Combined study of segmental movements and motion of the centre of mass during adaptation on a split-belt treadmill

BACKGROUND AND AIM: Walking on a split-belt treadmill (each of the two belts running at different speed) has been proposed as an experimental paradigm to investigate the flexibility of the neural control of gait and as a form of therapeutic exercise for hemi-paretic patients[1]. However the scarcity of dynamic investigations both for segmental aspects and for the entire body system, represented by the Centre of Mass (CoM), challenges the validity of the available findings on split-gait. Thus, the aim of the present study is to describe the dynamic adaptation of healthy subjects in terms of segmental and CoM motion, using Gait Analysis on Force Treadmill [2]. The study intends to clarify the effect of "split-gait", underlining its differences with pathologic claudication. METHODS: Ten healthy adults walked on a split-belt treadmill mounted on force sensors, with belts running either at the same speed ('Tied Condition', TC) or at different speeds ('Split Condition', SC, 0.4 vs 0.8 m/s). For the study of segmental motion, the surface Electromyography (sEMG), sagittal power and work provided by ankle, the main engine of body propulsion, were simultaneously recorded. For the study of the CoM motion, the Total Energy (Etot) and the percentage of Recovery (%R), the index of efficiency of the pendulum-like mechanism, were simultaneously analyzed. Various tied/split walking sequences were requested. The study was approved by the Local Ethic Committee. RESULTS: In the SC, the segmental motion analysis revealed a marked asymmetry between the two sides. The work provided by the ankle was 4.8 times higher (in the 0.4 vs 0.8 m/s conditions, respectively) compared with the slower side, and 1.2 times higher compared with the same speed in the TC (0.6 m/s) [3]. Paradoxically, the analysis of the CoM revealed an increased efficiency of the pendulum mechanism, with a higher %R in SC with respect to the TC at the same speed. CONCLUSIONS: Split gait entails its own pattern of locomotion, very different from pathologic claudication. The faster leg mimics the paretic limb temporally, but the unimpaired limb from the spatial and dynamic point of view[3]. This must be considered when a therapeutic application is designed. REFERENCES:1. Helm et al. Phys Med Rehabil Clin N Am. 2015;26(4):703-13. 2. Tesio et al. Am J Phys Med Rehabil. 2008;87(7):515-26. 3. Tesio et al. Int J Rehabil Res. 2018;41(4):304-315

Walking on a split-belt treadmill induces a higher power output and a shorter step length from the faster leg in healthy subjects, with opposite (after)-effect lasting less than 5 minutes after exercise

Walking on a split-belt treadmill has been claimed as a possible treatment of pathologic step asymmetries: in particular, the step lengthening on the affected side [1]. Placing the paretic limb on the slower belt would increase this asymmetry, reverting to long-lasting symmetry after exposure (after-effect). These studies neglected the underlying dynamics. Recently, it has been demonstrated that this paradigm entails an opposite spatial and dynamic asymmetry in healthy subjects. The stance on the faster belt is shortened, thus mimicking the paretic step temporally. On the contrary, the step is shorter and more muscle power is produced [2]. This challenges the rationale of the previous researches. The present study aims at extending these findings by investigating the after-effect both on spatiotemporal step parameters and power output from the plantar flexors on either belt. METHODS Ten healthy adults (21-34 years, 1.61-1.91 m tall, 5 women) participated in the study. After a brief familiarization, participants walked on a force-sensorized split-belt treadmill with one belt running at 0.4 m s-1 and the other belt running at 1.2 m s-1 (split condition) for 15 minutes and then, with no interruption, with the belts running at the same velocity (0.4 m s-1, tied condition) for other 5 minutes. The dominant lower limb was assigned to the faster belt. Kinematic data were recorded through an optoelectronic system as per the Davis anthropometric model. Joint sagittal power was computed by multiplying the moment generated by the ground reaction forces at the joints, times the rotation speed. All signals were simultaneously recorded [2]. The study was approved by the Local Ethics Committee. RESULTS Consistently with previous studies [3], during the split condition, the step length on the slower belt was longer, reaching gradually about 130% of the opposite step length. Ankle peak power attained about 15% of that observed on the opposite side. During the following tied condition, the step length on the formerly slower belt initially shortened by about 65% (after-effect), compared to the opposite step, and returned to values similar to that of the opposite side within 5 minutes. During this transition phase, ankle peak power gradually increased by up to 50% compared to baseline. On the formerly faster belt, step length did not change, while ankle peak power suddenly dropped to the contralateral level (Figure 1). Figure 1 Stride by stride plots (moving average, time-window 30 strides) of step length (upper panel) and ankle power (lower panel) from one representative subject (woman, 21 years, 1.65 m tall, body mass 60 kg) walking on a split-belt treadmill with the dominant lower limb on the faster belt (red) and the nondominant lower limb on the slower belt (blue). Strides from 1 to 867 refer to the split condition, and stride from 868 to 1025 refer to the following tied condition. DISCUSSION The increase in plantar flexor power on the faster belt, despite the shorter stance period and length, may reflect the priority need to counteract the backward drag from the faster belt, with respect to the slower one. This adaptation does not seem to lead to substantial learning, given that an after-effect, both on step length and ankle peak power, is only seen during the 5 minutes following split walking. In pathologic claudication, placing the affected lower limb on the faster belt might represent an effective form of \u201cforced-use\u201d [4], as far as enhanced power is requested. Long term effects remain questionable

