Training-Specific Neural Plasticity in Spinal Reflexes after Incomplete Spinal Cord Injury.

The neural plasticity of spinal reflexes after two contrasting forms of walking training was determined in individuals with chronic, motor-incomplete spinal cord injury (SCI). Endurance Training involved treadmill walking for as long as possible, and Precision Training involved walking precisely over obstacles and onto targets overground. Twenty participants started either Endurance or Precision Training for 2 months and then crossed over after a 2-month rest period to the other form of training for 2 months. Measures were taken before and after each phase of training and rest. The cutaneomuscular reflex (CMR) during walking was evoked in the soleus (SOL) and tibialis anterior muscles by stimulating the posterior tibial nerve at the ankle. Clonus was estimated from the EMG power in the SOL during unperturbed walking. The inhibitory component of the SOL CMR was enhanced after Endurance but not Precision Training. Clonus did not change after either form of training. Participants with lower reflex excitability tended to be better walkers (i.e., faster walking speeds) prior to training, and the reduction in clonus was significantly correlated with the improvement in walking speed and distance. Thus, reflex excitability responded in a training-specific way, with the reduction in reflex excitability related to improvements in walking function. Trial registration number is NCT01765153.

Gait Transitions in Human Infants: Coping with Extremes of Treadmill Speed.

Spinal pattern generators in quadrupedal animals can coordinate different forms of locomotion, like trotting or galloping, by altering coordination between the limbs (interlimb coordination). In the human system, infants have been used to study the subcortical control of gait, since the cerebral cortex and corticospinal tract are immature early in life. Like other animals, human infants can modify interlimb coordination to jump or step. Do human infants possess functional neuronal circuitry necessary to modify coordination within a limb (intralimb coordination) in order to generate distinct forms of alternating bipedal gait, such as walking and running? We monitored twenty-eight infants (7-12 months) stepping on a treadmill at speeds ranging between 0.06-2.36 m/s, and seventeen adults (22-47 years) walking or running at speeds spanning the walk-to-run transition. Six of the adults were tested with body weight support to mimic the conditions of infant stepping. We found that infants could accommodate a wide range of speeds by altering stride length and frequency, similar to adults. Moreover, as the treadmill speed increased, we observed periods of flight during which neither foot was in ground contact in infants and in adults. However, while adults modified other aspects of intralimb coordination and the mechanics of progression to transition to a running gait, infants did not make comparable changes. The lack of evidence for distinct walking and running patterns in infants suggests that the expression of different functional, alternating gait patterns in humans may require neuromuscular maturation and a period of learning post-independent walking.

Characteristics of the developing human locomotor system: Similarities to other mammals.

Similarities in the development of locomotion between young children and other mammals are explored by reanalysis of data accrued over ~18 years. Supported stepping in children was tested on a treadmill. Although the time course of development is more protracted in humans compared to other mammals, the same trends are seen. For example, the duration of the stepping cycle shortens rapidly in the first 5 months of life. Hypermetric flexion of the hip and knee during stepping is seen in children <3 mo old. Stability of the locomotor rhythm both with respect to cycle duration within a limb and coupling between limbs improves slowly. Finally, coordination between the left and right legs can be manipulated with training, indicating experience-dependent learning at a young age. The possible reasons for these remarkably similar trends in development are explored as a function of maturational time tables for neural structures.

Prior experience but not size of error improves motor learning on the split-belt treadmill in young children.

Children can modify learned motor skills, such as walking, to adapt to new environments. Movement errors in these new situations drive the learning. We used split-belt walking to determine whether size of the error affects the degree of learning. Twenty-two children (aged 2-5 y) walked on the split-belt treadmill on two separate days spaced 1 week apart. Twenty-eight adults served as controls. On Day 1, children experienced an abrupt change in belt speeds (from 1:1 to 2:1 differential) resulting in large errors, or a gradual change (same change in speed over 12-15 min), resulting in small errors. Learning was measured by the size of the aftereffect upon return to a 1:1 differential. On Day 2 (1 week later), the leg on the fast belt was reversed, as was the method of introducing the speed differential. We found that the error size did not affect learning. Unexpectedly, learning was greater on Day 2 compared to Day 1, especially for children under 4 y of age, despite the fact that the task was opposite to that of Day 1, and did not influence learning in adults. Hence, 11 additional children under 4 y of age were tested with belts running at the same speed on Day 1, and with a 2:1 speed differential (abrupt introduction) on Day 2. Surprisingly, learning was again greater on Day 2. We conclude that size of error during split-belt walking does not affect learning, but experience on a treadmill does, especially for younger children.

