Chemical composition analysis of Qingyan dropping pills using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

RATIONALE: This study developed a method for the rapid classification and identification of the chemical composition of Qingyan dropping pills (QDP) to provide the theoretical basis and data foundation for further in-depth research on the pharmacological substance basis of the formula and the selection of quality control indexes. METHODS: Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and data postprocessing technology were used to analyze the chemical composition of QDP. The fragmentation information on possible characteristic fragments and related neutral losses was summarized based on the literature and was compared with the MS data obtained from the assay, and thus a rapid classification and identification of chemical components in QDP could be achieved. RESULTS: A total of 73 compounds were identified, namely 24 flavonoids, 14 terpenoids, 30 organic acids and their esters, 3 alkaloids, and 2 phenylpropanoids. CONCLUSIONS: In this study, UHPLC-Q-TOF-MS and data postprocessing technology were used to realize the rapid classification and identification of the chemical constituents of QDP, which provided a comprehensive, efficient, and fast qualitative analysis method, a basis for further quality control and safe medication of QDP.

Probing functional conformation-state fluctuation dynamics in recognition binding between calmodulin and target peptide.

Conformational dynamics play a crucial role in protein functions. A molecular-level understanding of the conformational transition dynamics of proteins is fundamental for studying protein functions. Here, we report a study of real-time conformational dynamic interaction between calcium-activated calmodulin (CaM) and C28W peptide using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy and imaging. Plasma membrane Ca-ATPase protein interacts with CaM by its peptide segment that contains 28 amino acids (C28W). The interaction between CaM and the Ca-ATPase is essential for cell signaling. However, details about its dynamic interaction are still not clear. In our current study, we used Cyanine3 labeled CaM (N-domain) and Dylight 649 labeled C28W peptide (N-domain) to study the conformational dynamics during their interaction. In this study, the FRET can be measured when the CaM-C28W complex is formed and only be observed when such a complex is formed. By using single-molecule FRET efficiency trajectory and unique statistical approaches, we were able to observe multiple binding steps with detailed dynamic features of loosely bound and tightly bound state fluctuations. The C-domain of CaM tends to bind with C28W first with a higher affinity, followed by the binding of the CaM N-domain. Due to the comparatively high flexibility and low affinity of the N-domain and the presence of multiple anchor hydrophobic residues on the peptide, the N-domain binding may switch between selective and non-selective binding states, while the C-domain remains strongly bound with C28W. The results provide a mechanistic understanding of the CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics.

Optic Neuritis-Independent Retinal Atrophy in Neuromyelitis Optica Spectrum Disorder.

BACKGROUND: A limited number of studies have investigated the presence of ongoing disease activity independent of clinical relapses in neuromyelitis optica spectrum disorder (NMOSD), and data are conflicting. The objective of our study was to examine whether patients with aquaporin-4 (AQP4)-IgG seropositive NMOSD exhibit progressive retinal neuroaxonal loss, independently of optic neuritis (ON) attacks. METHODS: In this single-center, longitudinal study, 32 AQP4-IgG+ NMOSD patients and 48 healthy controls (HC) were followed with serial spectral-domain optical coherence tomography and visual acuity (VA) assessments. NMOSD patients with ON less than 6 months before baseline were excluded, whereas data from patients with ON during follow-up were censored at the last visit before ON. VA worsening was defined as a decrease in monocular letter acuity ≥5 letters for high-contrast VA and ≥7 letters for low-contrast VA. Analyses were performed with mixed-effects linear regression models adjusted for age, sex, and race. RESULTS: The median follow-up duration was 4.2 years (interquartile range: 1.8-7.5). Relative to HC, NMOSD eyes had faster peripapillary retinal nerve fiber layer (pRNFL) (ß = -0.25 µm/year faster, 95% confidence interval [CI]: -0.45 to -0.05, P = 0.014) and GCIPL thinning (ß = -0.09 µm/year faster, 95% CI: -0.17 to 0, P = 0.05). This difference seemed to be driven by faster pRNFL and GCIPL thinning in NMOSD eyes without a history of ON compared with HC (GCIPL: ß = -0.15 µm/year faster; P = 0.005; pRNFL: ß = -0.43 µm/year faster, P < 0.001), whereas rates of pRNFL (ß: -0.07 µm/year, P = 0.53) and GCIPL (ß = -0.01 µm/year, P = 0.90) thinning did not differ between NMOSD-ON and HC eyes. Nine NMOSD eyes had VA worsening during follow-up. CONCLUSIONS: In this longitudinal study, we observed progressive pRNFL and GCIPL atrophy in AQP4-IgG+ NMOSD eyes unaffected by ON. These results support that subclinical involvement of the anterior visual pathway may occur in AQP4-IgG+ NMOSD.