High-throughput microfluidic platform for adherent single cells non-viral gene delivery

The widespread use of gene therapy as a therapeutic tool relies on the development of DNA-carrying vehicles devoid of any safety concerns. In contrast to viral vectors, non-viral gene carriers show promise in this perspective, although their low transfection efficiency leads to the necessity to carry out further optimizations. In order to overcome the limitations of traditional macroscale approaches, which mainly consist of time-consuming and simplified models, a microfluidic strategy has been developed to carry out transfection studies on single cells in a high-throughput and deterministic fashion. A single cell trapping mechanism has been implemented, based on the dynamic variation of fluidic resistances. For this purpose, we designed a round-shaped culture chamber integrated with a bottom trapping junction, which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for optimization of the single cell trapping efficacy. The optimized chamber layout was integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, and (ii) a serial dilution generator with chaotic mixing properties, able to deliver to the chambers both soluble factors and non-diffusive particles (i.e., polymer/DNA complexes, polyplexes) under spatio-temporally controlled chemical patterns. The devices were experimentally validated and allowed the trapping of individual human glioblastoma–astrocytoma epithelial-like cells (U87-MG) with a trapping efficacy of about 40%. The cells were cultured within the device and underwent preliminary transfection experiments using 25 kDa linear polyethylenimine (lPEI)-based polyplexes, confirming the potentiality of the proposed platform for the future high-throughput screening of gene delivery vectors and for the optimization of transfection protocols

Microfluidic Platform for Adherent Single Cell High-Throughput Screening

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Traditionally, in vitro investigations on biology and physiology of cells rely on averaging the responses eliciting from heterogeneous cell populations, thus being unsuitable for assessing individual cell behaviors in response to external stimulations. In the last years, great interest has thus been focused on single cell analysis and screening, which represents a promising tool aiming at pursuing the direct and deterministic control over cause-effect relationships guiding cell behavior. In this regard, a high-throughput microfluidic platform for trapping and culturing adherent single cells was presented. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. A round-shaped culture chamber (Φ=250μm, h=25μm) was conceived presenting two connections with a main fluidic path: (i) an upper wide opening, and (ii) a bottom trapping junction which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for the optimization of the single cell trapping efficacy. The optimized chamber layouts were integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, and (ii) a chaoticmixer based serial dilution generator for delivering both soluble factors and non-diffusive molecules under spatio-temporally controlled chemical patterns. The devices were experimentally validated and allowed for trapping individual U87-MG (human glioblastoma-astrocytoma epithelial-like) cells and culturing them up to 3 days

The path curvature of the body centre of mass during walking as an index of balance control in patients with Multiple Sclerosis

BACKGROUND AND AIM: The path curvature of the centre of mass (CM), mechanically representative of the whole body system, may provide hints to detection of fall risk during walking. Here, an example is taken from results of an ongoing controlled study. It shows the comparison between the CM path in a healthy subject and in a fully autonomous patient with Multiple Sclerosis (MS). METHODS: A representative healthy subject (woman, 26 years, 1.55 m tall) and a MS patient (woman, 34 years, 1.65 m tall, with very mild left hemiparesis) are presented. Subjects walked on a force-sensorized treadmill (1) at 0.6 m/s. Data were averaged across 6 subsequent strides. The 3D displacements of the CM were computed via double integration of the ground reaction forces (Cavagna's Method). The path curvature of the CM during one stride was computed according to the Frenet-Serret formula (2). The instantaneous efficiency of the kinetic-potential, pendulum-like energy transfer of the CM was also computed (percent recovery, R: 100%=complete recovery, i.e. fully passive CM translation) (3). RESULTS: The left and right panels refer to the control and the MS subject, respectively. In the upper set of panels the human sketches on top of the figure help identifying the stride phases (% cycle) and give a frontal and a sagittal perspectives. The first and second rows of curves from the top give the instantaneous R and the path curvature of the CM during one stride. Each step begins with the single stance of the front leg (R=right; L=left). The horizontal bars under the curves mark the double and the single stance phases (continuous and dashed lines, respectively; grey tract=left step). The lower set of panels (closed curves) gives the planar projections of the CM path during the same stride. The space-time correspondence between the 2 sets of curves is facilitated by the shared A-D labeling of peak curvatures and the shared graphic conventions (dashed line=single stance; gray tract=left step). In both steps the curvature is peaking when R suddenly drops from 100 to 0, demonstrating that the passive pendulum-like mechanism of translation is briskly substituted by a short lasting, fully muscle-driven, propulsion. The highest peaks (A and C) are coincident with the lateral redirection during single stance. Of note, the patient's CM path is characterized by a 10-fold higher C peak (single stance, paretic-to-unaffected side redirection). This may be interpreted as a feature of "escape" limp, barely perceivable by clinical observation, when seen from the perspective of the body CM on the horizontal plane (bottom curves). CONCLUSIONS: Increased curvature peaks may reveal the attempt to shorten the stance on the affected side yet, placing at risk the lateral stability of the body. REFERENCES:[1] Tesio L, Rota V, Am J Phys Med Rehabil 2008;87:515-526 [2] Tesio L et al, J Biomech 2011;44:732-740 [3] Cavagna GA, J Appl Physiol 1975;39:174-179

