Mechanical performance of cervical intervertebral body fusion devices: A systematic analysis of data submitted to the Food and Drug Administration

Cervical intervertebral body fusion devices (IBFDs) are utilized to provide stability while fusion occurs in patients with cervical pathology. For a manufacturer to market a new cervical IBFD in the United States, substantial equivalence to a cervical IBFD previously cleared by FDA must be established through the 510(k) regulatory pathway. Mechanical performance data are typically provided as part of the 510(k) process for IBFDs. We reviewed all Traditional 510(k) submissions for cervical IBFDs deemed substantially equivalent and cleared for marketing from 2007 through 2014. To reduce sources of variability in test methods and results, analysis was restricted to cervical IBFD designs without integrated fixation, coatings, or expandable features. Mechanical testing reports were analyzed and results were aggregated for seven commonly performed tests (static and dynamic axial compression, compression-shear, and torsion testing per ASTM F2077, and subsidence testing per ASTM F2267), and percentile distributions of performance measurements were calculated. Eighty-three (83) submissions met the criteria for inclusion in this analysis. The median device yield strength was 10,117 N for static axial compression, 3680 N for static compression-shear, and 8.6 N m for static torsion. Median runout load was 2600 N for dynamic axial compression, 1400 N for dynamic compression-shear, and ±1.5 N m for dynamic torsion. In subsidence testing, median block stiffness (Kp) was 424 N/mm. The mechanical performance data presented here will aid in the development of future cervical IBFDs by providing a means for comparison for design verification purposes.     

Drinks containing anthocyanin-rich blackcurrant extract decrease postprandial blood glucose,insulin and incretin concentrations

Blackcurrants are rich in polyphenolic glycosides called anthocyanins, which may inhibit postprandial glycemia. The aim was to determine the dose-dependent effects of blackcurrant extract on postprandial glycemia. Men and postmenopausal women (14 M, 9 W, mean age 46 years, S.D.=14) were enrolled into a randomized, double-blind, crossover trial. Low sugar fruit drinks containing blackcurrant extract providing 150-mg (L-BE), 300-mg (M-BE) and 600-mg (H-BE) total anthocyanins or no blackcurrant extract (CON) were administered immediately before a high-carbohydrate meal. Plasma glucose, insulin and incretins (GIP and GLP-1) were measured 0–120 min, and plasma 8-isoprostane F_2α, together with arterial stiffness by digital volume pulse (DVP) was measured at 0 and 120 min. Early plasma glucose response was significantly reduced following H-BE (n=22), relative to CON, with a mean difference (95% CI) in area over baseline (AOB) 0-30 min of −0.34 mmol/l.h (−0.56, −0.11, P<.005); there were no differences between the intermediate doses and placebo. Plasma insulin concentrations (AOB 0–30 min) were similarly reduced. Plasma GIP concentrations (AOB 0–120 min) were significantly reduced following H-BE, with a mean difference of −46.6 ng/l.h (−66.7, −26.5, P<.0001) compared to CON. Plasma GLP-1 concentrations were reduced following H-BE at 90 min. There were no effects on 8-isoprostane F_2α or vascular function. Consumption of blackcurrant extract in amounts roughly equivalent to 100-g blackcurrants reduced postprandial glycemia, insulinemia and incretin secretion, which suggests that inclusion of blackcurrant polyphenols in foods may provide cardio-metabolic health benefits. This trial was registered at clinicaltrials.gov as NCT01706653.     

