A direct spinal cord-computer interface enables the control of the paralysed hand in spinal cord injury.

The paralysis of the muscles controlling the hand dramatically limits the quality of life of individuals living with spinal cord injury (SCI). Here, with a non-invasive neural interface, we demonstrate that eight motor complete SCI individuals (C5-C6) are still able to task-modulate in real-time the activity of populations of spinal motor neurons with residual neural pathways. In all SCI participants tested, we identified groups of motor units under voluntary control that encoded various hand movements. The motor unit discharges were mapped into more than 10 degrees of freedom, ranging from grasping to individual hand-digit flexion and extension. We then mapped the neural dynamics into a real-time controlled virtual hand. The SCI participants were able to match the cue hand posture by proportionally controlling four degrees of freedom (opening and closing the hand and index flexion/extension). These results demonstrate that wearable muscle sensors provide access to spared motor neurons that are fully under voluntary control in complete cervical SCI individuals. This non-invasive neural interface allows the investigation of motor neuron changes after the injury and has the potential to promote movement restoration when integrated with assistive devices.

NSF DARE-Transforming modeling in neurorehabilitation: Four threads for catalyzing progress.

We present an overview of the Conference on Transformative Opportunities for Modeling in Neurorehabilitation held in March 2023. It was supported by the Disability and Rehabilitation Engineering (DARE) program from the National Science Foundation's Engineering Biology and Health Cluster. The conference brought together experts and trainees from around the world to discuss critical questions, challenges, and opportunities at the intersection of computational modeling and neurorehabilitation to understand, optimize, and improve clinical translation of neurorehabilitation. We organized the conference around four key, relevant, and promising Focus Areas for modeling: Adaptation & Plasticity, Personalization, Human-Device Interactions, and Modeling 'In-the-Wild'. We identified four common threads across the Focus Areas that, if addressed, can catalyze progress in the short, medium, and long terms. These were: (i) the need to capture and curate appropriate and useful data necessary to develop, validate, and deploy useful computational models (ii) the need to create multi-scale models that span the personalization spectrum from individuals to populations, and from cellular to behavioral levels (iii) the need for algorithms that extract as much information from available data, while requiring as little data as possible from each client (iv) the insistence on leveraging readily available sensors and data systems to push model-driven treatments from the lab, and into the clinic, home, workplace, and community. The conference archive can be found at (dare2023.usc.edu). These topics are also extended by three perspective papers prepared by trainees and junior faculty, clinician researchers, and federal funding agency representatives who attended the conference.

Dynamic spinal reflex adaptation during locomotor adaptation.

The dynamics and interaction of spinal and supraspinal centers during locomotor adaptation remain vaguely understood. In this work, we use Hoffmann reflex measurements to investigate changes in spinal reflex gains during split-belt locomotor adaptation. We show that spinal reflex gains are dynamically modulated during split-belt locomotor adaptation. During first exposure to split-belt transitions, modulation occurs mostly on the leg ipsilateral to the speed change and constitutes rapid suppression or facilitation of the reflex gains, followed by slow recovery to baseline. Over repeated exposure, the modulation pattern washes out. We further show that reflex gain modulation strongly correlates with correction of leg asymmetry, and cannot be explained by speed modulation solely. We argue that reflex modulation is likely of supraspinal origins and constitutes an integral part of the neural substrate underlying split-belt locomotor adaptation.NEW & NOTEWORTHY This work presents direct evidence for spinal reflex modulation during locomotor adaptation. In particular, we show that reflexes can be modulated on-demand unilaterally during split-belt locomotor adaptation and speculate about reflex modulation as an underlying mechanism for adaptation of gait asymmetry in healthy adults.

Information about contact force and surface texture is mixed in the firing rates of cutaneous afferent neurons.