First Toxicological Analysis of the Pufferfish Sphoeroides pachygaster Collected in Italian Waters (Strait of Sicily): Role of Citizens Science in Monitoring Toxic Marine Species

Pufferfish (Tetraodontidae) inhabiting the Mediterranean Sea may represent an emerging public health risk due to the possible accumulation of marine neurotoxins such as tetrodotoxin (TTXs) and saxitoxin (STXs) in their tissues. In this study, the presence of pufferfish species in the Strait of Sicily (Lampedusa Island, Italy) was investigated using a citizen science (CS) approach, involving local fishermen. Samples (liver, intestine, gonads, muscle, skin) from 20 specimens were sent to the National Reference Laboratory on Marine Biotoxins for TTXs detection using a validated HILIC-MS/MS method on fish tissue. The presence of STXs was also screened in part of the specimens. Overall, 56 specimens identified as Sphoeroides pachygaster (Müller &Troschel, 1848) were collected. Data on their total length, body weight, fishing method and catch area (with relative depth temperature and salinity) were analyzed and compared with the S. pachygaster records reported in literature which were updated to 2022. All the analysed tissues were found to be negative for both TTXs and STXs. CS played an essential role in monitoring potentially toxic marine species in this investigation. Outcomes from this study, which is the first investigating S. pachygaster toxicity in Italian waters, may provide useful data for the proper assessment of this emerging risk

The Occurrence of Microplastics in Donax trunculus (Mollusca: Bivalvia) Collected along the Tuscany Coast (Mediterranean Sea)

Microplastics (MPs) (0.1 μm–5 mm particles) have been documented in oceans and seas. Bivalve molluscs (BMs) can accumulate MPs and transfer to humans through the food chain. BMs (especially mussels) are used to assess MPs’ contamination, but the genus Donax has not been thoroughly investigated. The aim of this study was to detect and characterize MPs in D. trunculus specimens collected along the Tuscan coast (Italy), and to assess the potential risk for consumers. The samples (~10 g of tissue and intervalval liquid from 35 specimens) were digested using a solution of 10% KOH, subjected to NaCl density separation, and filtered through 5 μm pore-size filters. All items were morphologically classified and measured, and their mean abundance (MA) was calculated. Furthermore, 20% of them were analyzed by Raman spectroscopy and, based on the obtained results, the MA was recalculated (corrected MA) and the annual human exposure was estimated. In the 39 samples analyzed, 85 items fibers (n = 45; 52.94%) and fragments (n = 40; 47.06%) were found. The MA was 0.23 ± 0.17 items/grww. Additionally, 83.33% of the items were confirmed as MPs (polyethylene and polyethylene terephthalate). Based on the correct MA (0.18 MPs/grww), D. trunculus consumers could be exposed to 19.2 MPs/per capita/year. The health risk level of MPs was classified as level III (moderate)

Measuring voluntary activation of the quadriceps femoris during isokinetic concentric contractions