Conformational states and fluctuations in endothelial nitric oxide synthase under calmodulin regulation.

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.

Unsupervised MR harmonization by learning disentangled representations using information bottleneck theory.

In magnetic resonance (MR) imaging, a lack of standardization in acquisition often causes pulse sequence-based contrast variations in MR images from site to site, which impedes consistent measurements in automatic analyses. In this paper, we propose an unsupervised MR image harmonization approach, CALAMITI (Contrast Anatomy Learning and Analysis for MR Intensity Translation and Integration), which aims to alleviate contrast variations in multi-site MR imaging. Designed using information bottleneck theory, CALAMITI learns a globally disentangled latent space containing both anatomical and contrast information, which permits harmonization. In contrast to supervised harmonization methods, our approach does not need a sample population to be imaged across sites. Unlike traditional unsupervised harmonization approaches which often suffer from geometry shifts, CALAMITI better preserves anatomy by design. The proposed method is also able to adapt to a new testing site with a straightforward fine-tuning process. Experiments on MR images acquired from ten sites show that CALAMITI achieves superior performance compared with other harmonization approaches.

Evidence of subclinical quantitative retinal layer abnormalities in AQP4-IgG seropositive NMOSD.

BACKGROUND: Prior studies have suggested that subclinical retinal abnormalities may be present in aquaporin-4 immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (NMOSD), in the absence of a clinical history of optic neuritis (ON). OBJECTIVE: Our aim was to compare retinal layer thicknesses at the fovea and surrounding macula between AQP4-IgG+ NMOSD eyes without a history of ON (AQP4-nonON) and healthy controls (HC). METHODS: In this single-center cross-sectional study, 83 AQP4-nonON and 154 HC eyes were studied with spectral-domain optical coherence tomography (OCT). RESULTS: Total foveal thickness did not differ between AQP4-nonON and HC eyes. AQP4-nonON eyes exhibited lower outer nuclear layer (ONL) and inner photoreceptor segment (IS) thickness at the fovea (ONL: -4.01 ± 2.03 µm, p = 0.049; IS: -0.32 ± 0.14 µm, p = 0.029) and surrounding macula (ONL: -1.98 ± 0.95 µm, p = 0.037; IS: -0.16 ± 0.07 µm, p = 0.023), compared to HC. Macular retinal nerve fiber layer (RNFL: -1.34 ± 0.51 µm, p = 0.009) and ganglion cell + inner plexiform layer (GCIPL: -2.44 ± 0.93 µm, p = 0.009) thicknesses were also lower in AQP4-nonON compared to HC eyes. Results were similar in sensitivity analyses restricted to AQP4-IgG+ patients who had never experienced ON in either eye. CONCLUSIONS: AQP4-nonON eyes exhibit evidence of subclinical retinal ganglion cell neuronal and axonal loss, as well as structural evidence of photoreceptor layer involvement. These findings support that subclinical anterior visual pathway involvement may occur in AQP4-IgG+ NMOSD.

Automatic cerebellum anatomical parcellation using U-Net with locally constrained optimization.

The cerebellum plays a central role in sensory input, voluntary motor action, and many neuropsychological functions and is involved in many brain diseases and neurological disorders. Cerebellar parcellation from magnetic resonance images provides a way to study regional cerebellar atrophy and also provides an anatomical map for functional imaging. In a recent comparison, a multi-atlas approach proved to be superior to other parcellation methods including some based on convolutional neural networks (CNNs) which have a considerable speed advantage. In this work, we developed an alternative CNN design for cerebellar parcellation, yielding a method that achieves the leading performance to date. The proposed method was evaluated on multiple data sets to show its broad applicability, and a Singularity container has been made publicly available.

Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy.

Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure-function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2-amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions.

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics.

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.

Growth of colloidal PbS nanosheets and the enhancement of their photoluminescence.

Dual photoluminescence peaks observed during the synthesis of colloidal PbS nanosheets reveal their growth mechanism - two-dimensional attachments of the quantum dots. Well-grown nanosheets show the photoluminescence linewidth of 95 meV at room temperature. Aged nanosheets in toluene have enhanced photoluminescence with intensity improved by an order of magnitude.

Manipulating and probing enzymatic conformational fluctuations and enzyme-substrate interactions by single-molecule FRET-magnetic tweezers microscopy.