Cutaneous mechanoreceptors in our hands gather information about the objects we handle. Tactile fibers encode mixed information about contact events and object properties. Neural coding in tactile afferents is typically studied by varying a single aspect of tactile stimuli, avoiding the confounds of real-world haptic interactions. We instead record responses of small populations of dorsal root ganglia (DRG) neurons to variable tactile stimuli and find that neurons primarily respond to force, though some texture information can be detected. Tactile nerve fibers convey information about many features of haptic interactions, including the force and speed of contact, as well as the texture and shape of the objects being handled. How we perceive these object features is relatively unaffected by the forces and movements we use when interacting with the object. Because signals related to contact events and object properties are mixed in the responses of tactile fibers, our ability to disentangle these different components of our tactile experience implies that they are demultiplexed as they propagate along the neuraxis. To understand how texture and contact mechanics are encoded together by tactile fibers, we studied the activity of multiple neurons recorded simultaneously in the cervical DRG of two anesthetized rhesus monkeys while textured surfaces were applied to the glabrous skin of the fingers and palm using a handheld probe. A transducer at the tip of the textured probe measured contact forces as tactile stimuli were applied at different locations on the finger-pads and palm. We examined how a sample population of DRG neurons encode force and texture and found that firing rates of individual neurons are modulated by both force and texture. In particular, slowly adapting (SA) neurons were more responsive to force than texture, and rapidly adapting (RA) neurons were more responsive to texture than force. Although force could be decoded accurately throughout the entire contact interval, texture signals were most salient during onset and offset phases of the contact interval.NEW & NOTEWORTHY Cutaneous mechanoreceptors in our hands gather information about the objects we handle. Tactile fibers encode mixed information about contact events and object properties. Neural coding in tactile afferents is typically studied by varying a single aspect of tactile stimuli, avoiding the confounds of real-world haptic interactions. We instead record responses of small populations of DRG neurons to variable tactile stimuli and find that neurons primarily respond to force, though some texture information can be detected.

Sensing and decoding the neural drive to paralyzed muscles during attempted movements of a person with tetraplegia using a sleeve array.

Motor neurons convey information about motor intent that can be extracted and interpreted to control assistive devices. However, most methods for measuring the firing activity of single neurons rely on implanted microelectrodes. Although intracortical brain-computer interfaces (BCIs) have been shown to be safe and effective, the requirement for surgery poses a barrier to widespread use that can be mitigated by instead using noninvasive interfaces. The objective of this study was to evaluate the feasibility of deriving motor control signals from a wearable sensor that can detect residual motor unit activity in paralyzed muscles after chronic cervical spinal cord injury (SCI). Despite generating no observable hand movement, volitional recruitment of motor units below the level of injury was observed across attempted movements of individual fingers and overt wrist and elbow movements. Subgroups of motor units were coactive during flexion or extension phases of the task. Single digit movement intentions were classified offline from the electromyogram (EMG) power [root-mean-square (RMS)] or motor unit firing rates with median classification accuracies >75% in both cases. Simulated online control of a virtual hand was performed with a binary classifier to test feasibility of real-time extraction and decoding of motor units. The online decomposition algorithm extracted motor units in 1.2 ms, and the firing rates predicted the correct digit motion 88 ± 24% of the time. This study provides the first demonstration of a wearable interface for recording and decoding firing rates of motor units below the level of injury in a person with motor complete SCI.NEW & NOTEWORTHY A wearable electrode array and machine learning methods were used to record and decode myoelectric signals and motor unit firing in paralyzed muscles of a person with motor complete tetraplegia. The myoelectric activity and motor unit firing rates were task specific, even in the absence of visible motion, enabling accurate classification of attempted single-digit movements. This wearable system has the potential to enable people with tetraplegia to control assistive devices through movement intent.

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in†vitro Applications.

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.

Advances in motion and electromyography based wearable technology for upper extremity function rehabilitation: A review.

