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Introduction: Foot progression angle (FPA) is a predictor of elevated regional plantar stresses and loads, which are indicators of dermal injury risk in individuals with diabetes mellitus and peripheral neuropathy (DMPN). FPA accounts for 15-45% of the variance in plantar stresses and loads in adults with DMPN. However, the biomechanical factors underlying an "out-toeing" gait pattern in this clinical population have not been examined. The primary purpose of this study was to identify static and dynamic predictors of foot progression angle magnitude in adults with and without DMPN., Methods: Thirty-three adults with and 12 adults without diabetes mellitus participated. Hip rotation, ankle dorsiflexion, and resting calcaneal stance position were measured using a standard goniometer. Kinematic and kinetic data were collected during walking., Results and Discussion: Static predictor variables did not significantly predict foot progression angle magnitude using multiple regression analysis. Of the dynamic predictor variables, thigh and shank lateral rotation accounted for 37% of foot progression angle variance (p<.01)., Conclusions: Our results show that dynamic measures of external rotation of proximal segments (thigh, shank) during gait are strong predictors of foot progression angle. Static measures of limited joint mobility and joint position do not predict foot progression angle. These findings suggest that targeting the thigh and shank rotation using verbal or tactile cueing may be a potential strategy when trying to alter walking movement patterns towards decreasing external (lateral) FPA to minimize risk of elevated regional plantar stresses in adults with DMPN at risk for ulceration.

Nitric oxide, a gaseous radical molecule, appears involved in many reactions in all living organisms. Fluorescent dyes like DAF-2 and related compounds are still widely used to monitor NO production inside or outside cells, although doubts about their specificity have recently been raised. We present evidence that DAF dyes do not only react with nitric oxide but also with peroxidase enzyme and hydrogen peroxide. Both are secreted in the case of elicitation of tobacco suspension cells with cryptogein, with a fluorescence increase mimicking NO release from cells. However, HPLC separation shows that fluorescence outside cells does not at all originate from DAF-2T, the product of DAF-2 and NO, but from other yet unidentified compounds. Inside cells, other DAF molecules are formed but only a minor part is DAF-2T. The chemical nature of the novel DAF derivatives still needs to be determined.

The microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) has been found in human plasma samples after intake of maritime pine bark extract (Pycnogenol). M1 has been previously shown to accumulate in endothelial and blood cells in vitro after facilitated uptake and to exhibit anti-inflammatory activity. The purpose of the present research approach was to systematically and comprehensively analyze the metabolism of M1 in human blood cells in vitro and in vivo. A metabolomic approach that had been successfully applied for drug metabolite profiling was chosen to detect 19 metabolite peaks of M1 which were subsequently further analyzed and validated. The metabolites were categorized into three levels of identification according to the Metabolomics Standards Initiative with six compounds each confirmed at levels 1 and 2 and seven putative metabolites at level 3. The predominant metabolites were glutathione conjugates which were rapidly formed and revealed prolonged presence within the cells. Although a formation of an intracellular conjugate of M1 and glutathione (M1-GSH) was already known two GSH conjugate isomers, M1-S-GSH and M1-N-GSH were observed in the current study. Additionally detected organosulfur metabolites were conjugates with oxidized glutathione and cysteine. Other biotransformation products constituted the open-chained ester form of M1 and a methylated M1. Six of the metabolites determined in in vitro assays were also detected in blood cells in vivo after ingestion of the pine bark extract by two volunteers. The present study provides the first evidence that multiple and structurally heterogeneous polyphenol metabolites can be generated in human blood cells. The bioactivity of the M1 metabolites and their contribution to the previously determined anti-inflammatory effects of M1 now need to be elucidated., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Accumulation of advanced glycation end products (AGEs) is thought to contribute to limited joint mobility in people with diabetes mellitus (DM), but the relationships among AGEs, shoulder structural changes, movement, and disability are not understood., Objective: The purpose of this study was to determine the differences and relationships among skin intrinsic fluorescence (SIF), a proxy measure of AGEs, biceps and supraspinatus tendon thickness, upper extremity movement, and disability in groups with and without DM., Design: This was a cross-sectional, case-control study., Methods: Fifty-two individuals participated: 26 with type 2 DM and 26 controls matched for sex, age, and body mass index. The main outcome measures were: SIF; biceps and supraspinatus tendon thickness; 3-dimensional peak shoulder motion; and Disability of the Arm, Shoulder and Hand (DASH) questionnaire scores., Results: Mean SIF measurements were 19% higher in the DM group compared with the control group (P<.05). Biceps tendons (mean and 95% confidence interval [CI]) (4.7 mm [4.4, 5.0] versus 3.2 mm [2.9, 3.5]) and supraspinatus tendons (6.4 mm [5.9, 6.8] versus 4.9 mm [4.4, 5.3]) were thicker and peak humerothoracic elevation (139° [135°, 146°] versus 150° [146°, 155°]) and glenohumeral external rotation (35° [26°, 46°] versus 51° [41°, 58°]) were reduced in the DM group compared with the control group (P<.05). In the DM group, SIF was correlated to biceps tendon thickness, DASH score, and shoulder motion (r=.44-.51, P<.05). The SIF score and shoulder strength explained 64% of the DASH scores (P<.01)., Limitations: Because this was a cross-sectional study design, a cause-effect relationship could not be established., Conclusions: Accumulation of AGEs in the connective tissues of individuals with DM appears to be associated with increased tendon thickness and decreased shoulder joint mobility and upper extremity function. Physical therapists should be aware of these possible metabolic effects on structure, movement, and disability when treating people with diabetes., (© 2015 American Physical Therapy Association.)

Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity., (© 2015 American Society of Plant Biologists. All rights reserved.)