Higher medially-directed joint reaction forces are a characteristic of dysplastic hips: A comparative study using subject-specific musculoskeletal models

Acetabular dysplasia is a known cause of hip osteoarthritis. In addition to abnormal anatomy, changes in kinematics, joint reaction forces (JRFs), and muscle forces could cause tissue damage to the cartilage and labrum, and may contribute to pain and fatigue. The objective of this study was to compare lower extremity joint angles, moments, hip JRFs and muscle forces during gait between patients with symptomatic acetabular dysplasia and healthy controls. Marker trajectories and ground reaction forces were measured in 10 dysplasia patients and 10 typically developing control subjects. A musculoskeletal model was scaled in OpenSim to each subject and subject-specific hip joint centers were determined using reconstructions from CT images. Joint kinematics and moments were calculated using inverse kinematics and inverse dynamics, respectively. Muscle forces and hip JRFs were estimated with static optimization. Inter-group differences were tested for statistical significance (p ≤ 0.05) and large effect sizes (d ≥ 0.8). Results demonstrated that dysplasia patients had higher medially directed JRFs. Joint angles and moments were mostly similar between the groups, but large inter-group effect sizes suggested some restriction in range of motion by patients at the hip and ankle. Higher medially-directed JRFs and inter-group differences in hip muscle forces likely stem from lateralization of the hip joint center in dysplastic patients. Joint force differences, combined with reductions in range of motion at the hip and ankle may also indicate compensatory strategies by patients with dysplasia to maintain joint stability.     

Spinal loads and trunk muscles forces during level walking – A combined in vivo and in silico study on six subjects

During level walking, lumbar spine is subjected to cyclic movements and intricate loading of the spinal discs and trunk musculature. This study aimed to estimate the spinal loads (T12–S1) and trunk muscles forces during a complete gait cycle.Six men, 24–33 years walk barefoot at self-selected speed (4–5 km/h). 3D kinematics and ground reaction forces were recorded using a motion capturing system and two force plates, implemented in an inverse dynamic musculoskeletal model to predict the spinal loads and trunk muscles forces. Additionally, the sensitivity of the intra-abdominal pressure and lumbar segment rotational stiffness was investigated.Peak spinal loads and trunk muscle forces were between the gait instances of heel strike and toe off. In L4–L5 segment, sensitivity analysis showed that average peak compressive, antero-posterior and medio-lateral shear forces were 130–179%, 2–15% and 1–6%, with max standard deviation (±STD) of 40%, 6% and 3% of the body weight. Average peak global muscles forces were 24–55% (longissimus thoracis), 11–23% (iliocostalis thoracis), 12–16% (external oblique), 17–25% (internal oblique) and 0–8% (rectus abdominus) of body weight whereas, the average peak local muscles forces were 11–19% (longissimus lumborum), 14–31% (iliocostalis lumborum) and 12–17% (multifidus). Maximum ± STD of the global and local muscles forces were 13% and 8% of the body weight.Large inter-individual differences were found in peak compressive and trunk muscles forces whereas the sensitivity analysis also showed a substantial variation.     

Metal-dependent thermal stability of recombinant endo-mannanase (ManB-1601) belonging to family GH 26 from Bacillus sp. CFR1601

A GH 26 endo-mannanase from Bacillus sp. CFR1601 was purified to homogeneity (M_w ∼39 kDa, specific activity 10,461.5 ± 100 IU/mg). Endo-mannanase gene (manb-1601, 1083 bp, accession No. KM404299) was expressed in Escherichia coli BL21 (DE3) and showed typical fingerprints of α/β proteins in the far-UV CD. A high degree of conservation among amino acid residues involved in metal chelation (His-1, 23 and Glu-336) and internal repeats (122–152 and 181–212) was observed in endo-mannanases reported from various Bacillus sp. Thermal inactivation kinetics suggested that metal ions are quintessential for stabilization of ManB-1601 structure as holoenzyme (E_a 87.4 kcal/mol, ΔH 86.7 kcal/mol, ΔS 186.6 cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (E_a 47 kcal/mol, ΔH 45.7 kcal/mol, ΔS 64.7 cal/k/mol). EDTA treatment of ManB-1601 not only lead to transitions in both secondary and tertiary structure but also promulgated the population of conformational state that unfolds at lower temperature. ManB-1601 followed a three-state process for thermal inactivation wherein loss of tertiary structure preceded the concurrent loss of secondary structure and activity.     