STUDY DESIGN: Scoping review. INTRODUCTION: With the recent advances in technologies, interactive wearable technologies including inertial motion sensors and e-textiles are emerging in the field of rehabilitation to monitor and provide feedback and therapy remotely. PURPOSE OF THE STUDY: This review article focuses on inertial measurement unit motion sensor and e-textiles-based technologies and proposes approaches to augment these interactive wearable technologies. METHODS: We conducted a comprehensive search of relevant electronic databases (eg, PubMed, the Cumulative Index to Nursing and Allied Health Literature, Embase, PsycINFO, The Cochrane Central Register of Controlled Trial, and the Physiotherapy Evidence Database). The scoping review included all study designs. RESULTS: Currently, there are a numerous research groups and companies investigating inertial motion sensors and e-textiles-based interactive wearable technologies. However, translation of these technologies to the clinic would need further research to increase ease of use and improve clinical validity of the outcomes of these technologies. DISCUSSION: The current review discusses the limitations of the interactive wearable technologies such as, limited clinical utility, bulky equipment, difficulty in setting up equipment inertial motion sensors and e-textiles. CONCLUSION: There is tremendous potential for interactive wearable technologies in rehabilitation. With the evolution of cloud computing, interactive wearable systems can remotely provide intervention and monitor patient progress using models of telerehabilitation. This will revolutionize the delivery of rehabilitation and make rehabilitation more accessible and affordable to millions of individuals.

What is the functional relevance of reorganization in primary motor cortex after spinal cord injury?

Motor output maps within primary motor cortex are widely distributed and modified by motor skill learning and neurological injury. Functions that these maps represent after spinal cord injury remain debatable. Moreover, the pattern of reorganization and whether it supports recovery of compromised motor function is not well understood. A deeper understanding of the pathophysiological mechanisms of motor dysfunction after spinal cord injury is necessary to develop and optimize repair strategies. There are three purposes for this review. The first is to synthesize available research on spontaneous reorganization with primary motor cortex following spinal cord injury. The second is to draw on existing evidence from the motor skill learning and brain injury literature to interpret the form and purpose of motor maps. The third purpose is to account for the existing research on intervention-induced reorganization of primary motor cortex following spinal cord injury. We conclude with directions for future work, emphasizing the need for multi-modal investigations that construct maps with both neuroimaging and non-invasive stimulation methods to derive a cohesive understanding of the effects of spinal cord injury on reorganization within primary motor cortex.

Altered modulation of sensorimotor rhythms with chronic paralysis.

After paralysis, the disconnection between the cortex and its peripheral targets leads to neuroplasticity throughout the nervous system. However, it is unclear how chronic paralysis specifically impacts cortical oscillations associated with attempted movement of impaired limbs. We hypothesized that µ- (8-13 Hz) and ß- (15-30 Hz) event-related desynchronization (ERD) would be less modulated for individuals with hand paralysis due to cervical spinal cord injury (SCI). To test this, we compared the modulation of ERD from magnetoencephalography (MEG) during attempted and imagined grasping performed by participants with cervical SCI (n = 12) and able-bodied controls (n = 13). Seven participants with tetraplegia were able to generate some electromyography (EMG) activity during attempted grasping, whereas the other five were not. The peak and area of ERD were significantly decreased for individuals without volitional muscle activity when they attempted to grasp compared with able-bodied subjects and participants with SCI,with some residual EMG activity. However, no significant differences were found between subject groups during mentally simulated tasks (i.e., motor imagery) where no muscle activity or somatosensory consequences were expected. These findings suggest that individuals who are unable to produce muscle activity are capable of generating ERD when attempting to move, but the characteristics of this ERD are altered. However, for people who maintain volitional muscle activity after SCI, there are no significant differences in ERD characteristics compared with able-bodied controls. These results provide evidence that ERD is dependent on the level of intact muscle activity after SCI.NEW & NOTEWORTHY Source space MEG was used to investigate sensorimotor cortical oscillations in individuals with SCI. This study provides evidence that individuals with cervical SCI exhibit decreased ERD when they attempt to grasp if they are incapable of generating muscle activity. However, there were no significant differences in ERD between paralyzed and able-bodied participants during motor imagery. These results have important implications for the design and evaluation of new therapies, such as motor imagery and neurofeedback interventions.

