into an anechoic water chamber showed a significant increase in power transmitted by the foams, principally at high frequencies. Material such as copper or resin bonded Kevlar incorporated as a facing upon the foam achieved substantial decoupling at high frequencies--low frequency transmission was largely unaffected. An acoustic transmission model replicated the coupling of the blast waves into the anechoic water chamber. The studies suggest that direct transmission of stress waves plays a dominant role in lung parenchymal injury from blast loading and that gross thoracic compression is not the primary injury mechanism. Acoustic decoupling principles may therefore be employed to reduce the direct stress coupled into the body and thus reduce the severity of lung injury--the most simple decoupler is a high acoustic impedance material as a facing upon a foam, but decoupling layers may be optimized using acoustic transmission models. Conventional impacts producing high body wall velocities will also lead to stress wave generation and transmission--stress wave effects may dominate the visceral response to the impact with direct compression and shear contributing little to the aetiology of the injury.     

Scale-up criteria of square tank surface aerator

Oxygen transfer rate and the corresponding power requirement to operate the rotor are vital for design and scale-up of surface aerators. Present study develops simulation or scale-up criterion correlating the oxygen transfer coefficient and power number along with a parameter governing theoretical power per unit volume (X, which is defined as equal to F(4/3)R(1/3), where F and R are impellers' Froude and Reynolds number, respectively). Based on such scale-up criteria, design considerations are developed to save energy requirements while designing square tank surface aerators. It has been demonstrated that energy can be saved substantially if the aeration tanks are run at relatively higher input powers. It is also demonstrated that smaller sized tanks are more energy conservative and economical when compared to big sized tanks, while aerating the same volume of water, and at the same time by maintaining a constant input power in all the tanks irrespective of their size. An example illustrating how energy can be reduced while designing different sized aerators is given. The results presented have a wide application in biotechnology and bioengineering areas with a particular emphasis on the design of appropriate surface aeration systems.     

Performance analysis of a cardiac assist device in counterpulsation.

Performance of a cardiac assist device pumping chamber in counterpulsation was evaluated using numerical simulations of the unsteady, three-dimensional flow inside the chamber and an analytical model of the force required to eject and fill the chamber. The wall shear stress within the device was similarly computed and modeled. The analytical model was scaled to match the numerical results and then used to predict performance at physiological operating conditions. According to these models for a stroke volume of 70 ml, between 0.4 and 1.0 W is required for counterpulsation at a frequency of 1.33 Hz against a restorative spring, depending on the spring constant chosen. The power and the maximum force calculated are within the ranges a trained skeletal muscle is capable of providing. Shear stress predictions show that platelet activation in the absence of surface effects and hemolysis due to high shear are unlikely to occur with this design. Furthermore, vortices that develop in the chamber during filling are predicted to increase blood mixing and provide favorable washing of the chamber walls. A computational-analytical approach such as this may have potential to aid rapid performance evaluation of new devices and streamline the design optimization process.     

Cryo‐EM reconstruction of Type VI secretion system baseplate and sheath distal end

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath–tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath–tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non‐contractile Vibrio cholerae sheaths by cryo‐electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F₂/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

Structural role of the polyglutamate portion of the folate found in T4D bacteriophage baseplate.

Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.     

超分辨显微成像技术在活细胞成像中的应用与发展

超分辨显微成像技术(super-resolution microscopy,SRM)可以绕过光学衍射极限对成像分辨率的限制,让以前观察不到的纳米级结构实现可视化,这一重大研究进展推动了现代生命科学和生物医学研究的进步与发展.细胞是生物体的基本组成单位,对活细胞内部的细微结构和动力学过程进行研究是掌握生命本质必不可少的途...     

Building a genome engineering toolbox in nonmodel prokaryotic microbes

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO₂, and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop “design rules” for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.     