Training to enhance walking in children with cerebral palsy: are we missing the window of opportunity?

The objective of this paper is to (1) identify from the literature a potential critical period for the maturation of the corticospinal tract (CST) and (2) report pilot data on an intensive, activity-based therapy applied during this period, in children with lesions to the CST. The best estimate of the CST critical period for the legs is when the child is younger than 2 years of age. Previous interventions for walking in children with CST damage were mainly applied after this age. Our preliminary results with training children younger than 2 years showed improvements in walking that exceeded all previous reports. Further, we refined techniques for measuring motor and sensory pathways to and from the legs, so that changes can be measured at this young age. Previous activity-based therapies may have been applied too late in development. A randomized controlled trial is now underway to determine if intensive leg therapy improves the outcome of children with early stroke.

Fusarium damage in small cereal grains from Western Canada. 2. Occurrence of fusarium toxins and their source organisms in durum wheat harvested in 2010.

Samples of Canadian western amber durum harvested in 2010 were obtained as part of the Canadian Grain Commission Harvest Sample Program, inspected, and graded according to Canadian guidelines. A subset of Fusarium -damaged samples were analyzed for Fusarium species as well as mycotoxins associated with these species, including deoxynivalenol and other trichothecenes, moniliformin, enniatins, and beauvericin. Overall, Fusarium avenaceum and F. graminearum were the top two most frequently recovered species. Phaeosphaeria nodorum (a.k.a. Septoria nodorum ), F. culmorum , F. poae , F. acuminatum , and F. sporotrichioides were observed in samples as well. All samples analyzed for mycotoxins contained quantifiable concentrations of enniatins, whereas beauvericin, deoxynivalenol, and moniliformin were measured in approximately 75% of the samples. Concentrations in Fusarium -damaged samples ranged from 0.011 to 34.2 mg/kg of enniatins plus beauvericin, up to 4.7 mg/kg of deoxynivalenol, and up to 6.36 mg/kg of moniliformin. Comparisons of enniatins, beauvericin, and moniliformin concentrations to the occurrence of various Fusarium species suggest the existence of an infection threshold above which these emerging mycotoxins are present at higher concentrations. The current grading factor of Fusarium -damaged kernels manages concentrations of these emerging mycotoxins in grain; lower provisional grades were assigned to samples that contained the highest concentrations of enniatins, beauvericin, and moniliformin.

Fusarium damage in cereal grains from Western Canada. 1. Phylogenetic analysis of moniliformin-producing fusarium species and their natural occurrence in mycotoxin-contaminated wheat, oats, and rye.

Harvest samples of common wheat (Triticum aestivum), oats (Avena sativa), and rye (Secale cereale) from producers in western Canada were analyzed for fungal infection by toxigenic Fusarium species and contamination by trichothecenes and moniliformin (MON). Fusarium graminearum and F. avenaceum were the two most frequently isolated species from samples of rye and wheat collected in 2010. F. poae and F. sporotrichioides were more commonly detected in randomly selected oat seeds. Other toxigenic Fusarium species including F. acuminatum, F. culmorum, and F. pseudograminearum as well as Phaeosphaeria nodorum (a.k.a. Septoria nodorum) were recovered primarily from fusarium-damaged kernels of wheat. Pure cultures of F. avenaceum, F. acuminatum, and other related species known to produce moniliformin were isolated from incubated seeds based on micro- and macromorphological criteria. The phylogenetic analysis inferred from partial DNA sequences of the acl1 and tef-1α genes revealed two major clades representing F. avenaceum and F. acuminatum, respectively. These clades comprised all Canadian isolates of the two species and a number of reference cultures studied earlier for their propensity to form moniliformin in vitro and in planta. However, some reference cultures previously reported to produce significant amounts of moniliformin formed minor phylogenetic lineages that represent rather distinct but closely related species. Concomitantly, cereal samples were analyzed for the presence of deoxynivalenol and moniliformin. These two Fusarium toxins were observed most frequently in common wheat, at concentrations up to 1.1 and 4.0 mg/kg, respectively. There was no apparent relationship between moniliformin concentrations and detection of F. avenaceum and F. acuminatum in rye and oat samples. Geographical analysis of the distribution of moniliformin and F. avenaceum and F. acuminatum across the Canadian Prairies also did not indicate a strong relationship.

