RESUMO
Significance: We explore the feasibility of using time-domain (TD) and continuous-wave (CW) functional near-infrared spectroscopy (fNIRS) to monitor brain hemodynamic oscillations during resting-state activity in humans, a phenomenon that is of increasing interest in the scientific and medical community and appears to be crucial to advancing the understanding of both healthy and pathological brain functioning. Aim: Our general object is to maximize fNIRS sensitivity to brain resting-state oscillations. More specifically, we aim to define comprehensive guidelines for optimizing main operational parameters in fNIRS measurements [average photon count rate, measurement length, sampling frequency, and source-detector distance (SSD)]. In addition, we compare TD and CW fNIRS performance for the detection and localization of oscillations. Approach: A series of synthetic TD and CW fNIRS signals were generated by exploiting the solution of the diffusion equation for two different geometries of the probed medium: a homogeneous medium and a bilayer medium. Known and periodical perturbations of the concentrations of oxy- and deoxy-hemoglobin were imposed in the medium, determining changes in its optical properties. The homogeneous slab model was used to determine the effect of multiple measurement parameters on fNIRS sensitivity to oscillatory phenomena, and the bilayer model was used to evaluate and compare the abilities of TD and CW fNIRS in detecting and isolating oscillations occurring at different depths. For TD fNIRS, two approaches to enhance depth-selectivity were evaluated: first, a time-windowing of the photon distribution of time-of-flight was performed, and then, the time-dependent mean partial pathlength (TMPP) method was used to retrieve the hemoglobin concentrations in the medium. Results: In the homogeneous medium case, the sensitivity of TD and CW fNIRS to periodical perturbations of the optical properties increases proportionally with the average photon count rate, the measurement length, and the sampling frequency and approximatively with the square of the SSD. In the bilayer medium case, the time-windowing method can detect and correctly localize the presence of oscillatory components in the TD fNIRS signal, even in the presence of very low photon count rates. The TMPP method demonstrates how to correctly retrieve the periodical variation of hemoglobin at different depths from the TD fNIRS signal acquired at a single SSD. For CW fNIRS, measurements taken at typical SSDs used for short-separation channel regression show notable sensitivity to oscillations occurring in the deep layer, challenging the assumptions underlying this correction method when the focus is on analyzing oscillatory phenomena. Conclusions: We demonstrated that the TD fNIRS technique allows for the detection and depth-localization of periodical fluctuations of the hemoglobin concentrations within the probed medium using an acquisition at a single SSD, offering an alternative to multi-distance CW fNIRS setups. Moreover, we offered some valuable guidelines that can assist researchers in defining optimal experimental protocols for fNIRS studies.
RESUMO
[This corrects the article on p. 5994 in vol. 14, PMID: 38021143.].
RESUMO
Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.
Assuntos
Biotina , Fluoretos , Compostos de Enxofre , Espectrometria de Massas em Tandem , Biotina/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/metabolismoRESUMO
In this work, we used a hybrid time domain near-infrared spectroscopy (TD-NIRS) and diffuse correlation spectroscopy (DCS) device to retrieve hemoglobin and blood flow oscillations of skeletal muscle microvasculature. We focused on very low (VLF) and low-frequency (LF) oscillations (i.e., frequency lower than 0.145â Hz), that are related to myogenic, neurogenic and endothelial activities. We measured power spectral density (PSD) of blood flow and hemoglobin concentration in four muscles (thenar eminence, plantar fascia, sternocleidomastoid and forearm) of 14 healthy volunteers to highlight possible differences in microvascular hemodynamic oscillations. We observed larger PSDs for blood flow compared to hemoglobin concentration, in particular in case of distal muscles (i.e., thenar eminence and plantar fascia). Finally, we compared the PSDs measured on the thenar eminence of healthy subjects with the ones measured on a septic patient in the intensive care unit: lower power in the endothelial-dependent frequency band, and larger power in the myogenic ones were observed in the septic patient, in accordance with previous works based on laser doppler flowmetry.