Heat acclimation enables plants to tolerate and survive short-term heat stress on hot days. In Arabidopsis thaliana, a genetically programmed heat shock response can be rapidly triggered in the temperature range of 32-38°C through activation of heat shock transcription factors (HSF). The heat shock response leads to heat acclimation and confers short-term protection against temperatures above 40°C. However, little is known about metabolic adjustments during heat acclimation.Untargeted metabolite analyses of A. thaliana seedlings revealed that levels of polyunsaturated triacylglycerols (TG) rapidly and dramatically increase during heat acclimation. TG accumulation was found to be temperature dependent in a temperature range of 32-50°C (optimum at 42°C) and reversible after a return from 37°C to normal growth temperatures. Heat-induced TGs accumulated in extra-chloroplastic compartments and increased in both roots and shoots to a similar extent. Analysis of mutants deficient in all four HSFA1 master regulator genes or the HSFA2 gene revealed that TG accumulation was not dependent on HSFs. Moreover, the TG response was not limited to heat stress because drought and salt stress also triggered an accumulation of TGs, but not short-term osmotic, cold, and high light stress. Lipid analysis revealed that heat-induced accumulation of TGs was not due to massive de novo fatty acid synthesis. It is hypothesized that TGs serve as transient stores for fatty acids that may be required for membrane remodelling during heat acclimation., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Objectives: To determine the severity of, and relationships between, upper extremity impairments, pain and disability in patients with diabetes mellitus, and to compare upper extremity impairments in patients with diabetes with non-diabetic controls., Design: Case-control, cross-sectional design., Setting: University-based, outpatient diabetes centre and physical therapy research clinic., Participants: Two hundred and thirty-six patients with diabetes attending an outpatient diabetes clinic completed the Shoulder Pain and Disability Index (SPADI) questionnaire. A detailed shoulder and hand examination was conducted on a subgroup of 29 volunteers with type 2 diabetes, and 27 controls matched for age, sex and body mass index., Interventions: None., Main Outcome Measures: SPADI score, passive shoulder range of motion (ROM) and strength, grip strength, hand sensation, dexterity and limited joint mobility of the hand., Results: Sixty-three percent (149/236) of patients with diabetes reported shoulder pain and/or disability [median SPADI score 10.0 (interquartile range 0.0 to 39.6)]. Compared with the control group, the subgroup of patients with diabetes had substantial reductions in shoulder ROM, shoulder muscle strength, grip and key pinch strength (P<0.05). Patients with diabetes had a greater prevalence of decreased sensation (26/27 vs 14/27) and limited joint mobility of the hand (17/27 vs 4/27) compared with the control group. Total SPADI score was negatively correlated (P<0.05) with shoulder ROM (r=-0.42 to -0.74) and strength measures (r=-0.44 to -0.63) in patients with diabetes., Conclusions: Upper extremity impairments in this sample of patients with diabetes were common, severe and related to complaints of pain and disability. Additional research is needed to understand the unique reasons for upper extremity problems in patients with diabetes, and to identify preventative treatments., (Copyright © 2014 Chartered Society of Physiotherapy. Published by Elsevier Ltd. All rights reserved.)

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Unlabelled: A major challenge for mass spectrometric-based lipidomics, aiming at describing all lipid species in a biological sample, lies in the computational and bioinformatic processing of the large amount of data that arises after data acquisition. Lipid-Pro is a software tool that supports the identification of lipids by interpreting large datasets generated by liquid chromatography--tandem mass spectrometry (LC-MS/MS) using the advanced data-independent acquisition mode MS(E). In the MS(E) mode, the instrument fragments all molecular ions generated from a sample and records time-resolved molecular ion data as well as fragment ion data for every detectable molecular ion. Lipid-Pro matches the retention time-aligned mass-to-charge ratio data of molecular- and fragment ions with a lipid database and generates a report on all identified lipid species. For generation of the lipid database, Lipid-Pro provides a module for construction of lipid species and their fragments using a flexible building block approach. Hence, Lipid-Pro is an easy to use analysis tool to interpret complex MS(E) lipidomics data and also offers a module to generate a user-specific lipid database., Availability and Implementation: Lipid-Pro is freely available at: http://www.neurogenetics.biozentrum.uni-wuerzburg.de/en/project/services/lipidpro/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aims: To determine leg intermuscular (IMAT) and subcutaneous (SQAT) adipose tissue and their relationships with muscle performance and function in obese adults with and without type 2 diabetes and peripheral neuropathy (T2DMPN)., Methods: Seventy-nine age-matched obese adults were studied, 13 T2DM, 54 T2DMPN, and 24 obese controls. Leg fat (%IMAT, %SQAT) volumes were quantified using MRI. Ankle plantar flexion (PF) torque and power were assessed with isokinetic dynamometry. Physical function was assessed with 9-item Physical Performance Test (PPT), 6-minute walk distance, single-limb balance, and time to ascend 10 stairs. One-way ANOVAs determined group differences, and multiple regression predicted PPT score from disease status, % IMAT, and PF power., Results: T2DMPN participants had 37% greater IMAT volumes and 15% lower SQAT volumes than controls (p =. 01). T2DMPN and T2DM showed reduced PF torque and power compared to controls. T2DMPN participants had lower PPT score, 6-minute walk, single-limb balance, and stair climbing than controls (all p<.05). %IMAT volume correlated inversely, and %SQAT correlated directly, with PPT. Leg %IMAT and disease status predicted 49% of PPT score., Conclusions: T2DMPN may represent a shift in adipose tissue accumulation from SQAT to IMAT depots, which is inversely associated with muscle performance and physical function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Limited joint mobility at the shoulder is an understudied problem in people with diabetes mellitus. The purpose of this study was to determine the differences in shoulder kinematics between a group with diabetes and those without diabetes., Methods: Fifty-two participants were recruited, 26 with diabetes and 26 non-diabetes controls (matched for age, BMI and sex). Three-dimensional position of the trunk, scapula and humerus were collected using electromagnetic tracking sensors during scapular plane elevation and rotation movements., Findings: Glenohumeral external rotation was reduced by 11.1°-16.3° (P<0.05) throughout the humerothoracic elevation range of motion, from neutral to peak elevation, in individuals with diabetes as compared to controls. Peak humerothoracic elevation was decreased by 10-14°, and peak external rotation with the arm abducted was decreased 22° in the diabetes group compared to controls (P<0.05). Scapulothoracic and glenohumeral internal rotation motions were not different between the two groups., Interpretation: Shoulder limited joint mobility, in particular decreased external rotation, was seen in individuals with diabetes as compared to control participants. Future research should investigate causes of diabetic limited joint mobility and strategies to improve shoulder mobility and prevent additional detrimental changes in movement and function., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: The purpose of this study is to develop a non-contrast magnetic resonance imaging (MRI) approach to evaluate skeletal muscle perfusion in the diabetic foot based on the concept of angiosomes of the foot., Methods: Five healthy volunteers and five participants with diabetes (HbA1c = 7.2 ± 1.8%) without a history of peripheral artery disease were examined. The non-contrast perfusion measurements were performed during a toe flexion challenge. Absolute perfusion maps were created and two regions (medial and lateral) on the maps were segmented based on angiosomes., Result: Regional difference in the perfusion of foot muscle was readily visualized in the MRI perfusion angiosomes during the challenge. In the participants with diabetes, the perfusion during toe flexion challenge was significantly lower than in healthy volunteers (P < 0.01). The average perfusion for the medial plantar region of the right foot was lower in subjects with diabetes (38 ± 9 ml/min/100 g) than in healthy subjects (93 ± 33 ml/min/100 g)., Conclusions: Non-contrast MRI perfusion angiosome maps demonstrate the feasibility of determining regional perfusion in foot muscles during toe challenge and may facilitate evaluation of muscle perfusion in diabetic feet., Key Points: • Non-contrast MRI perfusion angiosome maps measure regional perfusion in foot muscles non-invasively. • Foot perfusion response to challenge is reduced in persons with diabetes. • MRI perfusion angiosome maps may help evaluation of regional foot muscle perfusion.