Developmental constraints of quadrupedal coordination across crawling styles in human infants.

Human infants can crawl using several very different styles; this diversity appears at first glance to contradict our previous findings from hands-and-knees crawling, which suggested that there were strict limitations on coordination, imposed either mechanically or by the developing nervous system. To determine whether coordination was similarly restricted across crawling styles, we studied free crawling overground in 22 infants who used a number of different locomotor strategies. Despite the wide variety in the use of individual limbs and even the number of limbs used, the duration of the stance phase increased with duration of cycle, whereas the duration of the swing phase remained more constant. Additionally, all infants showed organized, rhythmic interlimb coordination. Alternating patterns (e.g., trotlike) predominated (86% of infants). Alternatively, yet much less frequently, all limbs used could work in synchrony (14% of infants). Pacelike patterns were never observed, even in infants that crawled with the belly remaining in contact with the ground so that stability was not a factor. To explore the robustness of the interlimb coordination, a perturbation that prolonged swing of the leg was imposed on 14 additional infants crawling on hands and knees overground or on the treadmill. The perturbation led to a resetting of the crawling pattern, but never to a change in the coordination of the limbs. The findings concur with those regarding other infant animals, together suggesting that the nervous system itself limits the coordination patterns available at a young age.

Unique characteristics of motor adaptation during walking in young children.

Children show precocious ability in the learning of languages; is this the case with motor learning? We used split-belt walking to probe motor adaptation (a form of motor learning) in children. Data from 27 children (ages 8-36 mo) were compared with those from 10 adults. Children walked with the treadmill belts at the same speed (tied belt), followed by walking with the belts moving at different speeds (split belt) for 8-10 min, followed again by tied-belt walking (postsplit). Initial asymmetries in temporal coordination (i.e., double support time) induced by split-belt walking were slowly reduced, with most children showing an aftereffect (i.e., asymmetry in the opposite direction to the initial) in the early postsplit period, indicative of learning. In contrast, asymmetries in spatial coordination (i.e., center of oscillation) persisted during split-belt walking and no aftereffect was seen. Step length, a measure of both spatial and temporal coordination, showed intermediate effects. The time course of learning in double support and step length was slower in children than in adults. Moreover, there was a significant negative correlation between the size of the initial asymmetry during early split-belt walking (called error) and the aftereffect for step length. Hence, children may have more difficulty learning when the errors are large. The findings further suggest that the mechanisms controlling temporal and spatial adaptation are different and mature at different times.

Development of a specific TaqMan real-time PCR assay for quantification of Fusarium graminearum clade 7 and comparison of fungal biomass determined by PCR with deoxynivalenol content in wheat and barley.

A Fusarium graminearum clade 7 specific real-time quantitative PCR (qPCR) assay was developed in this study based on unique polymorphisms in sequences of the mating type protein (MAT) gene. PCR amplification was not observed in eight phylogenetic lineages of the F. graminearum complex and four other closely related Fusarium species. Accuracy of the quantification of the real-time PCR assay was verified with wheat DNA spiked with F. graminearum clade 7 DNA. Wheat samples representing two Canadian wheat classes, CWRS (Canadian Western Red Spring) and CWRW (Canadian Western Red Winter) were used to determine the relationships among F. graminearum DNA, deoxynivalenol (DON) and Fusarium damaged kernel (FDK). The amount of DON and F. graminearum DNA remaining after removal of FDK varied among samples, but was sometimes substantial. Positive correlations were observed between F. graminearum clade 7 DNA (in picograms) and DON as well as FDK. There was also a strong correlation between FDK and DON in CWRS and CWRW wheat composite samples, but the inherent variability in individual producer samples precluded a definitive correlation. For barley, a positive correlation was observed between Fusarium DNA and DON values. Real-time PCR assays can be a valuable tool for barley as there are no reliable symptoms to visually assess the level of Fusarium head blight in this crop.

