Transforming eukaryotic cell culture with macromolecular crowding.

In multicellular organisms, the intracellular and extracellular spaces are considerably packed with a diverse range of macromolecular species. Yet, standard eukaryotic cell culture is performed in dilute, and deprived of macromolecules culture media, that barely imitate the density and complex macromolecular composition of tissues. Essentially, we drown cells in a sea of media and then expect them to perform physiologically. Herein, we argue the use of macromolecular crowding (MMC) in eukaryotic cell culture for regenerative medicine and drug discovery purposes.

Arterial input function estimation compensating for inflow and partial voluming in dynamic contrast-enhanced MRI.

Both inflow and the partial volume effect (PVE) are sources of error when measuring the arterial input function (AIF) in dynamic contrast-enhanced (DCE) MRI. This is relevant, as errors in the AIF can propagate into pharmacokinetic parameter estimations from the DCE data. A method was introduced for flow correction by estimating and compensating the number of the perceived pulse of spins during inflow. We hypothesized that the PVE has an impact on concentration-time curves similar to inflow. Therefore, we aimed to study the efficiency of this method to compensate for both effects simultaneously. We first simulated an AIF with different levels of inflow and PVE contamination. The peak, full width at half-maximum (FWHM), and area under curve (AUC) of the reconstructed AIFs were compared with the true (simulated) AIF. In clinical data, the PVE was included in AIFs artificially by averaging the signal in voxels surrounding a manually selected point in an artery. Subsequently, the artificial partial volume AIFs were corrected and compared with the AIF from the selected point. Additionally, corrected AIFs from the internal carotid artery (ICA), the middle cerebral artery (MCA), and the venous output function (VOF) estimated from the superior sagittal sinus (SSS) were compared. As such, we aimed to investigate the effectiveness of the correction method with different levels of inflow and PVE in clinical data. The simulation data demonstrated that the corrected AIFs had only marginal bias in peak value, FWHM, and AUC. Also, the algorithm yielded highly correlated reconstructed curves over increasingly larger neighbourhoods surrounding selected arterial points in clinical data. Furthermore, AIFs measured from the ICA and MCA produced similar peak height and FWHM, whereas a significantly larger peak and lower FWHM was found compared with the VOF. Our findings indicate that the proposed method has high potential to compensate for PVE and inflow simultaneously. The corrected AIFs could thereby provide a stable input source for DCE analysis.

Experimental and Monte Carlo based dosimetric investigation of a novel 3 mm radiosurgery 3 MV beam using the microSilicon detector.

BACKGROUND: The ZAP-X system is a novel gyroscopic radiosurgical system based on a 3 MV linear accelerator and collimator cones with a diameter between 4 and 25 mm. Advances in imaging modalities to detect small and early-stage pathologies allow for an early and less invasive treatment, where a smaller collimator matching the anatomical target could provide better sparing of surrounding healthy tissue. PURPOSE: A novel 3 mm collimator cone for the ZAP-X was developed. This study aims to investigate the usability of a commercial diode detector (microSilicon) for the dosimetric characterization of this small collimator cone; and to investigate the underlying small field perturbation effects. METHODS: Profile measurements in five depths as well as PDD and output ratio measurements were performed with a microSilicon detector and radiochromic EBT3 films. In addition, comprehensive Monte Carlo simulations were performed to validate the measurement observations and to quantify the perturbation effects of the microSilicon detector in these extremely small field conditions. RESULTS: It is shown that the microSilicon detector enables an accurate dosimetric characterization of the 3 mm beam. The profile parameters, such as the FWHM and 20%-80% penumbra width, agree within 0.1 to 0.2 mm between film and detector measurements. The output ratios agree within the measurement uncertainty between microSilicon detector and films, whereas the comparisons of the PDD results show good agreement with the Monte Carlo simulations. The analysis of the perturbation factors of the microSilicon detector reveals a small field correction factor of approximately 3% for the 3 mm circular beam and a correction factor smaller than 1.5% for field diameters above 3 mm. CONCLUSIONS: It could be shown that the microSilicon detector is well-suitable for the characterization of the new 3 mm circular beam of the ZAP-X system.

A CNN-based denoising method trained with images acquired with electron density phantoms for thin-sliced coronary artery calcium scans.

PURPOSE: This work proposed a convolutional neural network (CNN)-based method trained with images acquired with electron density phantoms to reduce quantum noise for coronary artery calcium (CAC) scans reconstructed with slice thickness less than 3 mm. METHODS: A DenseNet model was used to estimate quantum noise for CAC scans reconstructed with slice thickness of 0.5, 1.0 and 1.5 mm. Training data was acquired using electron density phantoms in three different sizes. The label images of the CNN model were real noise maps, while the input images of the CNN model were pseudo noise maps. Image denoising was conducted by subtracting the CNN output images from thin-sliced CAC scans. The efficacy of the proposed method was verified through both phantom study and patient study. RESULTS: By means of phantom study, the proposed method was proven effective in reducing quantum noise in CAC scans reconstructed with 1.5-mm slice thickness without causing significant texture change or variation in HU values. With regard to patient study, calcifications were more clear on the denoised CAC scans reconstructed with slice thickness of 0.5, 1.0 and 1.5 mm than on 3-mm slice images, while over-smooth changes were not observed in the denoised CAC scans reconstructed with 1.5-mm slice thickness. CONCLUSION: Our results demonstrated that the electron density phantoms can be used to generate training data for the proposed CNN-based denoising method to reduce quantum noise for CAC scans reconstructed with 1.5-mm slice thickness. Because anthropomorphic phantom is not a necessity, our method could make image denoising more practical in routine clinical practice.