Study Design: Controlled laboratory cross-sectional study. Objectives To assess strength differences of the hip rotator and abductor muscle groups in young adults with chronic hip joint pain (CHJP) and asymptomatic controls. A secondary objective was to determine if strength in the uninvolved hip of those with unilateral CHJP differs from that in asymptomatic controls., Background: Little is known about the relationship between hip muscle strength and CHJP in young adults., Methods: Thirty-five participants with CHJP and 35 matched controls (18 to 40 years of age) participated. Using handheld dynamometry, strength of the hip external rotators and internal rotators was assessed with the hip flexed to 90° and 0°. To assess external rotator and internal rotator strength, the hip was placed at the end range of external rotation and internal rotation, respectively. Strength of the hip abductors was assessed in sidelying, with the hip in 15° of abduction. Break tests were performed to determine maximum muscle force, and the average torque was calculated using the corresponding moment arm. Independent-sample t tests were used to compare strength values between (1) the involved limb in participants with CHJP and the corresponding limb in the matched controls, and (2) the uninvolved limb in participants with unilateral CHJP and the corresponding limb in the matched controls., Results: Compared to controls, participants with CHJP demonstrated weakness of 16% to 28% (P<.01) in all muscle groups tested in the involved hip. The uninvolved hip of 22 subjects with unilateral CHJP demonstrated weakness of 18% and 16% (P<.05) in the external rotators (0°) and abductors, respectively, when compared to the corresponding limb of the matched controls., Conclusion: The results of the present study demonstrate that persons with CHJP have weakness in the hip rotator and hip abductor muscles. Weakness also was found in the uninvolved hip of persons with CHJP.

Background: Diabetes-related medial column foot deformities contribute to high plantar pressure, joint instability, ulceration and amputation. Impaired foot function may be an early indicator of foot structural incompetence and contribute to deformity progression. This study examines the ability of single-limb heel rise multi-segmental kinematics and kinetics to identify midfoot and hindfoot dysfunction in those with diabetes-related medial column foot deformity., Methods: Single-limb heel rise foot kinematics and kinetics were examined in adults with diabetes mellitus and peripheral neuropathy with and without medial column foot deformity and age-, weight-matched controls., Findings: Hindfoot relative to shank plantarflexion, peak and excursion, were reduced in both diabetes groups compared to controls (P<0.017). Controls' initial forefoot relative to hindfoot position was plantarflexed 31° and plantarflexed an additional 13° during heel rise. The initial forefoot relative to hindfoot position for the diabetes group without deformity was similarly plantarflexed as controls (34°) while the diabetes deformity group was less plantarflexed (lower arch position: 23°, P<0.017). During the heel rise task both diabetes groups demonstrated less ability to plantarflex the forefoot relative to the hindfoot compared to controls (2 and 5° respectively, P<0.017). Ankle plantarflexion power was reduced in the diabetes deformity group compared to controls (P<0.017)., Interpretation: The single-limb heel rise task identified movement dysfunction in those with diabetes mellitus and peripheral neuropathy. Failure to plantarflex the forefoot relative to hindfoot may compromise midfoot joint stability and increase the risk of injury and arch collapse., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Honeybees show a remarkable behavioral plasticity at the transition from nursing inside the hive to foraging for nectar and/or pollen outside. This plasticity is important for age-related division of labor in honeybee colonies. The behavioral transition is associated with significant volume and synaptic changes in the mushroom bodies (MBs), brain centers for sensory integration, learning, and memory. We tested whether precocious sensory exposure to light leads to changes in the density of synaptic complexes [microglomeruli (MG)] in the MBs. The results show that exposure to light pulses over 3 days induces a significant decrease in the MG density in visual subregions (collar) of the MB. Earlier studies had shown that foragers have increased levels of juvenile hormone (JH) co-occurring with a decrease of vitellogenin (Vg). Previous work further established that RNAi-mediated knockdown of vg and ultraspiracle (usp) induced an upregulation of JH levels, which can lead to precocious foraging. By disturbing both Vg and JH pathways using gene knockdown of vg and usp, we tested whether the changes in the hormonal system directly affect MG densities. Our study shows that MG numbers remained unchanged when Vg and JH pathways were perturbed, suggesting no direct hormonal influences on MG densities. However, mass spectrometry detection of JH revealed that precocious light exposure triggered an increase in JH levels in the hemolymph (HL) of young bees. This suggests a dual effect following light exposure via direct effects on MG reorganization in the MB calyx and a possible positive feedback on HL JH levels., (© 2014 Wiley Periodicals, Inc.)

Background: The windlass mechanism, acting through the plantar fascia, stabilizes the arches of the foot during stance phase of gait. The purpose of this study was to compare changes in radiographic measurements of the medial longitudinal arch (MLA) between toe-flat and -extended positions in participants with and without diabetes mellitus (DM), peripheral neuropathy (PN), and a low MLA., Methods: Twelve participants with DMPN and low MLA and 12 controls received weightbearing radiographs in a toe-flat and toe-extended position. DMPN participants were subcategorized from radiographs into DMPN severe, evidence of severe joint changes, and DMPN low, absence of joint changes. Primary measurements of MLA were determined in each position and included Meary's angle, talar declination angle, first metatarsal declination angle, and navicular height., Results: The DMPN severe group had no difference between toe-flat and -extended positions for Meary's, talar declination, and first metatarsal declination angles (P > .35) while navicular height elevated (P < .05). The DMPN low group had no difference between toe-flat and -extended positions for talar declination angle (P = .38), while Meary's angle, first metatarsal declination angle, and navicular height elevated (P < .05). All measurements in the control group changed, consistent with arch height elevation, when toes were extended (P < .05)., Conclusion: The DMPN severe and low groups showed impaired ability to raise the arch from the toe-flat to -extended position. Further research is needed to examine the contribution of specific windlass mechanism components (ie, plantar fascia, ligament, foot joint integrity, and mobility) as they relate to progressive foot deformity in adults with DMPN., Level of Evidence: Level III, comparative series., (© The Author(s) 2014.)