Effect of changes in orientation and position of external loads on trunk muscle activity and kinematics in upright standing

Forces at different heights and orientations are often carried by hands while performing occupational tasks. Trunk muscle activity and spinal loads are likely dependent on not only moments but also the orientation and height of these forces. Here, we measured trunk kinematics and select superficial muscle activity of 12 asymptomatic subjects while supporting forces in hands in upright standing. Magnitude of forces in 5 orientations (−25°, 0°, 25°, 50° and 90°) and 2 heights (20 cm and 40 cm) were adjusted to generate flexion moments of 15, 30 and 45 N m at the L5-S1 disc centre. External forces were of much greater magnitude when applied at lower elevation or oriented upward at 25°. Spinal kinematics remained nearly unchanged in various tasks.Changes in orientation and elevation of external forces substantially influenced the recorded EMG, despite similar trunk posture and identical moments at the L5-S1. Greater EMG activity was overall recorded under larger forces albeit constant moment. Increases in the external moment at the L5-S1 substantially increased EMG in extensor muscles (p < 0.001) but had little effect on abdominals; e.g., mean longissimus EMG for all orientations increased by 38% and 75% as the moment level altered from 15 N m to 30 N m and to 45 N m while that in the rectus abdominus increased only by 2% and 4%, respectively. Under 45 N m moment and as the load orientation altered from 90° to 50°, 25°, 0° and −25°, mean EMG dropped by 3%, 12%, 12% and 1% in back muscles and by 17%, 17%, 19% and 13% in abdominals, respectively. As the load elevation increased from 20 cm to 40 cm, mean EMG under maximum moment decreased by 21% in back muscles and by 17% in abdominals.Due to the lack of EMG recording of deep lumbar muscles, changes in relative shear/compression components and different net moments at cranial discs despite identical moments at the caudal L5-S1 disc, complementary model studies are essential for a better comprehension of neuromuscular strategies in response to alterations in load height and orientation.     

Field and culture studies on the ecophysiology of the toxic dinoflagellate Alexandrium minutum (Halim) present in Greek coastal waters

In Greek coastal waters, the toxic dinoflagellate Alexandrium minutum (strain AJ879163) was detected for the first time in spring 2002. This species proliferated during spring–summer of 2002 and 2003 over a wide geographic range along the north-south Aegean Sea coastline, mostly at low concentrations (average: 10²–10³ cells L⁻¹) with one exception of higher abundance (average: 10⁵ cells L⁻¹). This study presents data on environmental (temperature, salinity, chl α, nutrients) and ecological (phytoplankton species composition, diversity, taxa dominance, community dissimilarities) parameters in the areas of A. minutum occurrence. A. minutum was isolated and grown in batch cultures used in a series of bioassay experiments for determination of its pigment composition by HPLC, half saturation constants (K_s) for nitrogen and phosphorus, and its response to different nitrogen to phosphorus (N:P) ratios.     

The use of ecological,fishing and environmental indicators in support of decision making in southern Benguela fisheries

Indicators have been recognised as a useful tool aiding the implementation of an ecosystem approach to fisheries in marine ecosystems. Studies, such as the IndiSeas project (www.indiseas.org), use a suite of indicators as a method of assessing the state and trends of several of the world's marine ecosystems. While it is well known that both fishing and climatic variability influence marine fisheries in the southern Benguela ecosystem there are currently few studies in support of fisheries management that make use of environmental indicators in order to include climatic impacts on marine fish populations. Trends in ecological, fishing and environmental indicators can be utilised in a way that allows an overall ecosystem trend to be determined, and can therefore be used to aid decision support within southern Benguela fisheries. In this study trends in indicators were determined using linear regressions across three time periods, Period 1: 1978–1993, Period 2: 1994–2003 and Period 3: 2004–2010. These time periods were selected based on the timing of regime shifts within the southern Benguela, including changes in upwelling, wind stress and temperature. Each ecological indicator received a score based on the direction and significance of the observed trend with respect to fishing. To account for the impacts of fishing and environmental drivers on ecological indicators, scores were adjusted by predetermined factors, depending on the extent and direction of trends in these indicators. Weightings were applied to correlated ecological indicators to account for their redundancy, and lessen their impact on overall ecosystem score. Mean weighted scores were then used to establish an overall ecosystem score for each time period. Ecosystem classification was determined as follows: 1–1.49 = improving, 1.5–2.49 = possibly improving, 2.5–3.49 = no improvement or deterioration, 3.5–4.49 = possible deterioration, 4.5–5 = deteriorating. The ecosystem was observed to neither deteriorate nor improve across Period 1 or 2 (mean weighted scores: 2.75 and 2.56 respectively), however, during Period 3 a possible improvement was observed (mean weighted score: 1.99). This study shows that the sequential analysis of suites of ecological, fishing and environmental indicators can be used in order to determine ecosystem trends, accounting for both the impacts of fishing and the environment on ecosystem components.     

