Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

Integration of diffusion tensor imaging parameters with mesh morphing for in-depth analysis of brain white matter fibre tracts.

Averaging is commonly used for data reduction/aggregation to analyse high-dimensional MRI data, but this often leads to information loss. To address this issue, we developed a novel technique that integrates diffusion tensor metrics along the whole volume of the fibre bundle using a 3D mesh-morphing technique coupled with principal component analysis for delineating case and control groups. Brain diffusion tensor MRI scans of high school rugby union players (n = 30, age 16-18) were acquired on a 3 T MRI before and after the sports season. A non-contact sport athlete cohort with matching demographics (n = 12) was also scanned. The utility of the new method in detecting differences in diffusion tensor metrics of the right corticospinal tract between contact and non-contact sport athletes was explored. The first step was to run automated tractography on each subject's native space. A template model of the right corticospinal tract was generated and morphed into each subject's native shape and space, matching individual geometry and diffusion metric distributions with minimal information loss. The common dimension of the 20 480 diffusion metrics allowed further data aggregation using principal component analysis to cluster the case and control groups as well as visualization of diffusion metric statistics (mean, ±2 SD). Our approach of analysing the whole volume of white matter tracts led to a clear delineation between the rugby and control cohort, which was not possible with the traditional averaging method. Moreover, our approach accounts for the individual subject's variations in diffusion tensor metrics to visualize group differences in quantitative MR data. This approach may benefit future prediction models based on other quantitative MRI methods.

Biomechanics of a collum-fixated short stem in total hip arthroplasty.

Background: Biomechanical reconstruction of the hip significantly impacts the clinical outcome and implant survival. Our knowledge is limited of the ability of neck-stabilised prostheses to restore hip biomechanics. We hypothesised that hip biomechanics, specifically leg length and global offset (GO), may be restored to an acceptable range using the Primoris™ stem. Methods and material: In this retrospective study, we analysed 152 patients who underwent total hip replacement (THA) using the short collum-fixated stem Primoris™.The primary outcomes were hip parameters measured by x-ray following THA using the Primoris™ stem. After surgery, the biomechanical parameters used were measured at the arthroplasty and the native contralateral side of the same x-ray. The X-rays were taken one year after the patient's surgery.1. GO.2. Leg length discrepancy (LLD).3. Neck shaft angle (NSA). Results: We recorded an average GO of -3.4 mm (standard deviation (SD) 7.2) and an average LLD of +3.8 mm (SD 6.4). Furthermore, we registered an average 14-degree NSA increase (SD 7.4). Conclusion: The Primoris™ neck-stabilised stem enabled hip anatomy restoration to a favourable range with respect to GO and LLD as the average difference fell within ±5 mm. However, the stem tended to be implanted in valgus.

A Wearable Open-Source electrical impedance tomography device.

Electrical impedance tomography (EIT) is medical imaging technique in which small electrical signals are used to map the electrical impedance distribution within the body. It is safe and non-invasive, which make it attractive for use in continuous monitoring or outpatient applications, but the high cost of commercial devices is an impediment to its adoption. Over the last 10 years, many research groups have developed their own EIT devices, but few designs for open-source EIT hardware are available. In this work, we present a complete open-source EIT system that is designed to be suitable for monitoring the lungs of free breathing subjects. The device is low-cost, wearable, and is designed to comply with the industry accepted safety standard for EIT. The device has been tested in two regimes: Firstly in terms of measurement uncertainty as a voltage measurement system, and secondly against a set of measures that have been proposed specifically for EIT hardware. The voltage measurement uncertainty of the device was measured to be - 0.7 % ± 0.36 mV. The EIT specific performance was measured in a phantom test designed to be as physiologically representative as practicable, and the device performed similarly to other published devices. This work will contribute to increased accessibility of EIT for study and will contribute to consensus on testing methodology for EIT devices.

Hit-to-Lead Identification and Validation of a Triaromatic Pleuromutilin Antibiotic Candidate.

Herein, we report the hit-to-lead identification of a drug-like pleuromutilin conjugate 16, based on a triaromatic hit reported in 2020. The lead arose as the clear candidate from a hit-optimization campaign in which Gram-positive antibacterial activity, solubility, and P-gp affinity were optimized. Conjugate 16 was extensively evaluated for its in vitro ADMET performance which, apart from solubility, was overall on par with lefamulin. This evaluation included Caco-2 cell permeability, plasma protein binding, hERG inhibition, cytotoxicity, metabolism in microsomes and CYP3A4, resistance induction, and time-kill kinetics. Intravenous pharmacokinetics of 16 proved satisfactory in both mice and pigs; however, oral bioavailability was limited likely due to insufficient solubility. The in vivo efficacy was evaluated in mice, systemically infected with Staphylococcus aureus, where 16 showed rapid reduction in blood bacteriaemia. Through our comprehensive studies, lead 16 has emerged as a highly promising and safe antibiotic candidate for the treatment of Gram-positive bacterial infections.