High light acclimation depends on retrograde control of nuclear gene expression. Retrograde regulation uses multiple signalling pathways and thus exploits signal patterns. To maximally challenge the acclimation system, Arabidopsis thaliana plants were either adapted to 8 (low light (L-light)) or 80 µmol quanta m(-2) s(-1) (normal light (N-light)) and subsequently exposed to a 100- and 10-fold light intensity increase, respectively, to high light (H-light, 800 µmol quanta m(-2) s(-1)), for up to 6 h. Both L → H- and N → H-light plants efficiently regulated CO2 assimilation to a constant level without apparent damage and inhibition. This experimental set-up was scrutinized for time-dependent regulation and efficiency of adjustment. Transcriptome profiles revealed that N-light and L-light plants differentially accumulated 2119 transcripts. After 6 h in H-light, only 205 remained differently regulated between the L → H- and N → H-light plants, indicating efficient regulation allowing the plants to reach a similar transcriptome state. Time-dependent analysis of transcripts as markers for signalling pathways, and of metabolites and hormones as possibly involved transmitters, suggests that oxylipins such as oxophytodienoic acid and jasmonic acid, metabolites and redox cues predominantly control the acclimation response, whereas abscisic acid, salicylic acid and auxins play an insignificant or minor role.

Conditions in the Democratic Republic of the Congo provide an ideal environment for leptospirosis and plague, both of which can cause severe pulmonary manifestations. In December 2004, an outbreak of lethal pneumonia occurred in a local mining camp, affecting 130 persons and killing 57 of them. Clinical signs, fast disease spread, and initial laboratory investigations suggested pneumonic plague. While leptospirosis had not recently been described in the region, it was considered as a differential diagnosis. Anti-Leptospira antibodies were detected by microscopic agglutination test (MAT). A confirmed case of leptospirosis was defined as having consistent clinical signs and any one of the following: seroconversion or four-fold increase in MAT titre for paired serum samples, or a MAT titre ≥ 1:400 for acute-phase serum samples. Twenty-nine of the 54 patients or convalescents tested for leptospirosis were seropositive. Two cases showed a confirmed infection for both plague and leptospirosis. While evidence supports the plague nature of this outbreak, the results suggest that some of the suspected plague cases might be due to leptospirosis. In any case, this diagnosis will have to be evoked in the future if a similar outbreak occurs in this region of Africa.

Lipocalins are a group of multifunctional proteins, recognized as carriers of small lipophilic molecules, which have been characterized in bacteria and animals. Two true lipocalins have been recently identified in plants, the temperature-induced lipocalin (TIL) and the chloroplastic lipocalin (CHL), the expression of which is induced by various abiotic stresses. Each lipocalin appeared to be specialized in the responses to specific stress conditions in Arabidopsis thaliana, with AtTIL and AtCHL playing a protective role against heat and high light, respectively. The double mutant AtCHL KO × AtTIL KO deficient in both lipocalins was more sensitive to temperature, drought and light stresses than the single mutants, exhibiting intense lipid peroxidation. AtCHL deficiency dramatically enhanced the photosensitivity of mutants (vte1, npq1) affected in lipid protection mechanisms (tocopherols, zeaxanthin), confirming the role of lipocalins in the prevention of lipid peroxidation. Seeds of the AtCHL KO × AtTIL KO double mutant were very sensitive to natural and artificial ageing, and again this phenomenon was associated with the oxidation of polyunsaturated lipids. The presented results show that the Arabidopsis lipocalins AtTIL and AtCHL have overlapping functions in lipid protection which are essential for stress resistance and survival., (© 2013 John Wiley & Sons Ltd.)

Objective: To assess alterations in the regional perfusion and oxygenation of the calf muscles in individuals with diabetes., Methods: Age-matched individuals with (n = 5) and without diabetes (n = 6) were investigated. Skeletal muscle perfusion, oxygen extraction fraction, and oxygen consumption rate were measured by newly developed noncontrast magnetic resonance imaging (MRI) techniques. The subjects lay supine on the MRI table with their foot firmly strapped to a custom-built isometric exercise device. The measurements were performed at rest and during an isometric plantar flexion muscle contraction., Results: Individuals without diabetes had up to a 10-fold increase in muscle perfusion, 25% elevation in muscle oxygen extraction fraction, and a 12-fold increase in oxygen consumption rate in the calf during the plantar flexion isometric contraction. In patients with diabetes, the increases in these parameters were only up to sixfold, 2%, and sixfold, respectively. Exercise oxygen consumption rate was inversely associated with blood HbA1c levels (r(2) = .91)., Conclusions: This is the first study to quantify regional skeletal muscle oxygenation in patients with diabetes using noncontrast MRI and warrants additional study. Attenuation of perfusion and oxygenation during exercise may have implications for understanding diabetic complications in the lower extremities., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ9-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.

Jasmonic acid methyl ester has been discussed as a stress signal in plants. To investigate the relevance of reversible methylation of jasmonic acid, stress responses of transgenic tomato lines with altered expression and activity of methyl jasmonate esterase were analysed. No consistent changes in levels of methyl jasmonate, 12-oxo-phytodienoic acid, jasmonic acid, jasmonic acid isoleucine and expression of the jasmonate-responsive genes AOC and PINII between control line and RNAi as well as overexpressing lines were detectable under basal and wound-induced conditions. In contrast, reduction as well as enhancement of methyl jasmonate esterase activity resulted in increased susceptibility to the fungal pathogen Sclerotinia sclerotiorum despite higher levels of the hormonal active jasmonic acid isoleucine conjugate. Results suggest that methyl jasmonate esterase has a function in vivo in plant defence, which appears not to be related to its in vitro capacity to hydrolyse methyl jasmonate.