Effect of Injectable Acellular Adipose Matrix on Soft Tissue Reconstruction in a Murine Model.

BACKGROUND: The extracellular matrix isolated from adipose tissue, known as acellular adipose matrix (AAM), represents a novel biomaterial. AAM functions as a scaffold that not only supports stem cell proliferation and differentiation but also induces adipogenesis and angiogenesis. This study aims to investigate the volumetric effects and microenvironmental changes associated with injectable AAM in comparison to conventional fat grafting. METHODS: AAM was manufactured from fresh human abdominoplasty fat using a mechanically modified method and then transformed into an injectable form. Lipoaspirate was harvested employing the Coleman technique. A weight and volume study was conducted on athymic nude mice by injecting either injectable AAM or lipoaspirate into the scalp (n=6 per group). After eight weeks, graft retention was assessed through weight measurement and volumetric analysis using micro-computed tomography (micro-CT) scanning. Histological analysis was performed using immunofluorescence staining for perilipin and CD31. RESULTS: Injectable AAM exhibited similar weight and volume effects in murine models. Histological analysis revealed comparable inflammatory cell presence with minimal capsule formation when compared to conventional fat grafts. Adipogenesis occurred in both AAM-injected and conventional fat graft models, with no significant difference in the blood vessel area (%) between the two. CONCLUSIONS: In summary, injectable AAM demonstrates effectiveness comparable to conventional fat grafting concerning volume effects and tissue regeneration in soft tissue reconstruction. This promising allogeneic injectable holds the potential to serve as a safe and effective "Off-the-Shelf" alternative in both aesthetic and reconstructive clinical practices. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Molecular-Scale Liquid Density Fluctuations and Cavity Thermodynamics.

Equilibrium density fluctuations at the molecular level produce cavities in a liquid and can be analyzed to shed light on the statistics of the number of molecules occupying observation volumes of increasing radius. An information theory approach led to the conclusion that these probabilities should follow a Gaussian distribution. Computer simulations confirmed this prediction across various liquid models if the size of the observation volume is not large. The reversible work required to create a cavity and the chance of finding no molecules in a fixed observation volume are directly correlated. The Gaussian formula for the latter probability is scrutinized to derive the changes in enthalpy and entropy, which arise from the cavity creation. The reversible work of cavity creation has a purely entropic origin as a consequence of the solvent-excluded volume effect produced by the inaccessibility of a region of the configurational space. The consequent structural reorganization leads to a perfect compensation of enthalpy and entropy changes. Such results are coherent with those obtained from Lee in his direct statistical mechanical study.

A cycle-consistent adversarial network for brain PET partial volume correction without prior anatomical information.

PURPOSE: Partial volume effect (PVE) is a consequence of the limited spatial resolution of PET scanners. PVE can cause the intensity values of a particular voxel to be underestimated or overestimated due to the effect of surrounding tracer uptake. We propose a novel partial volume correction (PVC) technique to overcome the adverse effects of PVE on PET images. METHODS: Two hundred and twelve clinical brain PET scans, including 50 18F-Fluorodeoxyglucose (18F-FDG), 50 18F-Flortaucipir, 36 18F-Flutemetamol, and 76 18F-FluoroDOPA, and their corresponding T1-weighted MR images were enrolled in this study. The Iterative Yang technique was used for PVC as a reference or surrogate of the ground truth for evaluation. A cycle-consistent adversarial network (CycleGAN) was trained to directly map non-PVC PET images to PVC PET images. Quantitative analysis using various metrics, including structural similarity index (SSIM), root mean squared error (RMSE), and peak signal-to-noise ratio (PSNR), was performed. Furthermore, voxel-wise and region-wise-based correlations of activity concentration between the predicted and reference images were evaluated through joint histogram and Bland and Altman analysis. In addition, radiomic analysis was performed by calculating 20 radiomic features within 83 brain regions. Finally, a voxel-wise two-sample t-test was used to compare the predicted PVC PET images with reference PVC images for each radiotracer. RESULTS: The Bland and Altman analysis showed the largest and smallest variance for 18F-FDG (95% CI: - 0.29, + 0.33 SUV, mean = 0.02 SUV) and 18F-Flutemetamol (95% CI: - 0.26, + 0.24 SUV, mean = - 0.01 SUV), respectively. The PSNR was lowest (29.64 ± 1.13 dB) for 18F-FDG and highest (36.01 ± 3.26 dB) for 18F-Flutemetamol. The smallest and largest SSIM were achieved for 18F-FDG (0.93 ± 0.01) and 18F-Flutemetamol (0.97 ± 0.01), respectively. The average relative error for the kurtosis radiomic feature was 3.32%, 9.39%, 4.17%, and 4.55%, while it was 4.74%, 8.80%, 7.27%, and 6.81% for NGLDM_contrast feature for 18F-Flutemetamol, 18F-FluoroDOPA, 18F-FDG, and 18F-Flortaucipir, respectively. CONCLUSION: An end-to-end CycleGAN PVC method was developed and evaluated. Our model generates PVC images from the original non-PVC PET images without requiring additional anatomical information, such as MRI or CT. Our model eliminates the need for accurate registration or segmentation or PET scanner system response characterization. In addition, no assumptions regarding anatomical structure size, homogeneity, boundary, or background level are required.