Dynamic in vivo 3D atlantoaxial spine kinematics during upright rotation

Diagnosing dysfunctional atlantoaxial motion is challenging given limitations of current diagnostic imaging techniques. Three-dimensional imaging during upright functional motion may be useful in identifying dynamic instability not apparent on static imaging. Abnormal atlantoaxial motion has been linked to numerous pathologies including whiplash, cervicogenic headaches, C2 fractures, and rheumatoid arthritis. However, normal C1/C2 rotational kinematics under dynamic physiologic loading have not been previously reported owing to imaging difficulties. The objective of this study was to determine dynamic three-dimensional in vivo C1/C2 kinematics during upright axial rotation. Twenty young healthy adults performed full head rotation while seated within a biplane X-ray system while radiographs were collected at 30 images per second. Six degree-of-freedom kinematics were determined for C1 and C2 via a validated volumetric model-based tracking process. The maximum global head rotation (to one side) was 73.6 ± 8.3°, whereas maximum C1 rotation relative to C2 was 36.8 ± 6.7°. The relationship between C1/C2 rotation and head rotation was linear through midrange motion (±20° head rotation from neutral) in a nearly 1:1 ratio. Coupled rotation between C1 and C2 included 4.5 ± 3.1° of flexion and 6.4 ± 8.2° of extension, and 9.8 ± 3.8° of contralateral bending. Translational motion of C1 relative to C2 was 7.8 ± 1.5 mm ipsilaterally, 2.2 ± 1.2 mm inferiorly, and 3.3 ± 1.0 mm posteriorly. We believe this is the first study describing 3D dynamic atlantoaxial kinematics under true physiologic conditions in healthy subjects. C1/C2 rotation accounts for approximately half of total head axial rotation. Additionally, C1 undergoes coupled flexion/extension and contralateral bending, in addition to inferior, lateral and posterior translation.     

Walking kinematics and kinetics following eccentric exercise-induced muscle damage

The goal of this investigation was to investigate how walking patterns are affected following muscle-damaging exercise by quantifying both lower limb kinematics and kinetics. Fifteen young women conducted a maximal isokinetic eccentric exercise (EE) muscle damage protocol (5 × 15) of the knee extensors and flexors of both legs at 60°/s. Three-dimensional motion data and ground reaction forces (GRFs) were collected 24 h pre-EE while the participants walked at their preferred self-selected walking speed (SWS). Participants were asked to perform two gait conditions 48 h post-EE. The first condition (COND1) was to walk at their own speed and the second condition (COND2) to maintain the SWS (±5%) they had 24 h pre-EE. Walking speed during COND1 was significantly lower compared to pre-exercise values. When walking speed was controlled during COND2, significant effects of muscle damage were noticed, among other variables, for stride frequency, loading rate, lateral and vertical GRFs, as well as for specific knee kinematics and kinetics. These findings provide new insights into how walking patterns are adapted to compensate for the impaired function of the knee musculature following muscle damage. The importance to distinguish the findings caused by muscle damage from those exhibited in response to changes in stride frequency is highlighted.     