A topological model of the baseplate of lactococcal phage Tuc2009

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three lactococcal phages have been determined recently, illustrating their modular assembly and assigning the nature of their bacterial receptor. These RBPs are attached to the baseplate, a large phage organelle, located at the tip of the tail. Tuc2009 baseplate is formed by the products of 6 open read frames, including the RBP. Because phage binding to its receptor induces DNA release, it has been postulated that the baseplate might be the trigger for DNA injection. We embarked on a structural study of the lactococcal phages baseplate, ultimately to gain insight into the triggering mechanism following receptor binding. Structural features of the Tuc2009 baseplate were established using size exclusion chromatography coupled to on-line UV-visible absorbance, light scattering, and refractive index detection (MALS/UV/RI). Combining the results of this approach with literature data led us to propose a "low resolution" model of Tuc2009 baseplate. This model will serve as a knowledge base to submit relevant complexes to crystallization trials.     

Identification of P48 and P54 as components of bacteriophage T4 baseplates.

The involvement of two bacteriophage T4 gene products in the initiation of T4 tail tube and sheath polymerization on mature baseplates has been studied by radioautography of acrylamide gels of various partially completed tail structures. The products of genes 48 and 54 (P48[the nomenclature P48 refers to the protein product of bacteriophage T4 gene 48] and P54), which are known to be required for the synthesis of mature baseplates, have been shown to be structural components of the baseplate. These gene products have molecular weights of 42,000 and 33,000, respectively. The addition of P54 to the baseplate not only permits the polymerization of the core protein, P19, onto the baseplate, but also caused the disappearance of a polypeptide of molecular weight about 15,000 from the supernatant fraction of infected cells. Another gene product, P27, has been identified in the crude extracts of infected cells. This gene product, which is required for the synthesis of baseplate structures, has the same mobility as one of the unidentified structural polypeptides of the baseplate and is therefore probably also a baseplate component.     

Adapting immunity with subunit vaccines: case studies with group A Streptococcus and malaria

Although vaccines have widely been regarded as the most cost-effective way to improve public health, for some organisms new technological advances in vaccine design and delivery, incurring additional developmental costs, will be essential. These organisms are typically those for which natural immunity is either slow to develop or does not develop at all. Clearly, such organisms have evolved strategies to evade immune responses and innovative approaches will be required to induce a type of immune response which is both different to that which develops naturally and is effective. This article describes some approaches to develop vaccines for two such organisms (malaria parasites and Streptococcus pyogenes (group A Streptococcus)) that are associated with widespread mortality and morbidity, mostly in the poorest countries of the world. At this stage, the challenges are primarily scientific, but if these hurdles are surmounted then the challenges will become financial ones--developing much needed vaccines for people least able to afford them.     

Biosafety: future priorities for research in health care

Currently the public interest in biosafety issues has focussed on the discussions surrounding the use of genetically modified organisms, very specifically on the use of transgenic plants in agriculture. Although many of the questions raised in connection with genetically modified organisms are of legitimate scientific interest, attention should be drawn back to a number of other more classical biosafety research areas, namely the problem of control of new and reemerging infectious diseases, the need for new vaccines, control of transport and routes of dissemination, biosafety information exchange and networking, where research results are dearly needed. In the area of modern biotechnology new applications such as gene therapy and transgenic animals will be on the list of future priorities for biosafety related activities and research.     

Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components

Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.     

A numerical model of heterogeneous surface strains in polymer scaffolds

In vitro bone tissue growth inside porous scaffolds can be enhanced by macroscopic cyclic compression of the construct, but the heterogeneous strain generated inside the construct must be investigated to determine appropriate levels of compression. For this purpose a linear micro-finite element (muFE) technique based on micro-computed tomography (muCT) was verified for the calculation of local displacements inside polymer scaffolds, from which local strains may be estimated. Local displacements in the axial direction at the surface of microstructures inside the scaffold in 60 locations were calculated with the muFE model, based on compression simulation of a muCT reconstruction of the scaffold. These displacements were compared with accurately measured displacements in the axial direction in the same polymer scaffold at the same 60 locations, using a micro-compression chamber and muCT reconstructions of the scaffold under two fixed levels of compression (5% and 0%). The correlation between the calculated and the measured displacements, after correction for the dependence of the axial displacement on the axial position, was r=0.786 (r2=0.617). From this we conclude that the linear muFE model is suitable to estimate local surface strains inside polymer scaffolds for tissue engineering applications. This technique can not only be used to determine appropriate parameters such as the level of macroscopic compression in experimental design, but also to investigate the cellular response to local surface strains generated inside three-dimensional scaffolds.