RESUMO
In clinical scenarios, the use of biomedical sensors, devices and multi-parameter assessments is fundamental to provide a comprehensive portrait of patients' state, in order to adapt and personalize rehabilitation interventions and support clinical decision-making. However, there is a huge gap between the potential of the multidomain techniques available and the limited practical use that is made in the clinical scenario. This paper reviews the current state-of-the-art and provides insights into future directions of multi-domain instrumental approaches in the clinical assessment of patients involved in neuromotor rehabilitation. We also summarize the main achievements and challenges of using multi-domain approaches in the assessment of rehabilitation for various neurological disorders affecting motor functions. Our results showed that multi-domain approaches combine information and measurements from different tools and biological signals, such as kinematics, electromyography (EMG), electroencephalography (EEG), near-infrared spectroscopy (NIRS), and clinical scales, to provide a comprehensive and objective evaluation of patients' state and recovery. This multi-domain approach permits the progress of research in clinical and rehabilitative practice and the understanding of the pathophysiological changes occurring during and after rehabilitation. We discuss the potential benefits and limitations of multi-domain approaches for clinical decision-making, personalized therapy, and prognosis. We conclude by highlighting the need for more standardized methods, validation studies, and the integration of multi-domain approaches in clinical practice and research.
RESUMO
Significance: Continuous wave near infrared spectroscopy (CW-NIRS) is widely exploited in clinics to estimate skeletal muscles and brain cortex oxygenation. Spatially resolved spectroscopy (SRS) is generally implemented in commercial devices. However, SRS suffers from two main limitations: the a priori assumption on the spectral dependence of the reduced scattering coefficient [µs'(λ)] and the modeling of tissue as homogeneous. Aim: We studied the accuracy and robustness of SRS NIRS. We investigated the errors in retrieving hemodynamic parameters, in particular tissue oxygen saturation (StO2), when µs'(λ) was varied from expected values, and when layered tissue was considered. Approach: We simulated hemodynamic variations mimicking real-life scenarios for skeletal muscles. Simulations were performed by exploiting the analytical solutions of the photon diffusion equation in different geometries: (1) semi-infinite homogeneous medium and constant µs'(λ); (2) semi-infinite homogeneous medium and linear changes in µs'(λ); (3) two-layered media with a superficial thickness s1=5, 7.5, 10 mm and constant µs'(λ). All simulated data were obtained at source-detector distances ρ=35, 40, 45 mm, and analyzed with the SRS approach to derive hemodynamic parameters (concentration of oxygenated and deoxygenated hemoglobin, total hemoglobin concentration, and tissue oxygen saturation, StO2) and their relative error. Results: Variations in µs'(λ) affect the estimated StO2 (up to ±10%), especially if changes are different at the two wavelengths. However, the main limitation of the SRS method is the presence of a superficial layer: errors strongly larger than 20% were retrieved for the estimated StO2 when the superficial thickness exceeds 5 mm. Conclusions: These results highlight the need for more sophisticated strategies (e.g., the use of multiple short and long distances) to reduce the influence of superficial tissues in retrieving hemodynamic parameters and warn the SRS users to be aware of the intrinsic limitation of this approach, particularly when exploited in the clinical environment.
Assuntos
Oxigênio , Espectroscopia de Luz Próxima ao Infravermelho , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Encéfalo/diagnóstico por imagem , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/química , Hemoglobinas/análiseRESUMO
Time-domain diffuse correlation spectroscopy (TD-DCS) has been introduced as an advancement of the "classical" continuous wave DCS (CW-DCS) allowing one to not only to measure depth-resolved blood flow index (BFI) but also to extract optical properties of the measured medium without using any additional diffuse optics technique. However, this method is a photon-starved technique, specially when considering only the late photons that are of primary interest which has limited its in vivo application. In this work, we present a TD-DCS system based on a superconducting nanowire single-photon detector (SNSPD) with a high quantum efficiency, a narrow timing response, and a negligibly low dark count noise. We compared it to the typically used single-photon avalanche diode (SPAD) detector. In addition, this system allowed us to conduct fast in vivo measurements and obtain gated pulsatile BFI on the adult human forehead.