A comparison of epigean and subterranean locomotion in the domestic ferret (Mustela putorius furo: Mustelidae: Carnivora)

Burrows are used by many mammals to escape predation, cache food and for parturition. Although the construction of burrows has been studied in some taxa, the locomotion while inside of them has received scant attention. In this study we collected simultaneous video and force data to characterize gaits, kinematics, peak ground reaction forces (GRFs) and external mechanical energy profiles in the domestic ferret, an animal that displays the typical morphology and behaviors associated with subterranean adaptations in mustelines. We compared kinematics and kinetics between locomotion in two experimental conditions: subterranean, simulated by a Plexiglass tunnel designed such that the ferrets’ peak back height was reduced by 40% and hip height by 25%, and epigean, or unconstrained overground. Despite the large change in posture, a striking number of gait and force variables were not statistically different between experimental conditions. In both subterranean and epigean conditions, the ferrets in our study traveled at similar average velocities (～0.8 m s^?1), preferred to use a lateral-sequence diagonal-couplet gait, and were more likely to demonstrate the in-phase fluctuations of external mechanical energy indicative of running mechanics (68% of all trials). The ferrets conformed to gait and mechanical patterns seen in a variety of other small (<1 kg) mammals rather than being unique, despite the divergent morphology of mustelines. Our results demonstrated biodynamically similar locomotion in both epigean and subterranean conditions and support the hypothesis that ferrets possess adaptations for tunnel locomotion.     

Influence of additional load on the moments of the agonist and antagonist muscle groups at the knee joint during closed chain exercise

The present study investigated the influence of additional loads on the knee net joint moment, flexor and extensor muscle group moments, and cocontraction index during a closed chain exercise. Loads of 8, 28, or 48 kg (i.e., respectively, 11.1 ± 1.5%, 38.8 ± 5.3%, and 66.4 ± 9.0% of body mass) were added to subjects during dynamic half squats. The flexor and extensor muscular moments and the amount of cocontraction were estimated at the knee joint using an EMG-and-optimization model that includes kinematics, ground reaction, and EMG measurements as inputs. In general, our results showed a significant influence of the Load factor on the net knee joint moment, the extensor muscular moment, and the flexor muscle group moment (all Anova p < .05). Hence we confirmed an increase in muscle moments with increasing load and moreover, we also showed an original “more than proportional” evolution of the flexor and extensor muscle group moments relative to the knee net joint moment. An influence of the Phase (i.e., descent vs. ascent) factor was also seen, revealing different activation strategies from the central nervous system depending on the mode of contraction of the agonist muscle group. The results of the present work could find applications in clinical fields, especially for rehabilitation protocols.     

The architecture and contraction time of intrinsic foot muscles

Although critical for effective human locomotion and posture, little data exists regarding the segmentation, architecture and contraction time of the human intrinsic foot muscles. To address this issue, the Abductor Hallucis (AH), Abductor Digiti Minimi (ADM), Flexor Digitorum Brevis (FDB) and Extensor Digitorum Brevis (EDB) were investigated utilizing a cadaveric dissection and a non-invasive whole muscle mechanomyographic (wMMG) technique. The segmental structure and architecture of formaldehyde-fixed foot specimens were determined in nine cadavers aged 60–80 years. The wMMG technique was used to determine the contraction time (Tc) of individual muscle segments, within each intrinsic foot muscle, in 12 volunteers of both genders aged between 19 and 24 years.While the pattern of segmentation and segmental –architecture (e.g. fibre length) and –Tc of individual muscle segments within the same muscle were similar, they varied between muscles. Also, the average whole muscle Tc of FDB was significantly (p < 0.05) shorter (faster) (Tc = 58 ms) than in all other foot muscles investigated (ADM Tc = 72 ms, EDB Tc = 72 ms and ABH Tc = 69 ms). The results suggest that the architecture and contraction time of the FDB reflect its unique direct contribution, through toe flexion, to postural stability and the rapid development of ground reaction forces during forceful activities such as running and jumping.     