Enzyme-substrate interaction plays a critical role in enzymatic reactions, forming the active enzyme-substrate complex, the transition state ready to react. Studying the enzyme-substrate interaction will help in the ultimate molecular-level characterization of the enzymatic transition state that defines the reaction pathway, energetics, and the dynamics. In our initial effort to experimentally investigate the enzyme-substrate interactions and the related conformational fluctuations, we have developed a new approach to manipulate the enzymatic conformation and enzyme-substrate interaction at a single-molecule level by using a combined magnetic tweezers and simultaneous fluorescence resonance energy transfer (FRET) spectroscopic microscopy. By a repetitive pulling-releasing manipulation of a Cy3-Cy5 dye labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule under the conditions with and without enzymatic substrates, we have probed and analyzed the enzymatic conformational dynamics. Our results indicate that the enzyme conformational flexibility can be regulated by enzyme-substrate interactions: (1) enzyme at its conformation-perturbed state has less flexibility when binding substrates, and (2) substrate binding to enzyme significantly changes the enzyme conformational flexibility, an experimental evidence of so called entropy trapping in the enzyme-substrate reactive transition state. Furthermore, our results provide a significant experimental analysis of the folding-binding enzyme-substrate interactions, a dynamic nature of the enzymatic active transition state formation process.

Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease.

In mammalian cells, DNA ligase 1 (LIG1) functions as the primary DNA ligase in both genomic replication and single-strand break repair. Several reported mutations in human LIG1, including R305Q, R641L, and R771W, cause LIG1 syndrome, a primary immunodeficiency. While the R641L and R771W mutations, respectively located in the nucleotidyl transferase and oligonucleotide binding domains, have been biochemically characterized and shown to reduce catalytic efficiency, the recently reported R305Q mutation within the DNA binding domain (DBD) remains mechanistically unexplored. The R641L and R771W mutations are known to decrease the catalytic activity of LIG1 by affecting both interdomain interactions and DNA binding during catalysis, without significantly impacting overall DNA affinity. To elucidate the molecular basis of the LIG1 syndrome-causing R305Q mutation, we purified this single-residue mutant protein and investigated its secondary structure, protein stability, DNA binding affinity, and catalytic efficiency. Our findings reveal that the R305Q mutation significantly impairs the function of LIG1 by disrupting the DBD-DNA interactions, leading to a 7-21-fold lower DNA binding affinity and a 33-300-fold reduced catalytic efficiency of LIG1. Additionally, the R305Q mutation slightly decreases LIG1's protein stability by 2 to 3.6 °C, on par with the effect observed previously with either the R641L or R771W mutant. Collectively, our results uncover a new mechanism whereby the R305Q mutation impairs LIG1-catalyzed nicked DNA ligation, resulting in LIG1 syndrome, and highlight the crucial roles of the DBD-DNA interactions in tight DNA binding and efficient LIG1 catalysis.

Dynamics of Center of Pressure Trajectory in Gait: Unilateral Transfemoral Amputees Versus Non-Disabled Individuals.

The primary goal of rehabilitation for individuals with lower limb amputation, particularly those with unilateral transfemoral amputation (uTFA), is to restore their ability to walk independently. Effective control of the center of pressure (COP) during gait is vital for maintaining balance and stability, yet it poses a significant challenge for individuals with uTFA. This study aims to study the COP during gait in individuals with uTFA and elucidate their unique compensatory strategies. This study involved 12 uTFA participants and age-matched non-disabled controls, with gait and COP trajectory data collected using an instrumented treadmill. Gait and COP parameters between the control limb (CL), prosthetic limb (PL), and intact limb (IL) were compared. Notably, the mediolateral displacement of COP in PL exhibited significant lateral displacement compared to the CL from 30% to 60% of the stance. In 20% to 45% of the stance, the COP forward speed of PL was significantly higher than that of the IL. Furthermore, during the initial 20% of the stance, the vertical ground reaction force of PL was significantly lower than that of IL. Additionally, individuals with uTFA exhibited a distinct gait pattern with altered duration of loading response, single limb support, pre-swing and swing phases, and step time. These findings indicate the adaptability of individuals with uTFA in weight transfer, balance control, and pressure distribution on gait stability. In conclusion, this study provides valuable insights into the unique gait dynamics and balance strategies of uTFA patients, highlighting the importance of optimizing prosthetic design, alignment procedures, and rehabilitation programs to enhance gait patterns and reduce the risk of injuries due to compensatory movements.

Analysis of Genetic Diversity and Population Structure of Endemic Endangered Goose (Anser cygnoides) Breeds Based on Mitochondrial CYTB.