Stable fixation of an ultra-short femoral neck-preserving hip prosthesis: a 5-year RSA, DXA, and clinical prospective outcome study of 48 patients.

BACKGROUND AND PURPOSE: We previously showed promising primary stability and preservation of bone stock with the ultra-short neck-loading hip implant in total hip arthroplasty (THA). The aim of this study was to evaluate clinical outcome, implant stability, and bone mineral density (BMD). METHODS: 50 patients were treated with the ultra-short neck Primoris hip implant at baseline and 48 were available for evaluation at 5-year follow-up. 5 different patient-reported outcome measures (PROMs) including hip-specific scores, disease-specific and generic quality of life outcome measures, and an activity score were used. Furthermore, implant stability using radiostereometric analysis (RSA) and assessment of periprosthetic BMD using dual-energy X-ray absorptiometry (DXA) were applied. RESULTS: By 1-year follow-up, all PROMs showed improvements and remained high at 5-year follow-up. After initial distal translation (subsidence) and negative rotation around the z-axis (varus tilt) the implant showed stable fixation at 5-year follow-up with no further migration beyond 12 months. In the regions of interest (ROI) 3 and 4, BMD remained stable. In ROI 2, further bone loss of 12% was found at 5-year follow-up. CONCLUSION: Clinical outcome including PROMs was satisfying throughout the 5-year follow-up period. The hip implant remains stable with both bone preservation and loss 5 years after surgery.

100 ancient genomes show repeated population turnovers in Neolithic Denmark.

Major migration events in Holocene Eurasia have been characterized genetically at broad regional scales1-4. However, insights into the population dynamics in the contact zones are hampered by a lack of ancient genomic data sampled at high spatiotemporal resolution5-7. Here, to address this, we analysed shotgun-sequenced genomes from 100 skeletons spanning 7,300 years of the Mesolithic period, Neolithic period and Early Bronze Age in Denmark and integrated these with proxies for diet (13C and 15N content), mobility (87Sr/86Sr ratio) and vegetation cover (pollen). We observe that Danish Mesolithic individuals of the Maglemose, Kongemose and Ertebølle cultures form a distinct genetic cluster related to other Western European hunter-gatherers. Despite shifts in material culture they displayed genetic homogeneity from around 10,500 to 5,900 calibrated years before present, when Neolithic farmers with Anatolian-derived ancestry arrived. Although the Neolithic transition was delayed by more than a millennium relative to Central Europe, it was very abrupt and resulted in a population turnover with limited genetic contribution from local hunter-gatherers. The succeeding Neolithic population, associated with the Funnel Beaker culture, persisted for only about 1,000 years before immigrants with eastern Steppe-derived ancestry arrived. This second and equally rapid population replacement gave rise to the Single Grave culture with an ancestry profile more similar to present-day Danes. In our multiproxy dataset, these major demographic events are manifested as parallel shifts in genotype, phenotype, diet and land use.

Population genomics of post-glacial western Eurasia.

Western Eurasia witnessed several large-scale human migrations during the Holocene1-5. Here, to investigate the cross-continental effects of these migrations, we shotgun-sequenced 317 genomes-mainly from the Mesolithic and Neolithic periods-from across northern and western Eurasia. These were imputed alongside published data to obtain diploid genotypes from more than 1,600 ancient humans. Our analyses revealed a 'great divide' genomic boundary extending from the Black Sea to the Baltic. Mesolithic hunter-gatherers were highly genetically differentiated east and west of this zone, and the effect of the neolithization was equally disparate. Large-scale ancestry shifts occurred in the west as farming was introduced, including near-total replacement of hunter-gatherers in many areas, whereas no substantial ancestry shifts happened east of the zone during the same period. Similarly, relatedness decreased in the west from the Neolithic transition onwards, whereas, east of the Urals, relatedness remained high until around 4,000 BP, consistent with the persistence of localized groups of hunter-gatherers. The boundary dissolved when Yamnaya-related ancestry spread across western Eurasia around 5,000 BP, resulting in a second major turnover that reached most parts of Europe within a 1,000-year span. The genetic origin and fate of the Yamnaya have remained elusive, but we show that hunter-gatherers from the Middle Don region contributed ancestry to them. Yamnaya groups later admixed with individuals associated with the Globular Amphora culture before expanding into Europe. Similar turnovers occurred in western Siberia, where we report new genomic data from a 'Neolithic steppe' cline spanning the Siberian forest steppe to Lake Baikal. These prehistoric migrations had profound and lasting effects on the genetic diversity of Eurasian populations.