Microstimulation of the lumbar DRG recruits primary afferent neurons in localized regions of lower limb.

Patterned microstimulation of the dorsal root ganglion (DRG) has been proposed as a method for delivering tactile and proprioceptive feedback to amputees. Previous studies demonstrated that large- and medium-diameter afferent neurons could be recruited separately, even several months after implantation. However, those studies did not examine the anatomical localization of sensory fibers recruited by microstimulation in the DRG. Achieving precise recruitment with respect to both modality and receptive field locations will likely be crucial to create a viable sensory neuroprosthesis. In this study, penetrating microelectrode arrays were implanted in the L5, L6, and L7 DRG of four isoflurane-anesthetized cats instrumented with nerve cuff electrodes around the proximal and distal branches of the sciatic and femoral nerves. A binary search was used to find the recruitment threshold for evoking a response in each nerve cuff. The selectivity of DRG stimulation was characterized by the ability to recruit individual distal branches to the exclusion of all others at threshold; 84.7% (n = 201) of the stimulation electrodes recruited a single nerve branch, with 9 of the 15 instrumented nerves recruited selectively. The median stimulation threshold was 0.68 nC/phase, and the median dynamic range (increase in charge while stimulation remained selective) was 0.36 nC/phase. These results demonstrate the ability of DRG microstimulation to achieve selective recruitment of the major nerve branches of the hindlimb, suggesting that this approach could be used to drive sensory input from localized regions of the limb. This sensory input might be useful for restoring tactile and proprioceptive feedback to a lower-limb amputee.

Microstimulation of afferents in the sacral dorsal root ganglia can evoke reflex bladder activity.

AIMS: Pudendal afferent fibers can be excited using electrical stimulation to evoke reflex bladder activity. While this approach shows promise for restoring bladder function, stimulation of desired pathways, and integration of afferent signals for sensory feedback remains challenging. At sacral dorsal root ganglia (DRG), the convergence of pelvic and pudendal afferent fibers provides a unique location for access to lower urinary tract neurons. Our goal in this study was to demonstrate the potential of microstimulation in sacral DRG for evoking reflex bladder responses. METHODS: Penetrating microelectrode arrays were inserted in the left S1 and S2 DRG of six anesthetized adult male cats. While the bladder volume was held at a level below the leak volume, single and multiple channel stimulation was performed using various stimulation patterns. RESULTS: Reflex bladder excitation was observed in five cats, for stimulation in either S1 or S2 DRG at 1 Hz and 30-33 Hz with a pulse amplitude of 10-50 µA. Bladder relaxation was observed during a few trials. Adjacent electrodes frequently elicited very different responses. CONCLUSIONS: These results demonstrate the potential of low-current microstimulation to recruit reflexive bladder responses. An approach such as this could be integrated with DRG recordings of bladder afferents to provide a closed-loop bladder neuroprosthesis.

Changes in Cerebellar Activation After Onabotulinumtoxin A Injections for Spasticity After Chronic Stroke: A Pilot Functional Magnetic Resonance Imaging Study.

OBJECTIVE: To investigate the effect of reducing spasticity via onabotulinumtoxin A (Obtx-A) injection on cerebellar activation after chronic stroke during unilateral gripping. DESIGN: Pre-post, case series. SETTING: Outpatient spasticity clinic. PARTICIPANTS: Individuals with chronic spasticity (N=4). INTERVENTIONS: Upper-limb Obtx-A injection. MAIN OUTCOME MEASURES: Functional magnetic resonance imaging (fMRI) was used to measure changes in cerebellar activation before and after upper-limb Obtx-A injection. During fMRI testing, participants performed the same motor task before and after injection, which was 15% and 30% of maximum voluntary isometric gripping measured before Obtx-A injection. RESULTS: After Obtx-A injection, cerebellar activation increased bilaterally during gripping with the paretic hand and during rest. During both pre- and postinjection scans, the paretic hand showed larger cerebellar activation during gripping compared with the nonparetic hand. Cerebellar activation during gripping with the nonparetic hand did not change significantly after Obtx-A injection. CONCLUSIONS: Reducing spasticity via Obtx-A injection may increase cerebellar activation both during gripping tasks with the paretic hand and during rest. To our knowledge, this is the first study that examines changes in cerebellar activation after spasticity treatment with Obtx-A.