BACKGROUND: It is known that Voluntary Activation (VA) of muscles may be lower during isokinetic concentric (CON) contractions than during isometric (ISOM) contractions, and that it may be further decreased in various motor impairments. OBJECTIVE: The aim of this study was to validate the Interpolated Twitch Technique (ITT) for quantifying VA of the Quadriceps femoris during CON contractions (knee extension at 60 and 120_/s, CON60 and CON120, respectively). METHODS: Pairs of electrical stimuli were delivered to the Quadriceps femoris of twenty-two healthy subjects at 50_ of knee flexion. Participants were instructed to exert five different levels of effort in decreasing order between 100% and 20% maximal voluntary efforts in ISOM and CON contractions. RESULTS: Through a linear regression model, a significant relationship between measures of VA and moment was observed for all the three contraction conditions: slopes (95% confidence intervals) = 1.04 (0.98\u20131.11), 0.96 (0.89\u20131.02) and 0.84 (0.78\u20130.91); intercepts = - 5.22 (\u2013 8.61\u2013 \u20131.83), 4.16 (0.71\u20137.62) and 14.58 (10.76\u201318.39), for ISOM, CON60 and CON120 contractions, respectively. This supported the validity of the method. CONCLUSIONS: It is concluded that ITT can be a valid method for measuring VA during CON contractions, potentially useful both in sports and rehabilitation studies

Limping on split-belt treadmills implies opposite kinematic and dynamic lower limb asymmetries

Walking on a split-belt treadmill (each of the two belts running at a different speed) has been proposed as an experimental paradigm to investigate the flexibility of the neural control of gait and as a form of therapeutic exercise. However, the scarcity of dynamic investigations challenges the validity of the available findings. The aim of the present study was to investigate the dynamic asymmetries of lower limbs of healthy adults during adaptation to gait on a split-belt treadmill. Ten healthy adults walked on a split-belt treadmill mounted on force sensors, with belts running either at the same speed ('tied' condition) or at different speeds ('split' condition, 0.4 vs. 0.8 or 0.8 vs. 1.2\u2009m/s). The sagittal power and work provided by ankle, knee and hip joints, joint rotations, muscle lengthening, and surface electromyography were recorded simultaneously. Various tied/split walking sequences were requested. In the split condition a marked asymmetry between the parameters recorded from each of the two lower limbs, in particular from the ankle joint, was recorded. The work provided by the ankle (the main engine of body propulsion) was 4.8 and 2.2 times higher (in the 0.4 vs. 0.8, and 0.8 vs. 1.2\u2009m/s conditions, respectively) compared with the slower side, and 1.2 and 1.1 times higher compared with the same speed in the tied condition. Compared with overground gait in hemiplegia, split gait entails an opposite spatial and dynamic asymmetry. The faster leg mimics the paretic limb temporally, but the unimpaired limb from the spatial and dynamic point of view. These differences challenge the proposed protocols of split gait as forms of therapeutic exercise

Crouch gait can be an effective form of forced-use/no constraint exercise for the paretic lower limb in stroke

In hemiplegic gait the paretic lower limb provides less muscle power and shows a briefer stance compared with the unaffected limb. Yet, a longer stance and a higher power can be obtained from the paretic lower limb if gait speed is increased. This supports the existence of a 'learned non-use' phenomenon, similar to that underlying some asymmetric impairments of the motion of the eyes and of the upper limbs. Crouch gait (CG) (bent-hip bent-knee, about 30 degrees minimum knee flexion) might be an effective form of 'forced-use' treatment of the paretic lower limb. It is not known whether it also stimulates a more symmetric muscle power output. Gait analysis on a force treadmill was carried out in 12 healthy adults and seven hemiplegic patients (1-127 months after stroke, median: 1.6). Speed was imposed at 0.3 m/s. Step length and single and double stance times, sagittal joint rotations, peak positive power, and work in extension of the hip, knee, and ankle (plantar flexion), and surface electromyography (sEMG) area from extensor muscles during the generation of power were measured on either side during both erect and crouch walking. Significance was set at P less than 0.05; corrections for multiplicity were applied. Patients, compared with healthy controls, adopted in both gait modalities and on both sides a shorter step length (61-84%) as well as a shorter stance (76-90%) and swing (63-83%) time. As a rule, they also provided a higher muscular work (median: 137%, range: 77-250%) paralleled by a greater sEMG area (median: 174%, range: 75-185%). In erect gait, the generation of peak extensor power across hip, knee, and ankle joints was in general lower (83-90%) from the paretic limb and higher (98-165%) from the unaffected limb compared with control values. In CG, peak power generation across the three lower limb joints was invariably higher in hemiparetic patients: 107-177% from the paretic limb and 114-231% from the unaffected limb. When gait shifted from erect to crouch, only for hemiplegic patients, at the hip, the paretic/unaffected ratio increased significantly. For peak power, work, sEMG area, and joint rotation, the paretic/unaffected ratio increased from 55 to 85%, 56 to 72%, 68 to 91%, and 67 to 93%, respectively. CG appears to be an effective form of forced-use exercise eliciting more power and work from the paretic lower limb muscles sustained by a greater neural drive. It also seems effective in forcing a more symmetric power and work from the hip extensor muscles, but neither from the knee nor the ankle