A pneumatic reconfigurable socket for transtibial amputees.

Many transtibial amputees rate the fit between their residual limb and prosthetic socket as the most critical factor in satisfaction with using their prosthesis. This study aims to address the issue of prosthetic socket fit by reconfiguring the socket shape at the interface of the residual limb and socket. The proposed reconfigurable socket shifts pressure from sensitive areas and compensates for residual limb volume fluctuations, the most important factors in determining a good socket fit. Computed tomography scan images are employed to create the phantom limb of an amputee and to manufacture the reconfigurable socket. The performance of the reconfigurable socket was evaluated both experimentally and numerically using finite element modelling. The study showed that the reconfigurable socket can reduce interface pressure at targeted areas by up to 61%.

A novel two-dimensional phantom for electrical impedance tomography using 3D printing.

Electrical impedance tomography (EIT) is an imaging method that can be used to image electrical impedance contrasts within various tissues of the body. To support development of EIT measurement systems, a phantom is required that represents the electrical characteristics of the imaging domain. No existing type of EIT phantom combines good performance in all three characteristics of resistivity resolution, spatial resolution, and stability. Here, a novel EIT phantom concept is proposed that uses 3D printed conductive material. Resistivity is controlled using the 3D printing infill percentage parameter, allowing arbitrary resistivity contrasts within the domain to be manufactured automatically. The concept of controlling resistivity through infill percentage is validated, and the manufacturing accuracy is quantified. A method for making electrical connections to the 3D printed material is developed. Finally, a prototype phantom is printed, and a sample EIT analysis is performed. The resulting phantom, printed with an Ultimaker S3, has high reported spatial resolution of 6.9 µm, 6.9 µm, and 2.5 µm for X, Y, and Z axis directions, respectively (X and Y being the horizontal axes, and Z the vertical). The number of resistivity levels that are manufacturable by varying infill percentage is 15 (calculated by dividing the available range of resistivities by two times the standard deviation of the manufacturing accuracy). This phantom construction technique will allow assessment of the performance of EIT devices under realistic physiological scenarios.

Quantifying changes in shoulder orientation between the prone and supine positions from magnetic resonance imaging.

BACKGROUND: Predicting breast tissue motion using biomechanical models can provide navigational guidance during breast cancer treatment procedures. These models typically do not account for changes in posture between procedures. Difference in shoulder position can alter the shape of the pectoral muscles and breast. A greater understanding of the differences in the shoulder orientation between prone and supine could improve the accuracy of breast biomechanical models. METHODS: 19 landmarks were placed on the sternum, clavicle, scapula, and humerus of the shoulder girdle in prone and supine breast MRIs (N = 10). These landmarks were used in an optimization framework to fit subject-specific skeletal models and compare joint angles of the shoulder girdle between these positions. FINDINGS: The mean Euclidean distance between joint locations from the fitted skeletal model and the manually identified joint locations was 15.7 mm ± 2.7 mm. Significant differences were observed between prone and supine. Compared to supine position, the shoulder girdle in the prone position had the lateral end of the clavicle in more anterior translation (i.e., scapula more protracted) (P < 0.05), the scapula in more protraction (P < 0.01), the scapula in more upward rotation (associated with humerus elevation) (P < 0.05); and the humerus more elevated (P < 0.05) for both the left and right sides. INTERPRETATION: Shoulder girdle orientation was found to be different between prone and supine. These differences would affect the shape of multiple pectoral muscles, which would affect breast shape and the accuracy of biomechanical models.

Toward Development of a Portable System for 3D Fluorescence Lymphography.