Diabetic foot deformity onset and progression maybe associated with abnormal foot and ankle motion. The modified Oxford multi-segmental foot model allows kinematic assessment of inter-segmental foot motion. However, there are insufficient anatomical landmarks to accurately representation the alignment of the hindfoot and forefoot segments during model construction. This is most notable for the sagittal plane which is referenced parallel to the floor, allowing comparison of inter-segmental excursion but not capturing important sagittal hind-to-forefoot deformity associated with diabetic foot disease and can potentially underestimate true kinematic differences. The purpose of the study was to compare walking kinematics using local coordinate systems derived from the modified Oxford model and the radiographic directed model which incorporated individual calcaneal and 1st metatarsal declination pitch angles for the hindfoot and forefoot. We studied twelve participants in each of the following groups: (1) diabetes mellitus, peripheral neuropathy and medial column foot deformity (DMPN+), (2) DMPN without medial column deformity (DMPN-) and (3) age- and weight-match controls. The modified Oxford model coordinate system did not identify differences between groups in the initial, peak, final, or excursion hindfoot relative to shank or forefoot relative to hindfoot dorsiflexion/plantarflexion during walking. The radiographic coordinate system identified the DMPN+ group to have an initial, peak and final position of the forefoot relative to hindfoot that was more dorsiflexed (lower arch phenotype) than the DMPN- group (p<.05). Use of radiographic alignment in kinematic modeling of those with foot deformity reveals segmental motion occurring upon alignment indicative of a lower arch., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Carbon fiber (CF) ankle-foot orthoses (AFOs) can improve gait by increasing ankle plantar-flexor power and improving plantar-flexor ankle joint moment and energy efficiency compared with posterior leaf spring AFOs made of thermoplastic. However, fabricating a CF AFO to optimize the performance of the individual user may require multiple AFOs and expensive fabrication costs. Finite element analysis (FEA) models were developed to predict the mechanical behavior of AFOs in this study. Three AFOs, two made of CF composite material and one made of thermoplastic material, were fabricated and then mechanically tested to produce force-displacement data. The FEA models were validated by comparing model predictions with mechanical testing data performed under the same loading and boundary conditions. The actual mechanical testing demonstrated that CF performs better than thermoplastic. The simulation results showed that FEA models produced accurate predictions for both types of orthoses. The relative error of the energy return ratio predicted by the CF AFO FEA model developed in this study is less than 3%. We conclude that highly accurate FEA models will allow orthotists to improve CF AFO fabrication without wasting resources (time and money) on trial and error fabrications that are expensive and do not consistently improve AFO and user performance.

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.

Endophytic fungi in cool-season grass species produce herbivore-toxic alkaloids, which are assumed to harm higher trophic levels along food chains. Previous studies have shown fitness disadvantages for higher trophic levels that feed on aphids that were exclusively reared on perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotyphodium lolii. However, it is unknown whether the alkaloids produced by the fungus-grass association can be assimilated by plant sap-sucking insects like aphids. Using an ultra high performance liquid chromatography method combined with mass spectrometry, we provide the first evidence that the alkaloids peramine and lolitrem B are present in aphids (Rhopalosiphum padi) and in aphid predators when the aphids are reared on endophyte-infected grass. We conclude that alkaloids can enter the plant sap of the grass and are responsible for longer pupal stages of the ladybird Harmonia axyridis and for fitness disadvantages of aphids and their predators as shown in previous studies.

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60-90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38-42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.

Background: Metatarsophalangeal joint deformity is associated with skin breakdown and amputation. The aims of this study were to compare intrinsic foot muscle deterioration ratios (ratio of adipose to muscle volume), and physical performance in subjects with diabetic neuropathy to controls, and determine their associations with 1) metatarsophalangeal joint angle and 2) history of foot ulcer., Methods: 23 diabetic, neuropathic subjects [59 (SD 10) years] and 12 age-matched controls [57 (SD 14) years] were studied. Radiographs and MRI were used to measure metatarsophalangeal joint angle and intrinsic foot muscle deterioration through tissue segmentation by image signal intensity. The Foot and Ankle Ability Measure evaluated physical performance., Findings: The diabetic, neuropathic group had a higher muscle deterioration ratio [1.6 (SD 1.2) vs. 0.3 (SD 0.2), P<0.001], and lower Foot and Ankle Ability Measure scores [65.1 (SD 24.4) vs. 98.3 (SD 3.3) %, P<0.01]. The correlation between muscle deterioration ratio and metatarsophalangeal joint angle was r=-0.51 (P=0.01) for all diabetic, neuropathic subjects, but increased to r=-0.81 (P<0.01) when only subjects with muscle deterioration ratios >1.0 were included. Muscle deterioration ratios in individuals with diabetic neuropathy were higher for those with a history of ulcers., Interpretation: Individuals with diabetic neuropathy had increased intrinsic foot muscle deterioration, which was associated with second metatarsophalangeal joint angle and history of ulceration. Additional research is required to understand how foot muscle deterioration interacts with other impairments leading to forefoot deformity and skin breakdown., (© 2013.)

Purpose: To describe a semi-automated program that will segment subcutaneous fat, muscle, and adipose tissue in the foot using MR imaging, determine the reliability of the program between and within raters, and determine the validity of the program using MR phantoms., Materials and Methods: MR images were acquired from 19 subjects with and without diabetes and peripheral neuropathy. Two raters segmented and measured volumes from single MR slices at the forefoot, midfoot, and hindfoot at two different times. Intra- and inter-rater correlation coefficients were determined. Muscle and fat MR phantoms of known volumes were measured by the program., Results: Most ICC reliability values were over 0.950. Validity estimates comparing MR estimates and known volumes resulted in r(2) values above 0.970 for all phantoms. The root mean square error was less than 5% for all phantoms., Conclusion: Subcutaneous fat, lean muscle, and adipose tissue volumes in the foot can be quantified in a reliable and valid way. This program can be applied in future studies investigating the relationship of these foot structures to functions in important pathologies, including the neuropathic foot or other musculoskeletal problems., (Copyright © 2013 Wiley Periodicals, Inc.)

Piriformosporaindica is a basidiomycete fungus colonizing roots of a wide range of higher plants, including crop plants and the model plant Arabidopsis thaliana. Previous studies have shown that P. indica improves growth, and enhances systemic pathogen resistance in leaves of host plants. To investigate systemic effects within the root system, we established a hydroponic split-root cultivation system for Arabidopsis. Using quantitative real-time PCR, we show that initial P. indica colonization triggers a local, transient response of several defense-related transcripts, of which some were also induced in shoots and in distal, non-colonized roots of the same plant. Systemic effects on distal roots included the inhibition of secondary P. indica colonization. Faster and stronger induction of defense-related transcripts during secondary inoculation revealed that a P. indica pretreatment triggers root-wide priming of defense responses, which could cause the observed reduction of secondary colonization levels. Secondary P. indica colonization also induced defense responses in distant, already colonized parts of the root. Endophytic fungi therefore trigger a spatially specific response in directly colonized and in systemic root tissues of host plants.