Interlimb coordination in human crawling reveals similarities in development and neural control with quadrupeds.

The study of quadrupeds has furnished most of our understanding of mammalian locomotion. To allow a more direct comparison of coordination between the four limbs in humans and quadrupeds, we studied crawling in the human, a behavior that is part of normal human development and mechanically more similar to quadrupedal locomotion than is bipedal walking. Interlimb coordination during hands-and-knees crawling is compared between humans and quadrupeds and between human infants and adults. Mechanical factors were manipulated during crawling to understand the relative contributions of mechanics and neural control. Twenty-six infants and seven adults were studied. Video, force plate, and electrogoniometer data were collected. Belt speed of the treadmill, width of base, and limb length were manipulated in adults. Influences of unweighting and limb length were explored in infants. Infants tended to move diagonal limbs together (trot-like). Adults additionally moved ipsilateral limbs together (pace-like). At lower speeds, movements of the four limbs were more equally spaced in time, with no clear pairing of limbs. At higher speeds, running symmetrical gaits were never observed, although one adult galloped. Widening stance prevented adults from using the pace-like gait, whereas lengthening the hind limbs (hands-and-feet crawling) largely prevented the trot-like gait. Limb length and unweighting had no effect on coordination in infants. We conclude that human crawling shares features both with other primates and with nonprimate quadrupeds, suggesting similar underlying mechanisms. The greater restriction in coordination patterns used by infants suggests their nervous system has less flexibility.

Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain.

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclaved wheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and <54 CFU/mL, respectively, in the initial sample. For L. monocytogenes, an improved detection limit of <62 CFU/mL was achieved using singleplex PCR. For autoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichment step. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis.

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.

Stimulation of the subthalamic nucleus changes the firing pattern of pallidal neurons.

To clarify the mechanism underlying improvement of parkinsonian signs by high-frequency electrical stimulation (HFS) of the subthalamic nucleus (STN), we investigated the effects of STN HFS on neuronal activity of the internal and external segment of the globus pallidus (GPi and GPe, respectively) in two rhesus monkeys rendered parkinsonian by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. A scaled-down version of the chronic stimulating electrode used in humans, consisting of four metal contacts 0.50 mm in length each separated by 0.50 mm, was implanted through a cephalic chamber targeting the STN. Histological reconstruction revealed that the cathode was located in the STN in both monkeys. Extracellular recordings from a total of 110 pallidal neurons during STN stimulation were performed. Poststimulus time histograms of single neurons triggered by 2 Hz STN stimulation pulses at 2.4-3.0 V revealed short-latency excitations at 2.5-4.5 and 5.5-7.0 msec after stimulation onset and inhibitions at 1.0-2.5, 4.5-5.5, and 7.0-9.0 msec for both GPe and GPi neurons. These short-latency responses were present with 136 Hz stimulation, at voltages effective for alleviation of parkinsonian signs, resulting in a significant increase in mean discharge rate and a stimulus-synchronized regular firing pattern. These results indicate that activation of the STN efferent fibers and resultant changes in the temporal firing pattern of neurons in GPe and GPi underlie the beneficial effect of HFS in the STN in Parkinson's disease and further support the role of temporal firing patterns in the basal ganglia in the development of Parkinson's disease and other movement disorders.

A Simple Medium to Aid the Identification of Fusarium moniliforme , F. proliferatum , and F. subglutinans 1.

Growth of Fusarium moniliforme , F. proliferatum , and F. subglutinans on Czapek solution agar containing 20% saccharose resulted in both cultural differences and enhanced micromorphological features. F. moniliforme could be reliably distinguished from the other two species based on differences in colony color and texture. These differences were intensified by lowering the pH of the media from 7.7 to 4.4 without adversely affecting micromorphology.