Closed-loop EMG-informed model-based analysis of human musculoskeletal mechanics on rough terrains.

This work aims at estimating the musculoskeletal forces acting in the human lower extremity during locomotion on rough terrains. We employ computational models of the human neuro-musculoskeletal system that are informed by multi-modal movement data including foot-ground reaction forces, 3D marker trajectories and lower extremity electromyograms (EMG). Data were recorded from one healthy subject locomoting on rough grounds realized using foam rubber blocks of different heights. Blocks arrangement was randomized across all locomotion trials to prevent adaptation to specific ground morphology. Data were used to generate subject-specific models that matched an individual's anthropometry and force-generating capacity. EMGs enabled capturing subject- and ground-specific muscle activation patterns employed for walking on the rough grounds. This allowed integrating realistic activation patterns in the forward dynamic simulations of the musculoskeletal system. The ability to accurately predict the joint mechanical forces necessary to walk on different terrains have implications for our understanding of human movement but also for developing intuitive human machine interfaces for wearable exoskeletons or prosthetic limbs that can seamlessly adapt to different mechanical demands matching biological limb performance.

Comparative analysis of MIR168 promoters in three plant species.

MicroRNAs (miRNAs) play important roles in the regulation of gene expression by post-transcriptionally targeting mRNAs for cleavage or translational repression. miR168 is a key miRNA because it regulates the expression of the slicer protein ARGONAUTE1 (AGO1), which catalyzes mRNA cleavage. Interestingly, plant miR168s are highly evolutionarily conserved; however, it is unclear whether MIR168 promoter elements and expression patterns are also conserved. Here, we isolated MIR168 promoters from monocot rice and dicot grape genomes. To determine the expression pattern, different promoters were fused to a beta-glucoronidase reporter gene and the resulting constructs were then transformed in Arabidopsis. The results revealed clear differences in the MIR168 promoter sequence of monocot and dicot plant species. Moreover, the pattern of MIR168 promoter expression differed between monocots and dicots. These results suggest that, unlike that of miR168, the MIR168 promoter is not conserved in monocots and dicots.

GST-PRIME: a genome-wide primer design software for the generation of gene sequence tags.

The availability of sequenced genomes has generated a need for experimental approaches that allow the simultaneous analysis of large, or even complete, sets of genes. To facilitate such analyses, we have developed GST-PRIME, a software package for retrieving and assembling gene sequences, even from complex genomes, using the NCBI public database, and then designing sets of primer pairs for use in gene amplification. Primers were designed by the program for the direct amplification of gene sequence tags (GSTs) from either genomic DNA or cDNA. Test runs of GST-PRIME on 2000 randomly selected Arabidopsis and Drosophila genes demonstrate that 93 and 88% of resulting GSTs, respectively, fulfilled imposed length criteria. GST-PRIME primer pairs were tested on a set of 1900 Arabidopsis genes coding for chloroplast-targeted proteins: 95% of the primer pairs used in PCRs with genomic DNA generated the correct amplicons. GST-PRIME can thus be reliably used for large-scale or specific amplification of intron-containing genes of multicellular eukaryotes.

Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis.

The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

Disruption of the Arabidopsis photosystem I gene psaE1 affects photosynthesis and impairs growth.

The psae1-1 mutant of Arabidopsis was identified on the basis of a decrease in the effective quantum yield of photosystem II, among a collection of plants subjected to transposon tagging with the Enhancer element. The steady-state redox level and the rate of re-oxidation of P700 are significantly altered in psae1-1 mutants. The responsible mutation was localised to psaE1, one of two Arabidopsis genes that encode subunit E of photosystem I. An additional mutant allele, psae1-2, was identified by reverse genetics. In wild-type plants, the psaE1 transcript is expressed at a higher level than psaE2 mRNA. In the mutants, however, the E1 transcript was barely detectable, and was expressed only in small groups of wild-type cells resulting from somatic reversions. As a consequence, the amount of PsaE protein present in the mutant is significantly reduced. Concomitantly, the levels of other stromal photosystem I subunits (PsaC and PsaD) are also affected. Mutant plants showed a marked increase in light sensitivity and photoinhibition. Additional effects of the psae1 mutation include light green pigmentation, an increase in chlorophyll fluorescence and a decrease of approximately 50% in growth rate under greenhouse conditions.

Chlorophyll binding to monomeric light-harvesting complex. A mutation analysis of chromophore-binding residues.

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q(y) transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.

The neoxanthin binding site of the major light harvesting complex (LHCII) from higher plants.

The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site-directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57 +/- 1.5 degrees with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

Multiple light-harvesting II polypeptides from maize mesophyll chloroplasts are distinct gene products.

The major light-harvesting complex of photosystem II in higher plants is known as LHCII. It is composed of a number of chlorophyll-binding proteins sharing epitopes with each other. The number of apoproteins resolved by fully denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis varies in different species. In order to know if this heterogeneity is caused by the expression of a number of homologous genes or if it is the product of post-translational modifications, we have resolved the six major apoproteins of Zea mays LHCII. Each protein is purified to homogeneity, subjected to direct protein sequencing and the sequences compared with those deduced from lhcb genes in maize and other organisms. All of the six proteins are distinct gene products, since they show differences in their primary structure. Three apoproteins are identified as products of type I lhcb genes and one each as type II and type III gene products. A sixth protein does not fit the requirements for any of the lhcb genes so far cloned and is therefore probably the product of an lhcb gene type not yet described. Our results clearly show that the major source of LHCII protein heterogeneity is the expression of many lhcb genes. Fractionation of maize LHCII by non-denaturing flat-bed isoelectric focusing resolves at least five major isoforms showing distinct differences in their polypeptide composition and also differing in their spectroscopic properties, thus suggesting that individual Lhcb gene products have distinct pigment-binding properties.