Objective: To determine the effects of weight-bearing (WB) versus nonweight-bearing (NWB) exercise for persons with diabetes mellitus (DM) and peripheral neuropathy (PN)., Design: Randomized controlled trial with evaluations at baseline and after intervention., Setting: University-based physical therapy research clinic., Participants: Participants with DM and PN (N=29) (mean age ± SD, 64.5±12.5y; mean body mass index [kg/m(2)] ± SD, 35.5±7.3) were randomly assigned to WB (n=15) and NWB (n=14) exercise groups. All participants (100%) completed the intervention and follow-up evaluations., Interventions: Group-specific progressive balance, flexibility, strengthening, and aerobic exercise conducted sitting or lying (NWB) or standing and walking (WB) occurred 3 times a week for 12 weeks., Main Outcome Measures: Measures included the 6-minute walk distance (6MWD) and daily step counts. Secondary outcome measures represented domains across the International Classification of Functioning, Disability and Health., Results: The WB group showed greater gains than the NWB group over time on the 6MWD and average daily step count (P<.05). The mean and 95% confidence intervals (CIs) between-group difference over time was 29m (95% CI, 6-51) for the 6MWD and 1178 (95% CI, 150-2205) steps for the average daily step count. The NWB group showed greater improvements than the WB group over time in hemoglobin A1c values (P<.05)., Conclusions: The results of this study indicate the ability of this population with chronic disease to increase 6MWD and daily step count with a WB exercise program compared with an NWB exercise program., (Copyright © 2013 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.)

Jasmonates are oxylipin signals that play important roles in the development of fertile flowers and in defense against pathogens and herbivores in leaves. The aim of this work was to understand the synthesis and function of jasmonates in roots. Grafting experiments with a jasmonate-deficient mutant demonstrated that roots produce jasmonates independently of leaves, despite low expression of biosynthetic enzymes. Levels of 12-oxo-phytodienoic acid, jasmonic acid, and its isoleucine derivative increased in roots upon osmotic and drought stress. Wounding resulted in a decrease of preformed 12-oxo-phytodienoic acid concomitant with an increase of jasmonic acid and jasmonoyl-isoleucine. 13-Lipoxygenases catalyze the first step of lipid oxidation leading to jasmonate production. Analysis of 13-lipoxygenase-deficient mutant lines showed that only one of the four 13-lipoxygenases, LOX6, is responsible and essential for stress-induced jasmonate accumulation in roots. In addition, LOX6 was required for production of basal 12-oxo-phytodienoic acid in leaves and roots. Loss-of-function mutants of LOX6 were more attractive to a detritivorous crustacean and more sensitive to drought, indicating that LOX6-derived oxylipins are important for the responses to abiotic and biotic factors.

Singlet oxygen (¹O₂) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, ¹O₂-induced transcriptomic changes result in acclimation to ¹O₂. Here, using a chlorophyll b-less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of ¹O₂ by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to ¹O₂ reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing ¹O₂ formation, indicating acclimation to ¹O₂. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in ¹O₂-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

Jasmonates and phytoprostanes are oxylipins that regulate stress responses and diverse physiological and developmental processes. 12-Oxo-phytodienoic acid (OPDA) and phytoprostanes are structurally related electrophilic cyclopentenones, which activate similar gene expression profiles that are for the most part different from the action of the cyclopentanone jasmonic acid (JA) and its biologically active amino acid conjugates. Whereas JA-isoleucine signals through binding to COI1, the bZIP transcription factors TGA2, TGA5, and TGA6 are involved in regulation of gene expression in response to phytoprostanes. Here root growth inhibition and target gene expression were compared after treatment with JA, OPDA, or phytoprostanes in mutants of the COI1/MYC2 pathway and in different TGA factor mutants. Inhibition of root growth by phytoprostanes was dependent on COI1 but independent of jasmonate biosynthesis. In contrast, phytoprostane-responsive gene expression was strongly dependent on TGA2, TGA5, and TGA6, but not dependent on COI1, MYC2, TGA1, and TGA4. Different mutant and overexpressing lines were used to determine individual contributions of TGA factors to cyclopentenone-responsive gene expression. Whereas OPDA-induced expression of the cytochrome P450 gene CYP81D11 was primarily regulated by TGA2 and TGA5, the glutathione S-transferase gene GST25 and the OPDA reductase gene OPR1 were regulated by TGA5 and TGA6, but less so by TGA2. These results support the model that phytoprostanes and OPDA regulate differently (i) growth responses, which are COI1 dependent but jasmonate independent; and (ii) lipid stress responses, which are strongly dependent on TGA2, TGA5, and TGA6. Identification of molecular components in cyclopentenone signalling provides an insight into novel oxylipin signal transduction pathways.

Defense signaling compounds and phytohormones play an essential role in the regulation of plant responses to various environmental abiotic and biotic stresses. Among the most severe stresses are herbivory, pathogen infection, and drought stress. The major hormones involved in the regulation of these responses are 12-oxo-phytodienoic acid (OPDA), the pro-hormone jasmonic acid (JA) and its biologically active isoleucine conjugate (JA-Ile), salicylic acid (SA), and abscisic acid (ABA). These signaling compounds are present and biologically active at very low concentrations from ng/g to μg/g dry weight. Accurate and sensitive quantification of these signals has made a significant contribution to the understanding of plant stress responses. Ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (MS/MS) has become an essential technique for the analysis and quantification of these compounds.

Nonenzymatic lipid oxidation is usually viewed as deleterious. But if this is the case, then why does it occur so frequently in cells? Here we review the mechanisms of membrane peroxidation and examine the genesis of reactive electrophile species (RES). Recent evidence suggests that during stress, both lipid peroxidation and RES generation can benefit cells. New results from genetic approaches support a model in which entire membranes can act as supramolecular sinks for singlet oxygen, the predominant reactive oxygen species (ROS) in plastids. RES reprogram gene expression through a class II TGA transcription factor module as well as other, unknown signaling pathways. We propose a framework to explain how RES signaling promotes cell "REScue" by stimulating the expression of genes encoding detoxification functions, cell cycle regulators, and chaperones. The majority of the known biological activities of oxygenated lipids (oxylipins) in plants are mediated either by jasmonate perception or through RES signaling networks.