Lymphoedema is a debilitating disease that results in chronic swelling of a body region due to a dysfunctional lymphatic system. Since a cure is yet to be identified for this disease, management is currently the best option for preventing disease progression and improving patient outcomes. Fluorescence lymphography is a popular approach for mapping the lymphatic vessels to provide information about the underlying lymphatic dysfunction. However, current clinical fluorescence lymphography tools do not enable the creation of comprehensive 3D maps of lymphatics throughout affected limbs. This work presents the development toward multi-camera 3D reconstruction with fluorescence imaging to overcome the current limitations in clinical tools. Pilot studies have been performed that identify suitable instrumentation for this multi-camera approach and techniques for creating a 3D fluorescence lymphography device are discussed.Clinical Relevance- This paper presents development toward new low-cost and portable clinical tools for lymphoedema diagnosis and to facilitate personalised treatment and self-management of this disease.

Measurement of Blood Dilution during Lancet-Free Blood Sampling.

In this paper, we report on a fluorescent and colorimetric system for measuring the dilution of capillary blood released by a needle-free jet injector. Jet injection uses a high-speed liquid jet to penetrate tissue, and in the process can release capillary blood that can be collected for performing blood tests. In this way, blood sampling can be performed without the use of a lancet. However, any injectate that mixes with the collected blood dilutes the sample and may significantly impact subsequent analyses. By adding the fluorescent marker indocyanine green to the injected liquid, the fraction of injectate mixed into the collected blood can be measured. The incorporation of colorimetry allows our system to also correct for the impact of hematocrit on fluorescence. The results from this system show that it can determine the dilution of blood that has been diluted by up to 10 %, the upper limit of dilution typically observed in lancet-free blood sampling via jet injection.Clinical Relevance- Blood samples can be collected by jet injection without significant dilution, avoiding the need for lancing.

An Instrument for High-throughput Testing of Heart Tissue In Vitro.

Cardiac trabeculae are small samples of heart muscle tissue that can be dissected and studied in vitro to better understand the underlying physiology of cardiac muscle. However, instruments for such experimentation often (1) involve delicate mounting of the muscle, (2) constrain investigations to one muscle at a time and, thus, (3) cannot retain the muscle in the same experimental configuration for post-experimental assessment including imaging analysis. Here, we present a novel device that allows trabeculae to be secured by a visible-light photo-initiated hydrogel, manipulated via a force sensor, and stimulated while being imaged. We use our robust, accurate image registration techniques to measure cantilever and gel deformation during trabecula contraction and thereby provide a measure of trabecula force production during twitches. A variety of experiments can then be conducted, with the potential for the trabecula to be fixed in place using hydrogel for further post-experiment analysis, as well as longitudinal evaluation. The device has multiple wells making it amenable to high-throughput testing.Clinical Relevance- These methods may allow longitudinal and high-throughput studies of cardiac tissue samples in health and disease.

Non-contact quantification of aortic stenosis and mitral regurgitation using carotid waveforms from skin displacements.

Objective. Early diagnosis of heart problems is essential for improving patient prognosis.Approach. We created a non-contact imaging system that calculates the vessel-induced deformation of the skin to estimate the carotid artery pressure displacement waveforms. We present a clinical study of the system in patients (n= 27) with no underlying condition, aortic stenosis (AS), or mitral regurgitation (MR).Main results. Displacement waveforms were compared to aortic catheter pressures in the same patients. The morphologies of the pressure and displacement waveforms were found to be similar, and pulse wave analysis metrics, such as our modified reflection indices (RI) and waveform duration proportions, showed no significant differences. Compared with the control group, AS patients displayed a greater proportion of time to peak (p= 0.026 andp= 0.047 for catheter and displacement, respectively), whereas augmentation index (AIx)was greater for the displacement waveform only (p= 0.030). The modified RI for MR (p= 0.047 andp= 0.004 for catheter and displacement, respectively) was lower than in the controls. AS and MR were also significantly different for the proportion of time to peak (p= 0.018 for the catheter measurements), RI (p= 0.045 andp= 0.002 for the catheter and displacement, respectively), and AIx (p= 0.005 for the displacement waveform).Significance. These findings demonstrate the ability of our system to provide insights into cardiac conditions and support further development as a diagnostic/telehealth-based screening tool.

Vacuum-Assisted Needle-Free Capillary Blood Sampling.