MEG-based neurofeedback for hand rehabilitation.

BACKGROUND: Providing neurofeedback (NF) of motor-related brain activity in a biologically-relevant and intuitive way could maximize the utility of a brain-computer interface (BCI) for promoting therapeutic plasticity. We present a BCI capable of providing intuitive and direct control of a video-based grasp. METHODS: Utilizing magnetoencephalography's (MEG) high temporal and spatial resolution, we recorded sensorimotor rhythms (SMR) that were modulated by grasp or rest intentions. SMR modulation controlled the grasp aperture of a stop motion video of a human hand. The displayed hand grasp position was driven incrementally towards a closed or opened state and subjects were required to hold the targeted position for a time that was adjusted to change the task difficulty. RESULTS: We demonstrated that three individuals with complete hand paralysis due to spinal cord injury (SCI) were able to maintain brain-control of closing and opening a virtual hand with an average of 63 % success which was significantly above the average chance rate of 19 %. This level of performance was achieved without pre-training and less than 4 min of calibration. In addition, successful grasp targets were reached in 1.96 ± 0.15 s. Subjects performed 200 brain-controlled trials in approximately 30 min excluding breaks. Two of the three participants showed a significant improvement in SMR indicating that they had learned to change their brain activity within a single session of NF. CONCLUSIONS: This study demonstrated the utility of a MEG-based BCI system to provide realistic, efficient, and focused NF to individuals with paralysis with the goal of using NF to induce neuroplasticity.

High-performance neuroprosthetic control by an individual with tetraplegia.

BACKGROUND: Paralysis or amputation of an arm results in the loss of the ability to orient the hand and grasp, manipulate, and carry objects, functions that are essential for activities of daily living. Brain-machine interfaces could provide a solution to restoring many of these lost functions. We therefore tested whether an individual with tetraplegia could rapidly achieve neurological control of a high-performance prosthetic limb using this type of an interface. METHODS: We implanted two 96-channel intracortical microelectrodes in the motor cortex of a 52-year-old individual with tetraplegia. Brain-machine-interface training was done for 13 weeks with the goal of controlling an anthropomorphic prosthetic limb with seven degrees of freedom (three-dimensional translation, three-dimensional orientation, one-dimensional grasping). The participant's ability to control the prosthetic limb was assessed with clinical measures of upper limb function. This study is registered with ClinicalTrials.gov, NCT01364480. FINDINGS: The participant was able to move the prosthetic limb freely in the three-dimensional workspace on the second day of training. After 13 weeks, robust seven-dimensional movements were performed routinely. Mean success rate on target-based reaching tasks was 91·6% (SD 4·4) versus median chance level 6·2% (95% CI 2·0-15·3). Improvements were seen in completion time (decreased from a mean of 148 s [SD 60] to 112 s [6]) and path efficiency (increased from 0·30 [0·04] to 0·38 [0·02]). The participant was also able to use the prosthetic limb to do skilful and coordinated reach and grasp movements that resulted in clinically significant gains in tests of upper limb function. No adverse events were reported. INTERPRETATION: With continued development of neuroprosthetic limbs, individuals with long-term paralysis could recover the natural and intuitive command signals for hand placement, orientation, and reaching, allowing them to perform activities of daily living. FUNDING: Defense Advanced Research Projects Agency, National Institutes of Health, Department of Veterans Affairs, and UPMC Rehabilitation Institute.

Computational modeling of dorsal root ganglion stimulation using an Injectrode.