Intra-operatively measured spastic semimembranosus forces of children with cerebral palsy

The knee kept forcibly in a flexed position is typical in cerebral palsy. Using a benchmark, we investigate intra-operatively if peak spastic hamstring force is measured in flexed knee positions. This tests the assumed shift of optimal length due to adaptation of spastic muscle and a decreasing force trend towards extension. Previously we measured spastic gracilis (GRA) and semitendinosus (ST) forces. Presently, we studied spastic semimembranosus (SM) and tested the following hypotheses: spastic SM forces are (1) high in flexed and (2) low in extended positions. We compared the data to those of GRA and ST to test (3) if percentages of peak force produced in flexed positions are different. During muscle lengthening surgery of 8 CP patients (9 years, 4 months; GMFCS levels = II–IV; limbs tested = 13) isometric SM forces were measured from flexion (120°) to full extension (0°). Spastic SM forces were low in flexed knee positions (only 4.2% (3.4%) and 10.7% (9.7%) of peak force at KA = 120° and KA = 90° respectively, indicating less force production compared to the GRA or ST) and high in extended knee positions (even 100% of peak force at KA = 0°). This indicates an absence of strong evidence for a shift of optimal muscle length of SM towards flexion.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Single nucleotide polymorphism (SNP) in alpha-lactalbumin gene of Indian Jamunapari breed of Capra hircus

Alpha-lactalbumin is a major whey protein found in milk. It influences lactose synthesis by modifying the substrate specificity of galactosyl-transferase, is important to milk synthesis since lactose, an impermeable disaccharide, is the major osmole of milk. The present study was undertaken to detect polymorphism in the full coding region of alpha-lactalbumin at the genetic level and to explore allelic variability of this gene. Samples of Jamunapari breed of goat (n = 50) were included under the present study. Jamunapari is the highest milk producer among local Indian goat breeds. PCR-SSCP of all four exons of alpha-lactalbumin (ALA) revealed a total of 9 gel phenotypes of Jamunapari breed of goat. These were sequenced, analyzed and deposited in GenBank, NCBI (accession nos. EU573193–EU573195). Nucleotide and amino acid variations were searched within breeds of Indian goats and homology between caprine, ovine, bovine, bubaline and human. In the present study we describe for the first time two novel gene variants on the goat alpha-lactalbumin gene exon 4.     

Purification and characterization of an extremely dimethylsulfoxide tolerant esterase from a salt-tolerant Bacillus species isolated from the marine environment of the Sundarbans

A Bacillus sp. isolated from the Sundarbans region of the Bay of Bengal (NCBI GenBank Accession no. AY723697) which can tolerate 10% (w/v) NaCl, produces esterase optimally in Marine Broth 2216 medium containing 1% (w/v) NaCl. The enzyme was purified 42.7-fold with 6.4% recovery, (specific activity 569.2 U/mg protein) by ammonium sulphate precipitation followed by anion and cation exchange chromatography. The serine type esterolytic enzyme has a molecular weight of 35.0 kDa and is denatured into polypeptides of molecular weights 20 kDa and 15 kDa. The esterase was most active at pH 8.0, the pH of the seawater at the site of collection and is stable in the pH range 6.0–9.0. The optimum temperature of activity of this esterase is 45 °C and the enzyme is very stable after 1 h pre-incubation at 50 °C. Our esterase shows about 100% activity when incubated with 1 M NaCl, the activity drops to about 50% when incubated with 2.5 M sodium chloride and the enzyme is completely inactivated when 4 M NaCl is present during reaction. The esterase is almost inactivated by Ca²⁺, Hg²⁺ and Fe³⁺ ions, reducing agents and detergent. Interestingly, Co²⁺, a known inhibitor of many enzymes, preserved 70% of the activity of this esterase. Specific activity of the esterase increases more than twofold in the presence of water-miscible organic solvents as compared to that in aqueous buffer. When incubated for a period of 10 days in the presence of 30–70% dimethylsufoxide (DMSO), the specific activity increased by approximately two–threefold compared to the enzyme in aqueous buffer throughout the period of study. Specific activity between 1283 and 525 U/mg was maintained by our enzyme when incubated with 50% DMSO for 10 days. The enzyme was most active on p-nitrophenyl acetate, ethyl acetate, alpha isomer of naphthyl acetate but shows relatively lesser activity towards triglycerides of fatty acids. Certain characteristics, such as molecular weight, effects of NaCl, metal ions (Zn²⁺ and Mg²⁺) and reactivity towards para-nitrophenyl and aliphatic esters were strikingly similar to already described marine bacterial derived esterases. Extreme stability in DMSO could make this enzyme a potential immobilized biocatalyst for application in non-aqueous based continuous bioprocesses. Higher specific activity and purification factor, better thermo tolerance and solvent stability would make our enzyme more attractive for biotechnological applications than the marine microbial derived esterases described so far.     