The analysis of the genetic diversity and historical dynamics of endemic endangered goose breeds structure has attracted great interest. Although various aspects of the goose breed structure have been elucidated, there is still insufficient research on the genetic basis of endemic endangered Chinese goose breeds. In this study, we collected blood samples from Lingxiang White (LX), Yan (YE), Yangjiang (YJ), Wuzong (WZ), Xupu (XP), and Baizi (BZ) geese (Anser cygnoides) and used Sanger sequencing to determine the partial sequence of the cytochrome b (CYTB) gene in a total of 180 geese. A total of 117 polymorphic sites were detected in the 707 bp sequence of the mtDNA CYTB gene after shearing and correction, accounting for approximately 16.55% of the entire sequence. The AT content (51.03%) of the processed sequence was slightly higher than the GC content (48.97%), indicating a preference for purine bases. The YJ, YE, and WZ breeds had the highest population genetic diversity, with a haplotype diversity greater than 0.9 (Hd > 0.9) and average population nucleotide difference of 8.01 (K > 8.01). A total of 81 haplotypes were detected and divided into six major branches. Among the six goose breeds, there were frequent genetic exchanges among LX, YJ, YE, and WZ geese (Nm > 15.00). We analyzed the distribution of base-mismatch differences in goose breeds and tested their historical dynamics for neutrality in Tajima's D and Fu's Fs. For YJ and WZ geese, Tajima's D > 0, but the difference was not significant (p > 0.05). The actual values for the two breeds exhibited multimodal Poisson distributions. The population patterns of the WZ and YJ geese are purportedly relatively stable, and the breeds have not experienced population expansions or bottleneck effects, which is consistent with the neutrality test results. This study provides new insights into the diverse genetic origins and historical dynamics that sustain endemic endangered goose breeds.

A proposed evidence-guided algorithm for the adjustment and optimization of multi-function articulated ankle-foot orthoses in the clinical setting.

Individuals with neuromuscular pathologies are often prescribed an ankle-foot orthosis (AFO) to improve their gait mechanics by decreasing pathological movements of the ankle and lower limb. AFOs can resist or assist excessive or absent muscular forces that lead to tripping, instability, and slow inefficient gait. However, selecting the appropriate AFO with mechanical characteristics, which limit pathological ankle motion in certain phases of the gait cycle while facilitating effective ankle movement during other phases, requires careful clinical decision-making. The aim of this study is to propose an explicit methodology for the adjustment of multi-function articulated AFOs in clinical settings. A secondary aim is to outline the evidence supporting this methodology and to identify gaps in the literature as potential areas for future research. An emerging class of AFO, the multi-function articulated AFO, offers features that permit more comprehensive, iterative, and reversible adjustments of AFO ankle alignment and resistance to ankle motion. However, no standard method exists for the application and optimization of these therapeutic devices in the clinical setting. Here we propose an evidence-guided methodology applicable to the adjustment of multi-function articulated AFOs in the clinical setting. Characteristic load-deflection curves are given to illustrate the idealized yet complex resistance-angle behavior of multi-function articulated AFOs. Research is cited to demonstrate how these mechanical characteristics can help mitigate specific pathologic ankle and knee kinematics and kinetics. Evidence is presented to support the effects of systematic adjustment of high resistance, alignable, articulated AFOs to address many typical pathomechanical patterns observed in individuals with neuromuscular disorders. The published evidence supporting most decision points of the algorithm is presented with identified gaps in the evidence. In addition, two hypothetical case examples are given to illustrate the application of the method in optimizing multi-function articulated AFOs for treating specific gait pathomechanics. This method is proposed as an evidence-guided systematic approach for the adjustment of multi-function articulated AFOs. It utilizes observed gait deviations mapped to specific changes in AFO alignment and resistance settings as a clinical tool in orthotic treatment for individuals with complex neuromuscular gait disorders.

Effects of 3D-printed ankle-foot orthoses on gait: a systematic review.