Objective.Minimally invasive neuromodulation therapies like the Injectrode, which is composed of a tightly wound polymer-coated Platinum/Iridium microcoil, offer a low-risk approach for administering electrical stimulation to the dorsal root ganglion (DRG). This flexible electrode is aimed to conform to the DRG. The stimulation occurs through a transcutaneous electrical stimulation (TES) patch, which subsequently transmits the stimulation to the Injectrode via a subcutaneous metal collector. However, it is important to note that the effectiveness of stimulation through TES relies on the specific geometrical configurations of the Injectrode-collector-patch system. Hence, there is a need to investigate which design parameters influence the activation of targeted neural structures.Approach.We employed a hybrid computational modeling approach to analyze the impact of Injectrode system design parameters on charge delivery and neural response to stimulation. We constructed multiple finite element method models of DRG stimulation, followed by the implementation of multi-compartment models of DRG neurons. By calculating potential distribution during monopolar stimulation, we simulated neural responses using various parameters based on prior acute experiments. Additionally, we developed a canonical monopolar stimulation and full-scale model of bipolar bilateral L5 DRG stimulation, allowing us to investigate how design parameters like Injectrode size and orientation influenced neural activation thresholds.Main results.Our findings were in accordance with acute experimental measurements and indicate that the minimally invasive Injectrode system predominantly engages large-diameter afferents (Aß-fibers). These activation thresholds were contingent upon the surface area of the Injectrode. As the charge density decreased due to increasing surface area, there was a corresponding expansion in the stimulation amplitude range before triggering any pain-related mechanoreceptor (Aδ-fibers) activity.Significance.The Injectrode demonstrates potential as a viable technology for minimally invasive stimulation of the DRG. Our findings indicate that utilizing a larger surface area Injectrode enhances the therapeutic margin, effectively distinguishing the desired Aßactivation from the undesired Aδ-fiber activation.

Supraspinal control of motoneurons after paralysis enabled by spinal cord stimulation.

Spinal cord stimulation (SCS) restores motor control after spinal cord injury (SCI) and stroke. This evidence led to the hypothesis that SCS facilitates residual supraspinal inputs to spinal motoneurons. Instead, here we show that SCS does not facilitate residual supraspinal inputs but directly triggers motoneurons action potentials. However, supraspinal inputs can shape SCS-mediated activity, mimicking volitional control of motoneuron firing. Specifically, by combining simulations, intraspinal electrophysiology in monkeys and single motor unit recordings in humans with motor paralysis, we found that residual supraspinal inputs transform subthreshold SCS-induced excitatory postsynaptic potentials into suprathreshold events. We then demonstrated that only a restricted set of stimulation parameters enables volitional control of motoneuron firing and that lesion severity further restricts the set of effective parameters. Our results explain the facilitation of voluntary motor control during SCS while predicting the limitations of this neurotechnology in cases of severe loss of supraspinal axons.

Neuroprosthetic technology for individuals with spinal cord injury.

CONTEXT: Spinal cord injury (SCI) results in a loss of function and sensation below the level of the lesion. Neuroprosthetic technology has been developed to help restore motor and autonomic functions as well as to provide sensory feedback. FINDINGS: This paper provides an overview of neuroprosthetic technology that aims to address the priorities for functional restoration as defined by individuals with SCI. We describe neuroprostheses that are in various stages of preclinical development, clinical testing, and commercialization including functional electrical stimulators, epidural and intraspinal microstimulation, bladder neuroprosthesis, and cortical stimulation for restoring sensation. We also discuss neural recording technologies that may provide command or feedback signals for neuroprosthetic devices. CONCLUSION/CLINICAL RELEVANCE: Neuroprostheses have begun to address the priorities of individuals with SCI, although there remains room for improvement. In addition to continued technological improvements, closing the loop between the technology and the user may help provide intuitive device control with high levels of performance.

Assistive robotics should seamlessly integrate humans and robots.