RESUMO
SIGNIFICANCE: Multi-laboratory initiatives are essential in performance assessment and standardization-crucial for bringing biophotonics to mature clinical use-to establish protocols and develop reference tissue phantoms that all will allow universal instrument comparison. AIM: The largest multi-laboratory comparison of performance assessment in near-infrared diffuse optics is presented, involving 28 instruments and 12 institutions on a total of eight experiments based on three consolidated protocols (BIP, MEDPHOT, and NEUROPT) as implemented on three kits of tissue phantoms. A total of 20 synthetic indicators were extracted from the dataset, some of them defined here anew. APPROACH: The exercise stems from the Innovative Training Network BitMap funded by the European Commission and expanded to include other European laboratories. A large variety of diffuse optics instruments were considered, based on different approaches (time domain/frequency domain/continuous wave), at various stages of maturity and designed for different applications (e.g., oximetry, spectroscopy, and imaging). RESULTS: This study highlights a substantial difference in hardware performances (e.g., nine decades in responsivity, four decades in dark count rate, and one decade in temporal resolution). Agreement in the estimates of homogeneous optical properties was within 12% of the median value for half of the systems, with a temporal stability of <5 % over 1 h, and day-to-day reproducibility of <3 % . Other tests encompassed linearity, crosstalk, uncertainty, and detection of optical inhomogeneities. CONCLUSIONS: This extensive multi-laboratory exercise provides a detailed assessment of near-infrared Diffuse optical instruments and can be used for reference grading. The dataset-available soon in an open data repository-can be evaluated in multiple ways, for instance, to compare different analysis tools or study the impact of hardware implementations.
Assuntos
Laboratórios , Óptica e Fotônica , Imagens de Fantasmas , Reprodutibilidade dos Testes , Análise EspectralRESUMO
Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the Cα-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds.
RESUMO
A high power setup for multichannel time-domain (TD) functional near infrared spectroscopy (fNIRS) measurements with high efficiency detection system was developed. It was fully characterized based on international performance assessment protocols for diffuse optics instruments, showing an improvement of the signal-to-noise ratio (SNR) with respect to previous analogue devices, and allowing acquisition of signals with sampling rate up to 20 Hz and source-detector distance up to 5 cm. A resting-state measurement on the motor cortex of a healthy volunteer was performed with an acquisition rate of 20 Hz at a 4 cm source-detector distance. The power spectrum for the cortical oxy- and deoxyhemoglobin is also provided.
Assuntos
Córtex Motor , Espectroscopia de Luz Próxima ao Infravermelho , Humanos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico/métodos , Razão Sinal-Ruído , Córtex Motor/diagnóstico por imagemRESUMO
The interest for Fused Deposition Modelling (FDM) in the field of Diffuse Optics (DO) is rapidly increasing. The most widespread FDM materials are polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS), thanks to their low cost and easiness-to-print. This is why, in this study, 3D printed samples of PLA and ABS materials were optically characterized in the range from the UV up to the IR wavelengths, in order to test their possible employment for probe construction in DO applications. To this purpose, measurements with Near Infrared Spectroscopy and Diffuse Correlation Spectroscopy techniques were considered. The results obtained show how the material employed for probe construction can negatively affect the quality of DO measurements.