This systematic review aimed to explore comprehensive evidence on the efficacy of the 3D-printed ankle-foot orthoses (AFOs) on gait parameters in individuals with neuromuscular and/or musculoskeletal ankle impairments. Electronic databases including PubMed, Scopus, Web of Science, Embase, ProQuest, Cochrane, and EBSCOhost were searched from inception to August 2023. Ten studies that had participants with ankle impairments, as a result of stroke, cerebral palsy, mechanical trauma, muscle weakness, or Charcot-Marie-Tooth disease, investigated the immediate effects of the 3D-printed AFOs on gait parameters were included. Methodological rigor was evaluated using the modified Downs & Black index. The gait parameters included lower extremity joint angles, moments, and work/power, plantar pressures, spatiotemporal measures, and patient satisfaction were improved with the 3D-printed AFOs when compared to the no-AFO (i.e. barefoot, or shoe-only) conditions. 3D-printed AFOs revealed similar functional efficacy as conventional AFOs. Notably, the level of patient satisfaction regarding fitting and comfort was higher with the 3D-printed AFOs. Although the study on the effects of the 3D-printed AFOs are limited, emerging evidence indicates their effectiveness in improving gait biomechanics and functions. To further confirm their effects, rigorous randomized control studies with larger sample sizes and longer follow-ups on the effects are warranted in the future.

Transfemoral prosthetic simulators versus amputees: ground reaction forces and spatio-temporal parameters in gait.

This study aimed to compare the ground reaction forces (GRFs) and spatio-temporal parameters as well as their asymmetry ratios in gait between individuals wearing a transfemoral prosthetic simulator (TFSim) and individuals with unilateral transfemoral amputation (TFAmp) across a range of walking speeds (2.0-5.5 km h-1). The study recruited 10 non-disabled individuals using TFSim and 10 individuals with unilateral TFAmp using a transfemoral prosthesis. Data were collected using an instrumented treadmill with built-in force plates, and subsequently, the GRFs and spatio-temporal parameters, as well as their asymmetry ratios, were analysed. When comparing the TFSim and TFAmp groups, no significant differences were found among the gait parameters and asymmetry ratios of all tested metrics except the vertical GRFs. The TFSim may not realistically reproduce the vertical GRFs during the weight acceptance and push-off phases. The structural and functional variations in prosthetic limbs and components between the TFSim and TFAmp groups may be primary contributors to the difference in the vertical GRFs. These results suggest that TFSim might be able to emulate the gait of individuals with TFAmp regarding the majority of spatio-temporal and GRF parameters. However, the vertical GRFs of TFSim should be interpreted with caution.

Combined topographic, spectroscopic, and model analyses of inhomogeneous energetic coupling of linear light harvesting complex II aggregates in native photosynthetic membranes.

Light harvesting by LH1 and LH2 antenna proteins in the photosynthetic membranes of purple bacteria has been extensively studied in recent years for the fundamental understanding of the energy transfer dynamics and mechanism. Here we report the inhomogeneous structural organization of the LH2 complexes in photosynthetic membranes, giving evidence for the existence of energetically coupled linear LH2 aggregates in the native photosynthetic membranes of purple bacteria. Focusing on systematic model analyses, we combined AFM imaging and spectroscopic analysis with energetic coupling model analysis to characterize the inhomogeneous linear aggregation of LH2. Our AFM imaging results reveal that the LH2 complexes form linear aggregates with the monomer number varying from one to eight and each monomer tilted along the aggregated structure in photosynthetic membranes. The spectroscopic results support the attribution of aggregated LH2 complexes in the photosynthetic membranes, and the model calculation values for the absorption, emission and lifetime are consistent with the experimentally determined spectroscopic values, further proving a molecular-level understanding of the energetic coupling and energy transfer among the LH2 complexes in the photosynthetic membranes.

Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics.

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Coordination of Lower Limb During Gait in Individuals With Unilateral Transfemoral Amputation.

Understanding the lower-limb coordination of individuals with unilateral transfemoral amputation (uTFA) while walking is essential to understand their gait mechanisms. Continuous relative phase (CRP) analysis provides insights into gait coordination patterns of the neuromusculoskeletal system based on movement kinematics. Fourteen individuals with uTFA and their age-matched non-disabled individuals participated in this study. Kinematic data of the lower limbs of the participants were collected during walking. The joint angles, segment angles, and CRP values of the thigh-shank and shank-foot couplings were investigated. The curves among the lower limbs of the participants were compared using a statistical parametric mapping test. Compensatory strategies were found in the lower limbs from coordination patterns. In thigh-shank coupling, although distinct coordination traits in stance and swing phases among the lower limbs were found, the lower limbs in both groups were discovered to remain in a similar coordination pattern during gait. For individuals with uTFA, in shank-foot coupling, intact limbs demonstrated a short period of foot-leading pattern which was significantly different from that of the other limbs during mid-stance to compensate for the weaker force generation by prosthetic limbs. The findings offer normative coordination patterns on the walking of individuals with uTFA, which could benefit prosthetic gait rehabilitation and development.