Honeybee queens are generated on purpose by extensive feeding with a glandular secretion termed royal jelly. Major royal jelly proteins (MRJPs) are the dominant proteinaceous component of royal jelly. One of them, MRJP1, was found to play a central role in honeybee queen development. Genes encoding MRJPs were reported to originate from a single originator, and several of them have evolved nutritive function. Phylogenetic analysis provides evidence that the same originator has multiplied independently in Nasonia and ant lineages. Here we show that bumblebees represent a transition species preserving a single-copy pre-multiplication stage of MRJP evolution. By exploring the single-copy BtRJPL gene, we found striking similarities with MRJPs of the honeybee such as gene structure and expression regulation. At the same time it turned out that BtRJPL does not fulfill criteria for functioning as a nutritive protein. Instead we found evidence that BtRJPL is involved in food digestion or modification, which appears to be the original MRJP function, at least in this lineage. Thus, the evolutionary pattern of MRJPs in hymenopterans constitutes an excellent example of a functional diversification combined with the origin of new properties followed by intensive gene duplication events., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: In people with diabetes and peripheral neuropathy (DM+PN), injury risk is not clearly known for weight bearing (WB) vs. non-weight bearing (NWB) exercise. In-shoe peak plantar pressures (PPP) often are used as a surrogate indicator of injury to the insensitive foot., Objective: Compare PPPs in people with DM+PN during selected WB and NWB exercises., Methods: Fifteen subjects with DM+PN participated. PPPs were recorded for the forefoot, midfoot, and heel during level walking and compared to; WB exercises--treadmill walking, heel and toe raises, sit to stands, stair climbing, single leg standing; and NWB exercises--stationary bicycling, balance ball exercise and plantar flexion exercise., Results: Compared to level walking; mean forefoot PPP during treadmill walking was 13% higher, but this difference was eliminated when walking speed was used as a covariate. Mean PPPs were similar or substantially lower for other exercises, except for higher forefoot PPP with heel raise exercises., Conclusions: Slow progression and regular monitoring of insensitive feet are recommended for all exercises, but especially for heel raises, and increases in walking speed. The remaining WB and NWB exercises pose no greater risk to the insensitive foot due to increases in PPP compared to level walking., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.

Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO· production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H(2)O(2) plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H(2)O(2) also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H(2)O(2). DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers. In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H(2)O(2), and another, minor peak eluted at or close to DAF-2T. Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO· dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO· production., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: High forefoot plantar pressure is associated with plantar ulcers in people with diabetes and peripheral neuropathy. The purpose of this pilot study was to determine the safety and efficacy of botulinum toxin A injected into the gastrocnemius-soleus muscles to reduce muscle strength and plantar pressure., Methods: This double blind, randomized clinical trial studied 17 people with diabetes mellitus, peripheral neuropathy and a forefoot plantar ulcer. Subjects were randomized into one of three groups receiving gastrocnemius-soleus muscle injections on the involved side with; 1) Saline (n = 5, weight =99± 21 kg), 2) 200-units of Botox® (n = 7, weight = 101± 5 kg), or 3) 300-units of Botox® (n = 5, weight = 129± 22 kg). Botox® dose was converted to units/kg, the majority received between 1.9 and 2.4 units/kg (n = 11) and one 3.2 units/kg. Plantarflexor peak torque and forefoot peak plantar pressure were quantified prior and 2 weeks post-injection., Results: There were no complications from the injections. Plantarflexor peak torque on the involved side increased in the placebo and 300 groups (3± 4 Nm and 6± 10 Nm, respectively) and decreased -8± 11 Nm in the 200 group. There was no relationship between units/kg of Botox® for each subject and change in plantarflexor peak torque. Forefoot peak plantar pressure did not change in the placebo and 300 groups (0± 11 and 0± 5 N/cm(2), respectively) and decreased -4± 16 N/cm2 (4%) for the 200 group., Conclusion: There were no adverse events associated with the Botox® injections. This study was unable to determine the dose to consistently reduce plantarflexor strength and forefoot plantar pressure. Additional research is needed to investigate diabetes mellitus specific physiological changes and their impact of BoNT-A effectiveness in order to guide appropriate dosing.

Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of β-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).

Background: The goal of rehabilitation following lower extremity amputation is to restore the highest level of independent function. As much as possible, this includes the functional use of a prosthetic device fitted to the residual limb. Early prosthetic fit depends, in turn, on rapid healing of the amputation site., Methods: We hypothesized that compliance with a novel custom-designed amputation protection and compression system (CAPCS) to the residual limb can accelerate and improve the likelihood of successful prosthesis use. We conducted a retrospective study of all patients who were offered CAPCS by certified prosthetists (Hanger Prosthetics and Orthotics, Bethesda, MD) during the period between April 2004 and November 2009. Variables included age, sex, indication for amputation, and compliance with CAPCS. Compliance was defined as consistent observed wearing of the CAPCS as directed. The primary end point was the fitting of a prosthetic device to the amputated limb, with time to prosthetic fit being the secondary outcome., Results: Out of 100 patients who were offered CAPCS (n = 100) during the study period, 76% were considered compliant (n = 76). Sixty five patients (65%) were ultimately fitted with prosthetic limbs. In multivariate analysis, we found that patients who had compliant use of CAPCS were significantly more likely to be successfully fit with prosthesis (72 vs. 42%, p = 0.005). At 100 days post amputation, the cumulative incidence of prosthesis fitting was significantly higher in CAPCS compliant patients (69.7 vs. 22.2%, p = 0.012)., Conclusions: Compliant use of a CAPCS following amputation is associated with earlier and more frequent use of a prosthetic. Based on this limited data set, a conclusion can be drawn that the potential exists to significantly improve functional outcomes after amputation, but well-designed prospective studies are needed to confirm this association., (Published by Elsevier Inc.)

Background and Purpose: The exercise guidelines for people with diabetes mellitus and peripheral neuropathy (DM+PN) have recently changed to allow moderate-intensity weight-bearing exercise, but there are few reports in the literature describing appropriate weight-bearing exercise for those with DM+PN. This case report describes a successful and safe progressive exercise program for an individual with DM+PN., Case Description: The patient was a 76-year-old man with a 30-year history of DM+PN. He participated in a 12-week, moderate-intensity, progressive exercise program (heart rate approximately 75% of maximum heart rate; rate of perceived exertion=11-13; 3 times per week) involving walking on a treadmill, balance exercises, and strengthening exercises for the lower extremities using body weight resistance., Outcomes: Measurements were taken before and after the 12 weeks of exercise. The patient's Six-Minute Walk Test distance increased from 1,200 to 1,470 ft. His Physical Performance Test score did not change. His Foot and Ankle Ability Measure questionnaire score improved from 89 to 98. Dorsiflexor and plantar-flexor peak torque increased (dorsiflexor peak torque: right side=4.5-4.6 N·m, left side=2.8-3.8 N·m; plantar-flexor peak torque: right side=44.7-62.4 N·m, left side=40.8-56.0 N·m), as did his average daily step count (6,176-8,273 steps/day). Close monitoring of the plantar surface of the feet indicated that the exercise program was well tolerated and there were no adverse events., Discussion and Conclusion: This case report describes a moderate-intensity exercise program that was successful in increasing some measures of muscle strength, physical function, and activity without causing injury in an individual with DM+PN.