Assuntos
Acrilonitrila , Butadienos , Elastômeros , Fenômenos Ópticos , Poliésteres , Impressão Tridimensional , Estirenos , Raios Infravermelhos , Espectroscopia de Luz Próxima ao Infravermelho , Raios UltravioletaRESUMO
We propose a quantitative and systematic investigation of the differential pathlength factor (DPF) behavior for skeletal muscles and its dependence on different factors, such as the subcutaneous adipose tissue thickness (ATT), the variations of the tissue absorption (µa ) and reduced scattering (µ's ) coefficients, and the source-detector distance. A time domain (TD) NIRS simulation study is performed in a two-layer geometry mimicking a human skeletal muscle with an overlying adipose tissue layer. The DPF decreases when µa increases, while it increases when µ's increases. Moreover, a positive correlation between DPF and ATT is found. These results are supported by an in-vivo TD NIRS study on vastus lateralis and biceps brachii muscles of eleven subjects at rest, showing a high inter-subject and inter-muscle variability.
RESUMO
Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.
RESUMO
In time-domain diffuse optical spectroscopy, the simultaneous acquisition of the time-of-flight distribution (DTOF) of photons traveling in a diffusive medium and of the instrument response function (IRF) is necessary to perform quantitative measurements of optical properties (absorption and reduced scattering coefficients) while taking into account the non-idealities of a real system (e.g. temporal resolution and time delays). The IRF acquisition can be a non-trivial and time-consuming operation that requires directly facing the injection and collection fibers. Since this operation is not always possible, a new IRF measurement scheme is here proposed where the IRF is acquired in reflectance geometry from a corrugate reflective surface. Validation measurements on a set of reference homogenous phantoms have been performed, resulting in an error in the optical properties estimation lower than 10% with respect to the typical IRF configuration. Thus, the proposed method proved to be a reliable approach that after a preliminary calibration can be exploited in a laboratory and clinical set-ups, leading to faster and more accurate measurements and reducing the operator-dependent performance.
RESUMO
Epoxidation chemistry often suffers from the challenging handling of peracids and thus requires in situ preparation. Here, we describe a two-phase enzymatic system that allows the effective generation of peracids and directly translate their activity to the epoxidation of olefins. We demonstrate the approach by application to lipid and olefin epoxidation as well as sulfide oxidation. These methods offer useful applications to synthetic modifications and scalable green processes.
Assuntos
Alcenos/química , Compostos de Epóxi/química , Lipídeos/química , Sulfetos/química , Estrutura Molecular , OxirreduçãoRESUMO
Large vessel occlusion (LVO) stroke might cause different degrees of hemodynamic impairment that affects microcirculation and contributes to metabolic derangement. Time-domain near-infrared spectroscopy (TD-NIRS) estimates the oxygenation of microcirculation of cerebral outer layers. We measure hemoglobin species and tissue oxygen saturation ( StO 2 ) of anterior circulation stroke patients, classified as LVO or lacunar, and assess the differences compared with controls and according to LVO recanalization status. Fiducial markers categorize the brain region below each TD-NIRS probe as ischemic or nonstroke areas. The study includes 47 consecutive acute ischemic stroke patients and 35 controls. The ischemic area has significantly higher deoxy-hemoglobin (HbR) and total hemoglobin (HbT) compared with controls in both recanalized and nonrecanalized patients but lower StO 2 only in recanalized patients. Recanalized patients have significantly lower mean StO 2 in the ipsilateral hemisphere compared with nonrecanalized patients. This is the first study to report TD-NIRS measurements in acute ischemic stroke patients. TD-NIRS is able to detect significant differences in hemoglobin species in LVO stroke compared with controls and according to recanalization status. This preliminary data might suggest that StO 2 can serve as a surrogate functional marker of the metabolic activity of rescued brain tissue.
RESUMO
Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles. The former are considered primary metabolites that are linear hydrocarbon acids, while the latter are complex aromatic or polyunsaturated secondary metabolites. Though the study of bacterial FAS has benefitted from decades of biochemical and structural investigations, type II PKSs have remained less understood. Here we review the recent approaches to understanding the protein-protein and protein-substrate interactions in these pathways, with an emphasis on recent chemical biology and structural applications. New approaches to the study of FAS have highlighted the critical role of the acyl carrier protein (ACP) with regard to how it stabilizes intermediates through sequestration and selectively delivers cargo to successive enzymes within these iterative pathways, utilizing protein-protein interactions to guide and organize enzymatic timing and specificity. Recent tools that have shown promise in FAS elucidation should find new approaches to studying type II PKS systems in the coming years.