BACKGROUND: Poor glycemic management persists among people practicing insulin therapy in relation to type 1 and 2 diabetes despite a clear relationship with negative health outcomes. Skin penetration by jet injection has recently been shown as a viable method for inducing blood release from fingertips. This study examines the use of vacuum to enhance the volume of blood released and quantifies any dilution of the collected blood. METHODS: A single-blind crossover study involving 15 participants, each receiving four different interventions, was conducted wherein each participant served as their own control. Each participant experienced fingertip lancing and fingertip jet injection, both with and without applied vacuum. Participants were divided into three equal groups to explore different vacuum pressures. RESULTS: This study found that glucose concentration in blood collected under vacuum following jet injection and lancing were equivalent. We found that applying a 40 kPa vacuum following jet injection produced a 35-fold increase in the collected volume. We determined the limited extent to which the injectate dilutes blood collected following jet injection. The mean dilution of blood collected by jet injection was 5.5%. We show that jet injection is as acceptable to patients as lancing, while being equally suited for conducting glucose measurements. CONCLUSIONS: Vacuum significantly enhances the volume of capillary blood released from the fingertip without any difference in pain. The blood collected by jet injection with vacuum is equivalent to that from lancing for glucose measurement purposes.

On lightmyography based muscle-machine interfaces for the efficient decoding of human gestures and forces.

Conventional muscle-machine interfaces like Electromyography (EMG), have significant drawbacks, such as crosstalk, a non-linear relationship between the signal and the corresponding motion, and increased signal processing requirements. In this work, we introduce a new muscle-machine interfacing technique called lightmyography (LMG), that can be used to efficiently decode human hand gestures, motion, and forces from the detected contractions of the human muscles. LMG utilizes light propagation through elastic media and human tissue, measuring changes in light luminosity to detect muscle movement. Similar to forcemyography, LMG infers muscular contractions through tissue deformation and skin displacements. In this study, we look at how different characteristics of the light source and silicone medium affect the performance of LMG and we compare LMG and EMG based gesture decoding using various machine learning techniques. To do that, we design an armband equipped with five LMG modules, and we use it to collect the required LMG data. Three different machine learning methods are employed: Random Forests, Convolutional Neural Networks, and Temporal Multi-Channel Vision Transformers. The system has also been efficiently used in decoding the forces exerted during power grasping. The results demonstrate that LMG outperforms EMG for most methods and subjects.

Minimally invasive capillary blood sampling methods.

INTRODUCTION: Whole blood samples, including arterial, venous, and capillary blood, are regularly used for disease diagnosis and monitoring. The global Covid-19 pandemic has highlighted the need for a more resilient screening capacity. Minimally invasive sampling techniques, such as capillary blood sampling, are routinely used for point of care testing in the home healthcare setting and clinical settings such as the Intensive Care Unit with less pain and wounding than conventional venepuncture. AREAS COVERED: In this manuscript, we aim to provide a overview of state-of-the-art of techniques for obtaining samples of capillary blood. We first review both established and novel methods for releasing blood from capillaries in the skin. Next, we provide a comparison of different capillary blood sampling methods based on their mechanism, testing site, puncture size, cost, wound geometry, healing, and perceptions of pain. Finally, we overview established and new methods for enhancing capillary blood collection. EXPERT OPINION: We expect that microneedles will prove to be a preferred option for paediatric blood collection. The ability of microneedles to collect a capillary blood sample without pain will improve paediatric healthcare outcomes. Jet injection may prove to be a useful method for facilitating both blood collection and drug delivery.

Jet-Induced Blood Release From Human Fingertips: A Single-Blind, Randomized, Crossover Trial.

BACKGROUND: Lancet pricks are often poorly received by individuals with diabetes; jet injection may allow lancet-free blood sampling. We examine whether the technique of jet injection can release sufficient blood from the fingertip to enable measurement of blood glucose concentration. In addition, we assess the effect of jet shape and cross-sectional area on fluid release, blood dilution, and perceived pain. METHODS: A randomized, single-blind, crossover study was conducted on 20 healthy volunteers who received interventions on four fingertips: a lancet prick, and jet injection of a small quantity of saline solution through three differently shaped and sized nozzles. Released fluid volume, blood concentration, and glucose concentration were assessed immediately after the intervention. Pain perception and duration, and any skin reactions, were evaluated both immediately and 24 hours after the intervention. RESULTS: Jet injection released sufficient blood from the fingertip to conduct a glucose measurement. A slot-shaped nozzle released the most blood, although less than a lancet, with slightly higher pain. The blood glucose levels estimated from the extracted fluid showed a mean absolute percentage error of 25%. There was no consistent evidence that a jet injection leads to different skin reactions at the intervention site relative to a lancet prick. CONCLUSIONS: Fingertip penetration by jet injection can release a volume of fluid sufficient for blood glucose measurement. Jet injection with a slot-shaped nozzle and/or a nozzle with larger outlet area helps to release more fluid. This technique may enable blood sampling, glucose concentration measurement, and insulin delivery to be performed in a single device.