Muscle-specific indices to characterise the functional behaviour of human lower-limb muscles during locomotion

The mechanical output of a muscle may be characterised by having distinct functional behaviours, which can shift to satisfy the varying demands of movement, and may vary relative to a proximo-distal gradient in the muscle-tendon architecture (MTU) among lower-limb muscles in humans and other terrestrial vertebrates. We adapted a previous joint-level approach to develop a muscle-specific index-based approach to characterise the functional behaviours of human lower-limb muscles during movement tasks. Using muscle mechanical power and work outputs derived from experimental data and computational simulations of human walking and running, our index-based approach differentiated known distinct functional behaviours with varying mechanical demands, such as greater spring-like function during running compared with walking; with anatomical location, such as greater motor-like function in proximal compared with the distal lower-limb muscles; and with MTU architecture, such as greater strut-like muscles fibre function compared with the MTU in the ankle plantarflexors. The functional indices developed in this study provide distinct quantitative measures of muscle function in the human lower-limb muscles during dynamic movement tasks, which may be beneficial towards tuning the design and control strategies of physiologically-inspired robotic and assistive devices.     

Drosophila as a screening tool to study human neurodegenerative diseases

In an aging society, research involving neurodegenerative disorders is of paramount importance. Over the past few years, research on Alzheimer's and Parkinson's diseases has made tremendous progress. Experimental studies, however, rely mostly on transgenic animal models, preferentially using mice. Although experiments on mice have enormous advantages, they also have some inherent limitations, some of which can be overcome by the use of Drosophila melanogaster as an experimental animal. Among the major advantages of using the fly is its small genome, which can also be modified very easily. The fact that its genome lends itself to diverse alterations (e. g. mutagenesis, transposons) has made the fly a useful organism to perform large‐scale and genome‐wide screening approaches. This has opened up an entirely new field of experimental research aiming to elucidate genetic interactions and screen for modifiers of disease processes in vivo. Here, we provide a brief overview of how flies can be used to analyze molecular mechanisms underlying human neurodegenerative diseases.     

Transplantation as a tool to study progenitors within the vertebrate nervous system

In recent years, many studies have focused on the fate and potential of neural progenitors in vertebrates. While much progress has been made, many questions remain about the mechanisms which lead to neural diversity, in terms of both the regionalization of the nervous system and specification of cell fates within those regions. Studies aimed at addressing these questions have fallen into three main categories: in vivo lineage tracings, in vitro differentiation analyses, and in vivo cell transplantation studies. This body of work has pointed to the existence of both pluripotent and unipotent neural progenitors, and has suggested that both cell intrinsic and extrinsic cues play a role in the determination of neural cell fate. In addition, the existence of neural “stem cells” maintained into adulthood has been suggested. This review will focus on transplantation studies in mammals, and will emphasize how this method has been useful as a means of determining the changing potential of neural precursors and their environments within the developing nervous system. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 152–161, 1998     

Solid-phase handling of hydrophobins: immobilized hydrophobins as a new tool to study lipases

Hydrophobins are fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. This attribute can be used to introduce hydrophobic foci on the surface of hydrophilic supports where hydrophobins are attached by covalent binding. In this paper, we report the binding of Pleurotus ostreatus hydrophobins to a hydrophilic matrix (agarose) to construct a support for noncovalent immobilization and activation of lipases from Candida antarctica, Humicola lanuginosa, and Pseudomonas flourescens. Lipase immobilization on agarose-bound hydrophobins proceeded at very low ionic strength and resulted in increased lipase activity and stability. The enzyme could be desorbed from the support using moderate concentrations of Triton X-100, and its enantioselectivity was similar to that of lipases interfacially immobilized on conventional hydrophobic supports. These results suggest that lipase adsorption on hydrophobins follows an "interfacial activation" mechanism; immobilization on hydrophobins offers new possibilities for lipase study and modulation and reveals a new application for fungal hydrophobins.     

Mice 3D testicular organoid system as a novel tool to study Zika virus pathogenesis

Zika virus (ZIKV) poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage. However, deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury. Organoids are relatively simple in vitro models, which could mimic the pathological changes of corresponding organs. In this study, we constructed a 3D testicular organoid model using primary testicular cells from adult BALB/c mice. Similar to the testis, this organoid system has a blood-testis barrier (BTB)-like structure and could synthesize testosterone. ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis. Therefore, our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.     

