Identifying the Effects of Assistive and Resistive Guidance on the Gait of Elderly People Using a Smart Walker.

Progression of technology has expanded applications of smart walkers in clinical fields. However, it is essential to investigate the effects of different types of gait guidance in order to introduce smart walkers more widely throughout these fields. The purpose of this study was to identify the effects of assistive and resistive guidance on the gait of elderly people using a smart walker. Gait parameters, surface electromyography of lower limb muscles, and trunk acceleration were measured. The assistive guidance force significantly increased gait speed, step length, and cadence while increasing trunk acceleration variability. The same amount of resistive guidance force did not change gait parameters; instead, however, it restrained the speed-dependent increase of trunk acceleration variability in the mediolateral direction. An analysis of muscle activity suggested that the lower limb muscle activity could be increased by varying gait parameters including speed, step length, and cadence.

Characterization of a 24-kb plasmid pCGR2 newly isolated from Corynebacterium glutamicum.

A 24-kb plasmid with 21 open reading frames (ORFs) was newly isolated from Corynebacterium glutamicum ATCC 14997 and named pCGR2. Three of its ORFs were indispensable for stable autonomous replication of pCGR2 in C. glutamicum: in the absence of selective pressure, deletion derivatives of pCGR2 containing the three ORFs showed stability in C. glutamicum for over 50 generations. The first of these ORFs encoded replicase repA whose gene product revealed high amino acid sequence similarity to corresponding gene products of C. glutamicum pCG1-family plasmids in general, and to that of pTET3 plasmid repA in particular. The other two ORFs were located upstream of repA and exhibited high sequence similarity to pTET3 parA and parB, respectively. Interestingly, plasmids based on the pCGR2 were compatible not only with those based on different family plasmids (pBL1, pCASE1) but also with those based on pCG1-family plasmid. Plasmids comprising pCGR2 repA showed a copy number of four in C. glutamicum. The number increased to 240 upon introduction of a mutation within the repA origin of the putative promoter for counter-transcribed RNA. This 60-fold increase in copy number should immensely contribute towards enhanced expression of desired genes in C. glutamicum.

Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences.

Systematic screening of secretion proteins using an approach based on the completely sequenced genome of Corynebacterium glutamicum R revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-form alpha-amylase derived from Geobacillus stearothermophilus. These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secreted alpha-amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency.

Identification of new secreted proteins and secretion of heterologous amylase by C. glutamicum.

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5-5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form alpha-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.

Characterization of a new 2.4-kb plasmid of Corynebacterium casei and development of stable corynebacterial cloning vector.

A new plasmid pCASE1 was isolated from Gram-positive Corynebacterium casei JCM 12072. It comprised a 2.4-kb nucleotide sequence with three ORFs, two of which were indispensable for autonomous replication in Corynebacterium glutamicum. Homology search identified these two ORFs as repA and repB, areas coding proteins involved in plasmid replication. repA sequence showed high similarity to theta-replicating Escherichia coli ColE2-P9 plasmids and even higher similarity to plasmids derived from Gram-positive bacteria belonging to a subfamily of this ColE2-P9 group. An E. coli-C. glutamicum shuttle vector was constructed with pCASE1 fragment including repA and repB to transform C. glutamicum and showed compatibility with corynebacterial plasmids from different plasmid families. The copy number of the shuttle vector in C. glutamicum was 13 and the vector showed stability for 102 generations with no selective pressure.

Effect of lignocellulose-derived inhibitors on growth of and ethanol production by growth-arrested Corynebacterium glutamicum R.

In cellulosic ethanol production, pretreatment of a biomass to facilitate enzymatic hydrolysis inevitably yields fermentation inhibitors such as organic acids, furans, and phenols. With representative inhibitors included in the medium at various concentrations, individually or in various combinations, ethanol production by Corynebacterium glutamicum R under growth-arrested conditions was investigated. In the presence of various inhibitors, the 62 to 100% ethanol productivity retained by the C. glutamicum R-dependent method far exceeded that retained by previously reported methods.