A putative proline iminopeptidase of Thermotoga maritima is a leucine aminopeptidese with lysine-p-nitroanilide hydrolyzing activity

A putative aminopeptidase P gene (TM0042, Swissport Q9WXP9, GeneBank AAD35136) of Thermotoga maritima was cloned and expressed in Escherichia coli BL21 (RIL). The enzyme was purified by the combination of ion exchange chromatography; Q-Sepharose and Mono-Q column. The purified recombinant T. maritima aminopeptidase P enzyme, gave a homogenous protein band with an apparent molecular weight of 40 kDa in SDS-PAGE analysis. The enzyme was purified 23-fold with the specific activity of 16.5 unit/mg with the final recovery of 22%. The enzyme was thermostable up to 90 °C for 30 min. An optimal activity was observed at 90 °C at pH 7.5. The purified enzyme was stable between pH 6.5 and 8 at 80 °C with the optimum of pH 7.5. Based on the amino acid sequence, the enzyme belongs to M 24B family of metalloenzymes. None of the divalent cations enhance the activity of the enzyme while Pb²⁺, Cu²⁺, Co²⁺, Cd²⁺, and Zn²⁺ were inhibitory to the enzyme activity. Divalent cation of Mg²⁺ showed 100% enzyme activity, to a lesser extent, Ca²⁺ and Mn²⁺ whereas strong inhibition of enzyme activity was observed with Zn²⁺ and Cd²⁺. The enzyme designated as putative aminopeptidase P was very low activity in hydrolyzing proline-p-nitroanilide. Kinetic studies on the purified enzyme confirmed that the enzyme is a leucine aminopeptidase. Enzyme also hydrolyzes lysine-p-nitroanilide with efficiency comparable to that of leucine-p-nitroanilide. This is the first report of leucine aminopeptidase with lysine-p-nitroanilide hydrolyzing activity, which belongs to the M 24B family of metalloenzymes.     

Lower limb joint stiffness and muscle co-contraction adaptations to instability footwear during locomotion

Unstable shoes (US) continually perturb gait which can train the lower limb musculature, but muscle co-contraction and potential joint stiffness strategies are not well understood. A shoe with a randomly perturbing midsole (IM) may enhance these adaptations. This study compares ankle and knee joint stiffness, and ankle muscle co-contraction during walking and running in US, IM and a control shoe in 18 healthy females. Ground reaction forces, three-dimensional kinematics and electromyography of the gastrocnemius medialis and tibialis anterior were recorded. Stiffness was calculated during loading and propulsion, derived from the sagittal joint angle-moment curves. Ankle co-contraction was analysed during pre-activation and stiffness phases. Ankle stiffness reduced and knee stiffness increased during loading in IM and US whilst walking (ankle, knee: p = 0.008, 0.005) and running (p < 0.001; p = 0.002). During propulsion, the opposite joint stiffness re-organisation was found in IM whilst walking (both joints p < 0.001). Ankle co-contraction increased in IM during pre-activation (walking: p = 0.001; running: p < 0.001), and loading whilst walking (p = 0.003), not relating to ankle stiffness. Results identified relative levels of joint stiffness change in unstable shoes, providing new evidence of how stability is maintained at the joint level.