Assuntos
Ácido Graxo Sintase Tipo II/metabolismo , Policetídeo Sintases/metabolismo , Mapas de Interação de Proteínas , Cristalografia por Raios X , Ciclização , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/genética , Helicobacter pylori/metabolismo , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Policetídeo Sintases/química , Policetídeo Sintases/genética , Proteínas/análise , Proteínas/química , Proteínas/genética , Especificidade por SubstratoRESUMO
Measurement of muscle oxidative metabolism is of interest for monitoring the training status in athletes and the rehabilitation process in patients. Time domain near infrared spectroscopy (TD NIRS) is an optical technique that allows the non-invasive measurement of the hemodynamic parameters in muscular tissue: concentrations of oxy- and deoxy-hemoglobin, total hemoglobin content, and tissue oxygen saturation. In this paper, we present a novel TD NIRS medical device for muscle oxidative metabolism. A custom-printed 3D probe, able to host optical elements for signal acquisition from muscle, was develop for TD NIRS in vivo measurements. The system was widely characterized on solid phantoms and during in vivo protocols on healthy subjects. In particular, we tested the in vivo repeatability of the measurements to quantify the error that we can have by repositioning the probe. Furthermore, we considered a series of acquisitions on different muscles that were not yet previously performed with this custom probe: a venous-arterial cuff occlusion of the arm muscle, a cycling exercise, and an isometric contraction of the vastus lateralis.
Assuntos
Espectroscopia de Luz Próxima ao Infravermelho , Exercício Físico , Humanos , Músculo Esquelético , Estresse Oxidativo , Oxigênio , Consumo de OxigênioRESUMO
The reproducibility of cerebral time-domain near-infrared spectroscopy (TD-NIRS) has not been investigated so far. Besides, reference intervals of cerebral optical properties, of absolute concentrations of deoxygenated-hemoglobin (HbR), oxygenated-hemoglobin (HbO), total hemoglobin (HbT) and tissue oxygen saturation (StO2) and their variability have not been reported. We have addressed these issues on a sample of 88 adult healthy subjects. TD-NIRS measurements at 690, 785, 830 nm were fitted with the diffusion model for semi-infinite homogenous media. Reproducibility, performed on 3 measurements at 5 minutes intervals, ranges from 1.8 to 6.9% for each of the hemoglobin species. The mean ± SD global values of HbR, HbO, HbT, StO2 are respectively 24 ± 7 µM, 33.3 ± 9.5 µM, 57.4 ± 15.8 µM, 58 ± 4.2%. StO2 displays the narrowest range of variability across brain regions.
RESUMO
We report the development of a compact probe for time-domain (TD) functional near-infrared spectroscopy (fNIRS) based on a fast silicon photomultiplier (SiPM) that can be put directly in contact with the sample without the need of optical fibers for light collection. We directly integrated an avalanche signal amplification stage close to the SiPM, thus reducing the size of the detection channel and optimizing the signal immunity to electromagnetic interferences. The whole detection electronics was placed in a plastic screw holder compatible with the electroencephalography standard cap for measurement on brain or with custom probe holders. The SiPM is inserted into a transparent and insulating resin to avoid the direct contact of the scalp with the 100-V bias voltage. The probe was integrated in an instrument for TD fNIRS spectroscopy. The system was characterized on tissue phantoms in terms of temporal resolution, responsivity, linearity, and capability to detect deep absorption changes. Preliminary in vivo tests on adult volunteers were performed to monitor hemodynamic changes in the arm during a cuff occlusion and in the brain cortex during a motor task.