Purpose. People with obesity, diabetes, and peripheral neuropathy have high levels of intermuscular adipose tissue (IMAT) volume which has been inversely related to physical function. We determined if IMAT is muscle specific, if calf IMAT is different between a healthy obese group (HO), a group with diabetes mellitus (D), and a group with diabetes mellitus and peripheral neuropathy (DN), and if IMAT volume or the ratio of IMAT/muscle volume is related to physical function in these groups. Methods. 10 healthy obese people, 11 with type 2 diabetes, 24 with diabetes and peripheral neuropathy, had assessments of muscle morphology, physical function and muscle performance. Results. The gastrocnemius muscle had a higher ratio of IMAT/muscle volume than any other muscle or compartment. There were no differences between groups in calf muscle or IMAT volumes. Calf IMAT was inversely related to physical performance on the 6-minute walk test (r = -0.47) and physical performance test (r = -0.36). IMAT/muscle volume was inversely related to physical performance (PPT, r = -0.44; 6 MW r = -0.48; stair power, r = -0.30). Conclusions. IMAT accumulation varies in calf muscles, is highest in the gastrocnemius muscle, and is associated with poor physical performance.

Honeybee populations are severely threatened by parasites and diseases. Recent outbreaks of Colony Collapse Disorder (CCD) has caused loss of more than 35% of bee colonies in the USA, and this is thought to at least in part be due to parasites and/or disease. Interestingly, the honeybee possesses of a limited set of immune genes compared to other insects. Non-canonical immune genes of honeybee are of interest because they may provide greater insights into the peculiar nature of the immune system of this social insect. Previous analyses of bee haemolymph upon bacterial challenge identified a novel leucine-rich repeat protein termed IRP30. Here we show that IRP30 behaves as a typical secreted immune protein. It is expressed simultaneously with carboxylesterase upon treatment with bacteria or other elicitors of immune response. Furthermore we characterize the gene and the mRNA encoding this protein and the IRP30 protein itself. Its regulation and evolution reveal that IRP30 belongs to a protein family, distributed broadly among Hymenoptera, suggesting its ancient function in immune response. We document an interesting case of a recent IRP30 loss in the ant Atta cephalotes and hypothesize that a putative IRP30 homolog of Nasonia emerged by convergent evolution rather than diverged from a common ancestor., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: To describe a new semiautomated method for segmenting and measuring the volume of the muscle, bone, and adipose (subcutaneous and intermuscular) tissue in calf muscle compartments using magnetic resonance (MR) images and determine the intrarater and interrater reproducibility of the measures., Materials and Methods: Proton-density weighted MR images were acquired from the right calf of 21 subjects. Three raters segmented and measured the volumes of bones, adipose tissue, and five individual muscle compartments. Two raters repeated the segmentations. The intra- and interrater reproducibility of the measures (intraclass correlation coefficients; ICC) were determined using generalizability theory., Results: All ICC values were greater than 0.96. The average standard error of the mean (SEM) of all measures was 1.21 cm(3) and none were greater than 2.3 cm(3) . Essentially all variation (≥97% for all measures) was due to subject differences, indicating low error in the measurements., Conclusion: The volumetric measurements for the bones, adipose tissue, and muscle in each of the compartments using MRI were highly reproducible. MRI can provide quantitative, reproducible volumetric measures of bone, adipose tissue, and individual muscle compartments in the calf. We believe these methods can be used to quantify specific muscle or adipose volumetric measures for other clinical or research purposes., (Copyright © 2011 Wiley Periodicals, Inc.)

The jasmonate receptor COI1 is known to facilitate plant defense responses against necrotrophic pathogens, including the ascomycete Sclerotinia sclerotiorum. However, it is not known to what extent jasmonates contribute to defense nor have COI1-independent defense pathways been sufficiently characterized. Here we show that the susceptibility to S. sclerotiorum of the aos mutant, deficient in biosynthesis of jasmonic acid (JA) and its precursor 12-oxophytadienoic acid, was elevated to a level reminiscent of that of hypersusceptible coi1 mutants. In contrast, susceptibility of the JA-deficient opr3 mutant was comparable with that of the wild type. A set of 99 genes responded similarly to infection with S. sclerotiorum in wild-type and coi1 mutant leaves. Expression of this COI1-independent gene set correlated with known differences in gene expression between wild-type plants and a mutant in the transcriptional repressor auxin response factor 2 (arf2). Susceptibility to S. sclerotiorum was reduced in two arf2 mutants early during infection, implicating ARF2 as a negative regulator of defense responses against this pathogen. Hypersusceptibility of an axr1 mutant to S. sclerotiorum confirmed the contribution of auxin action to defense responses against this fungal pathogen.

Sphingolipids are implied in several regulatory processes, including cell death. Levels of the free sphingobase t18:0 (phytosphingosine) increase in Arabidopsis in response to the bacterial pathogen Pseudomonas syringae. To gain information on sphingobase-induced signaling, we determined kinetics of leaf reactive oxygen species (ROS) levels and cell death in response to specific sphingobases. t18:0, d18:0 and d17:1, but not d20:0, induced ROS and cell death within 1.5-2h. Early sphingobase-induced ROS production was independent of cell death induction and required the NADPH oxidase Respiratory Burst Oxidase Homolog D (RBOHD). Specific sphingobases can therefore induce cell death and require RBOHD for early ROS induction in plants., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Plants, like almost all living organisms, spontaneously emit photons of visible light. We used a highly sensitive, low-noise cooled charge coupled device camera to image spontaneous photon emission (autoluminescence) of plants. Oxidative stress and wounding induced a long-lasting enhancement of plant autoluminescence, the origin of which is investigated here. This long-lived phenomenon can be distinguished from the short-lived chlorophyll luminescence resulting from charge recombinations within the photosystems by pre-adapting the plant to darkness for about 2 h. Lipids in solvent were found to emit a persistent luminescence after oxidation in vitro, which exhibited the same time and temperature dependence as plant autoluminescence. Other biological molecules, such as DNA or proteins, either did not produce measurable light upon oxidation or they did produce a chemiluminescence that decayed rapidly, which excludes their significant contribution to the in vivo light emission signal. Selective manipulation of the lipid oxidation levels in Arabidopsis mutants affected in lipid hydroperoxide metabolism revealed a causal link between leaf autoluminescence and lipid oxidation. Addition of chlorophyll to oxidized lipids enhanced light emission. Both oxidized lipids and plants predominantly emit light at wavelengths higher than 600 nm; the emission spectrum of plant autoluminescence was shifted towards even higher wavelengths, a phenomenon ascribable to chlorophyll molecules acting as luminescence enhancers in vivo. Taken together, the presented results show that spontaneous photon emission imaged in plants mainly emanates from oxidized lipids. Imaging of this signal thus provides a simple and sensitive non-invasive method to selectively visualize and map patterns of lipid oxidation in plants., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)