A Constitutive Expression System for Cellulase Secretion in Escherichia coli and Its Use in Bioethanol Production

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.     

Improvement in infected wound healing in type 1 diabetic rat by the synergistic effect of photobiomodulation therapy and conditioned medium

We investigated the effects of photobiomodulation therapy (PBMT) and conditioned medium (CM) of human bone marrow mesenchymal stem cells (hBM-MSC) individually and/or in combination on the stereological parameters and the expression of basic fibroblast growth factor (bFGF), hypoxia-inducible factor (HIF-1α), and stromal cell–derived factor-1α (SDF-1α) in a wound model infected with methicillin-resistant Staphylococcus aureus (MRSA) in diabetic rats. CM was provided by culturing hBM-MSCs. Type 1 diabetes mellitus (T1DM) was induced in 72 rats, divided into four groups, harboring 18 rats each: group 1 served as a control group, group 2 received PBMT, group 3 received CM, and group 4 received CM + PBMT. On days 4, 7, and 15, six animals from each group were euthanized and the skin samples were separated for stereology examination and gene expression analysis by real-time polymerase chain reaction. In the CM + PBMT, CM, and PBMT groups, significant decreases were induced in the number of neutrophils (1460 ± 93, 1854 ± 138, 1719 ± 248) and macrophages (539 ± 69, 804 ± 63, 912 ± 41), and significant increases in the number of fibroblasts (1073 ± 116, 836 ± 75, 912 ± 41) and angiogenesis (15 230 ± 516, 13 318 ± 1116, 14 041 ± 867), compared with those of the control group (2690 ± 371, 1139 ± 145, 566 ± 90, 12 585 ± 1219). Interestingly, the findings of the stereological examination in the CM + PBMT group were statistically more significant than those in the other groups. In the PBMT group, in most cases, the expression of bFGF, HIF-1α, and SDF-1α, on day 4 (27.7 ± 0.14, 28.8 ± 0.52, 27.5 ± 0.54) and day 7 (26.8 ± 1.4, 29.6 ± 1.4, 28.3 ± 1.2) were more significant than those in the control (day 4, 19.3 ± 0.42, 25.5 ± 0.08, 22.6 ± 0.04; day 7, 22.3 ± 0.22, 28.3 ± 0.59, 24.3 ± 0.19) and other treatment groups. The application of PBMT + CM induced anti-inflammatory and angiogenic activities, and hastened wound healing process in a T1 DM model of MRSA infected wound.     

Estimation of Quasi-Stiffness and Propulsive Work of the Human Ankle in the Stance Phase of Walking

Characterizing the quasi-stiffness and work of lower extremity joints is critical for evaluating human locomotion and designing assistive devices such as prostheses and orthoses intended to emulate the biological behavior of human legs. This work aims to establish statistical models that allow us to predict the ankle quasi-stiffness and net mechanical work for adults walking on level ground. During the stance phase of walking, the ankle joint propels the body through three distinctive phases of nearly constant stiffness known as the quasi-stiffness of each phase. Using a generic equation for the ankle moment obtained through an inverse dynamics analysis, we identify key independent parameters needed to predict ankle quasi-stiffness and propulsive work and also the functional form of each correlation. These parameters include gait speed, ankle excursion, and subject height and weight. Based on the identified form of the correlation and key variables, we applied linear regression on experimental walking data for 216 gait trials across 26 subjects (speeds from 0.75–2.63 m/s) to obtain statistical models of varying complexity. The most general forms of the statistical models include all the key parameters and have an R² of 75% to 81% in the prediction of the ankle quasi-stiffnesses and propulsive work. The most specific models include only subject height and weight and could predict the ankle quasi-stiffnesses and work for optimal walking speed with average error of 13% to 30%. We discuss how these models provide a useful framework and foundation for designing subject- and gait-specific prosthetic and exoskeletal devices designed to emulate biological ankle function during level ground walking.     

Estimation of Quasi-Stiffness of the Human Knee in the Stance Phase of Walking

Biomechanical data characterizing the quasi-stiffness of lower-limb joints during human locomotion is limited. Understanding joint stiffness is critical for evaluating gait function and designing devices such as prostheses and orthoses intended to emulate biological properties of human legs. The knee joint moment-angle relationship is approximately linear in the flexion and extension stages of stance, exhibiting nearly constant stiffnesses, known as the quasi-stiffnesses of each stage. Using a generalized inverse dynamics analysis approach, we identify the key independent variables needed to predict knee quasi-stiffness during walking, including gait speed, knee excursion, and subject height and weight. Then, based on the identified key variables, we used experimental walking data for 136 conditions (speeds of 0.75–2.63 m/s) across 14 subjects to obtain best fit linear regressions for a set of general models, which were further simplified for the optimal gait speed. We found R² > 86% for the most general models of knee quasi-stiffnesses for the flexion and extension stages of stance. With only subject height and weight, we could predict knee quasi-stiffness for preferred walking speed with average error of 9% with only one outlier. These results provide a useful framework and foundation for selecting subject-specific stiffness for prosthetic and exoskeletal devices designed to emulate biological knee function during walking.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

Secretions of the interaural gland contain information about individuality and colony membership in the Bechstein's bat

Mammals use chemical signals for individual and kin recognition, to establish social hierarchies, mark territories and choose mates. The nocturnal and social lifestyle of bats suggests that, besides acoustic signals, they also use scent to communicate. We investigated in the communally breeding Bechstein's bat, Myotis bechsteinii, whether secretions of the facial interaural gland contain information that can be used for individual and colony recognition. Since female Bechstein's bats live in closed societies and show cooperative behaviour, we predicted they would recognize colony members. We analysed interaural gland secretions, which we repeatedly sampled from 85 females belonging to four free-ranging colonies. Gas chromatography/mass spectrometry profiles were individually specific and differed between colonies. Comparing odour profiles between colonies we found a relation between chemical similarity and the mitochondrial haplotype of colony members. Within colonies there was no correlation between mass spectrometer profile similarity and genetic relatedness. Our results suggest that female Bechstein's bats may use interaural gland secretions for individual and colony recognition but not to infer kinship directly.Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.      

A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17–30 and LFampin268–284, a shorter fragment of LFampin265–284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10⁵ CFU/ml, could be killed by 5–10 μM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.