Evaluation of human hepatocyte incubation as a new tool for metabolism study of androstenedione and norandrostenedione in a doping control perspective

A novel and simple method has been developed for the simultaneous quantification of tryptophan, kynurenine and indole derivatives as well as four catecholamines, including dopamine, noradrenaline, homovanillic acid and 3,4-dihydroxyphenylacetic acid. The method utilises isocratic reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase and the detection potentials, was investigated. Separation of 13 compounds was achieved by a mobile phase consisting of 10% methanol in 50 mM sodium phosphate-acetate buffer, pH 4.10, containing 0.42 mM octanesulphonic acid. The calibration curve was linear over the range 12 pg to 300 ng on-column. The detection limits (SIN 3) depended on the working potential and were found to be between 10 and 100 pg injected. The method was reproducible with intra-day RSDs of 0.3 to 1.5% and inter-day RSDs of 0.5 to 4%.     

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months. Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations. The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions.IHH cells retain several differentiated features of normal hepatocytes. They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 µg albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 µg/ml per day), and apolipoprotein A-I (1 µg/ml per day). However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range.In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells. Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.     

A microfluidic gradient mixer-flow chamber as a new tool to study biofilm development under defined solute gradients

Understanding the dynamics of biofilm development in response to chemical cues and signals is required toward the development of controllable biofilm-mediated bioprocesses. In this study, we report a new biofilm growth system that integrates a microfluidic gradient mixer with a biofilm growth chamber. The biofilm growth system allows biofilms to grow under defined solute gradients and enables nondestructive monitoring of the biofilm development dynamics in response to the defined gradients. The solute gradients generated in the system were simulated and then validated experimentally. We then demonstrated the applicability of the biofilm growth system in studying biofilm development under defined solute gradients. Specifically, we examined biofilm development of Shewanella oneidensis and Comamonas testosteroni under a defined calcium and nitrate gradient, respectively. Using two C. testosteroni strains (WDL7 and I2), we further demonstrated the applicability of our biofilm growth system to study the development of coculture biofilms under a defined solute gradient. Our results show that the biofilm growth system we have developed here can be a promising tool to reveal the dynamics of biofilm development in response to chemical cues and signals as well as the interorganism interactions in coculture biofilms.     

Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi

Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus.     

Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes

Chromatin organization spans a wide range of structural complexity. Substructures at the 10-200nm scale are poorly characterized, especially in living cells, due to the limitations of electron microscopy and standard optical microscopy. Recently developed super-resolution fluorescence microscopy methods represent an exciting opportunity to access those substructures, and recent progress with these techniques has yielded insights into chromatin organization at different condensation stages. Recent studies have focused on confronting the challenges that are specific to chromatin super-resolution imaging, such as the high packing density of mitotic chromosomes and difficulties in interpreting interphase chromatin images. Building on these first results and with ongoing rapid technical advances in super-resolution fluorescence imaging there is great potential to uncover new features with unprecedented detail.     

Secretomers as a new tool for the monitoring of CTL responses

Efforts to follow tumor-specific immune responses in patients are often thwarted by lack of knowledge of the appropriate tumor antigens and the CTL epitopes of those antigens. There is, therefore, a growing need for techniques to monitor tumor-specific immune responses in settings where tumor antigens, and antigenic epitopes, remain unidentified. Here we describe a novel system to follow tumor-specific CTL immune responses. A truncated, soluble murine class I MHC (H-2D^b) molecule was fused with a rat IgG_2a Fc, in order to allow secretion of the complex. Tumor-specific CTL could then be detected as a result of the complex fastening to specific T cell receptors (TCR). These constructs were inserted into the genome of a recombinant adenovirus vector. Infection of tumor cells with these adenovirus constructs results in the secretion of the complexes into the culture supernatant. These soluble divalent class I MHC molecules were used to detect and activate specific CTL populations.     

Use of TSAR as a new tool to analyze the molecular dynamics trajectories of proteins

There is a lack of tools to analyze simulations of protein molecular dynamics quantitatively. Our aim is to use calmodulin, a prototypical calcium-binding protein, to describe a strategy and some tools for extracting relevant information from dynamics calculations. Our main conclusions are as follows:

• •• Autocorrelation vectors may be used to represent a 3D conformation in an n-dimensional space, where n is variable (n ⩽ 20–30).
• •• On such a transformation, classic statistical tools (PCA, clustering, etc.) may be used to differentiate or characterize dynamics trajectories quantitatively.
• •• TSAR, an integrated package used for quantitative structure-activity relationships, is well suited (after minor modifications) for such a purpose.