Better integration of assistive robots with humans and adoption of a user-centric approach in their development will improve performance.

Using a high-frequency carrier does not improve comfort of transcutaneous spinal cord stimulation.

Objective.Spinal cord neuromodulation has gained much attention for demonstrating improved motor recovery in people with spinal cord injury, motivating the development of clinically applicable technologies. Among them, transcutaneous spinal cord stimulation (tSCS) is attractive because of its non-invasive profile. Many tSCS studies employ a high-frequency (10 kHz) carrier, which has been reported to reduce stimulation discomfort. However, these claims have come under scrutiny in recent years. The purpose of this study was to determine whether using a high-frequency carrier for tSCS is more comfortable at therapeutic amplitudes, which evoke posterior root-muscle (PRM) reflexes.Approach.In 16 neurologically intact participants, tSCS was delivered using a 1 ms long monophasic pulse with and without a high-frequency carrier. Stimulation amplitude and pulse duration were varied and PRM reflexes were recorded from the soleus, gastrocnemius, and tibialis anterior muscles. Participants rated their discomfort during stimulation from 0 to 10 at PRM reflex threshold.Main Results.At PRM reflex threshold, the addition of a high-frequency carrier (0.87 ± 0.2) was equally comfortable as conventional stimulation (1.03 ± 0.18) but required approximately double the charge to evoke the PRM reflex (conventional: 32.4 ± 9.2µC; high-frequency carrier: 62.5 ± 11.1µC). Strength-duration curves for tSCS with a high-frequency carrier had a rheobase that was 4.8× greater and a chronaxie that was 5.7× narrower than the conventional monophasic pulse, indicating that the addition of a high-frequency carrier makes stimulation less efficient in recruiting neural activity in spinal roots.Significance.Using a high-frequency carrier for tSCS is equally as comfortable and less efficient as conventional stimulation at amplitudes required to stimulate spinal dorsal roots.

Multi-enzyme catalysed processes using purified and whole-cell biocatalysts towards a 1,3,4-substituted tetrahydroisoquinoline.

In this work, two multi-enzyme catalysed processes to access a 1,3,4-substituted tetrahydroisoquinoline (THIQ), using either purified enzymes or lyophilised whole-cell catalysts, are presented. A key focus was the first step in which the reduction of 3-hydroxybenzoic acid (3-OH-BZ) into 3-hydroxybenzaldehyde (3-OH-BA) was catalysed by a carboxylate reductase (CAR) enzyme. Incorporation of the CAR-catalysed step enables substituted benzoic acids as the aromatic components, which can potentially be obtained from renewable resources by microbial cell factories. In this reduction, the implementation of an efficient cofactor regeneration system of both ATP and NADPH was crucial. Two different recycling approaches, either using purified enzymes or lyophilised whole-cells, were established and compared. Both of them showed high conversions of the acid into 3-OH-BA (>80%). However, the whole-cell system showed superior performance because it allowed the combination of the first and second steps into a one-pot cascade with excellent HPLC yields (>99%, enantiomeric excess (ee) ≥ 95%) producing the intermediate 3-hydroxyphenylacetylcarbinol. Moreover, enhanced substrate loads could be achieved compared to the system employing only purified enzymes. The third and fourth steps were performed in a sequential mode to avoid cross-reactivities and the formation of several side products. Thus, (1R,2S)-metaraminol could be formed with high HPLC yields (>90%, isomeric content (ic) ≥ 95%) applying either purified or whole-cell transaminases from Bacillus megaterium (BmTA) or Chromobacterium violaceum (Cv2025). Finally, the cyclisation step was performed using either a purified or lyophilised whole-cell norcoclaurine synthase variant from Thalictrum flavum (ΔTfNCS-A79I), leading to the formation of the target THIQ product with high HPLC yields (>90%, ic > 90%). As many of the educts applied are from renewable resources and a complex product with three chiral centers can be gained by only four highly selective steps, a very step- and atom efficient approach to stereoisomerically pure THIQ is shown.