Finally, this type of strategy is able to point out the effects of the solvent screening parameters of the Amber software on the dynamics trajectories of calmodulin.     

Computer animation as a tool to study preferences in the cichlid Pelvicachromis taeniatus

Four choice experiments were conducted with both sexes of the cichlid Pelvicachromis taeniatus using computer-manipulated stimuli of digital images differing in movement, body shape or colouration. The results show that computer animations can be useful and flexible tools in studying preferences of a cichlid with complex and variable preferences for different visual cues.     

A new type of chemically modified tRNA as a tool for the study of tRNA-aminoacyl-tRNA synthetase interaction

tRNA(Phe) in which the adenine and cytosine rings in the aminoacyl arm and in the anticodon loop were converted to alkylating derivatives by mild treatment with methyl chlorotetrolate was used to study the tRNA(Phe)-yeast phenylalanyl-tRNA(Phe) synthetase interaction. At neutral pH, modified tRNA inhibited the enzyme competitively. At pH 9 this binding is accompanied by irreversible inactivation of the enzyme due to alkylation of the alpha subunit of the synthetase. Such a derivatization of tRNA could probably be used to investigate the interaction of other tRNAs with their cognate synthetases.     

Comparative systems biology of human and mouse as a tool to guide the modeling of human placental pathology

Placental abnormalities are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting ～5% of all pregnancies. An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities, but there have been no systematic attempts to assess their molecular similarities or differences. We collected protein and mRNA expression data through shot‐gun proteomics and microarray expression analysis of the highly vascular exchange region, microdissected from the human and mouse near‐term placenta. Over 7000 ortholog genes were detected with 70% co‐expressed in both species. Close to 90% agreement was found between our human proteomic results and 1649 genes assayed by immunohistochemistry for expression in the human placenta in the Human Protein Atlas. Interestingly, over 80% of genes known to cause placental phenotypes in mouse are co‐expressed in human. Several of these phenotype‐associated proteins form a tight protein–protein interaction network involving 15 known and 34 novel candidate proteins also likely important in placental structure and/or function. The entire data are available as a web‐accessible database to guide the informed development of mouse models to study human disease.     

Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization

Soluble organic matrix (SOM) synthesis and secretion were investigated in two scleractinian corals using antibodies raised against this organic matrix. Results demonstrate that even if other cell types, including zooxanthellae, can supply precursors for SOM synthesis, only calicoblastic cells facing the skeleton are directly responsible for the synthesis and secretion of the SOM components. Results also indicate that, as is the case for other biominerals, skeleton formation is biologically controlled and not chemically dominated as originally believed. In addition to advancing the understanding of mechanisms of coral biomineralization, these antibodies could have numerous applications: for example as markers of skeletogenesis, as tools for cell culture, and in comparative studies among calcifying organisms.     

Probiogenomics as a tool to obtain genetic insights into adaptation of probiotic bacteria to the human gut

Bifidobacteria and lactobacilli are widely exploited as health-promoting bacteria in many functional foods. However, the molecular mechanisms as to how these bacteria positively impact on host health are far from completely understood. For this reason these microorganisms represent a growing area of interest with respect to their genomics, molecular biology and genetics. Recent genome sequencing of a large number of strains of bifidobacteria and lactobacilli has allowed access to the complete genetic makeup of representative members of these bacteria. Here, we will discuss how the analysis of genomic data has helped us to understand the mechanisms by which these bacteria adapt to the specific environment of the gastrointestinal tract, while also revealing genetic functions that mediate specific host-microbe interactions.     

The use of a syncytium model of the crystalline lens of the eye as a new tool to study the light flashes phenomenon seen by astronauts

A syncytium model to study some electrical properties of the eye is proposed to study the phenomenon of anomalous light flashes (LF) perceived by astronauts in orbit. The crystalline lens is modelled as an ellipsoidal syncytium with a variable relative dielectric constant. The corresponding mathematical model is a boundary value problem for a system of two coupled elliptic partial differential equations in the two unknown syncytial electrical potentials. A numerical method to compute an approximate solution of this mathematical model is used, and some numerical results are shown. The model can be regarded as a new tool to study the LF phenomenon. In particular, the energy lost in the syncytium by a transversing cosmic charged particle is calculated and the results obtained with the syncytium model are compared with those obtained using the previously available Geant 3.21 simulation program. In addition, the interaction of antimatter–syncytium is studied, and the Creme96 computer program is used to evaluate the cosmic ray fluxes encountered by the International Space Station in its standard mission.