Facility patient volume and survival among individuals diagnosed with malignant central nervous system tumors.

PURPOSE: Prior research indicates that the volume of central nervous system (CNS) tumor patients seen by a facility is associated with outcomes. However, most studies have focused on short-term survival and specific CNS tumor subtypes. Our objective was to examine whether facility CNS tumor patient volume is associated with longer-term CNS tumor survival overall and by subtype. METHODS: We obtained National Cancer Database (NCDB) data including individuals diagnosed with CNS tumors from 2004 to 2016. Analyses were stratified by age group (0-14, 15-39, 40-64, and ≥ 65 years) and tumor type. We used Cox Proportional Hazards (PH) regression and restricted mean survival time (RMST) analyses to examine associations between survival and facility patient volume percentile category adjusting for potential confounding factors. RESULTS: Our analytic dataset included data from 130,830 individuals diagnosed with malignant first primary CNS tumors. We found a consistently reduced hazard rate of death across age groups for individuals reported by higher vs. lower (> 95th vs. ≤ 70th percentile) volume facilities (hazard ratio (HR)0-14 = 0.78, 95% confidence interval (CI) 0.64-0.95; HR15-39 = 0.87, 95% CI 0.78-0.96; HR40-64 = 0.82, 95% CI 0.76-0.88; HR≥65 = 0.80, 95% CI 0.75-0.86). Significantly longer survival times within 5 years for higher vs. lower volume facilities were observed ranging from 1.20 months (15-39) to 3.08 months (40-64) higher. Associations varied by CNS tumor subtype for all age groups. CONCLUSIONS: These results suggest facility factors influence CNS tumor survival with longer survival for patients reported by higher volume facilities. Understanding these factors will be critical to developing strategies that eliminate modifiable differences in survival times.

Adapting a practical EPR dosimetry protocol to measure output factors in small fields with alanine.

PURPOSE: Modern radiotherapy techniques often deliver small radiation fields. In this work, a practical Electron Paramagnetic Resonance (EPR) dosimetry protocol is adapted and applied to measure output factors (OF) in small fields of a 6 MV radiotherapy system. Correction factors and uncertainties are presented and OFs are compared to the values obtained by following TRS-483 using an ionization chamber (IC). METHODS: Irradiations were performed at 10 cm depth inside a water phantom positioned at 90 cm source to surface distance with a 6 MV flattening filter free photon beam of a Halcyon radiotherapy system. OFs for different nominal field sizes (1 × 1, 2 × 2, 3 × 3, 4 × 4, normalized to 10 × 10 cm2 ) were determined with a PinPoint 3D (PTW 31022) IC following TRS-483 as well as with alanine pellets with a diameter of 4 mm and a height of 2.4 mm. EPR readout was performed with a benchtop X-band spectrometer. Correction factors due to volume averaging and due to positional uncertainties were derived from 2D film measurements. RESULTS: OFs obtained from both dosimeter types agreed within 0.7% after applying corrections for the volume averaging effect. For the used alanine pellets, volume averaging correction factors of 1.030(2) for the 1 × 1 cm2 field and <1.002 for the larger field sizes were determined. The correction factor for positional uncertainties of 1 mm was in the order of 1.018 for the 1 × 1 cm2 field. Combined relative standard uncertainties uc for the OFs resulting from alanine measurements were estimated to be below 1.5% for all field sizes. For IC measurements, uc was estimated to be below 1.0%. CONCLUSIONS: A practical EPR dosimetry protocol is adaptable for precisely measuring OFs in small fields down to 1 × 1 cm2 . It is recommended to consider the effect of positional uncertainties for field sizes <2 × 2 cm2 .

Biomedical advances and future prospects of high-precision three-dimensional radiotherapy and four-dimensional radiotherapy.

Biomedical advances of external-beam radiotherapy (EBRT) with improvements in physical accuracy are reviewed. High-precision (±1 mm) three-dimensional radiotherapy (3DRT) can utilize respective therapeutic open doors in the tumor control probability curve and in the normal tissue complication probability curve instead of the one single therapeutic window in two-dimensional EBRT. High-precision 3DRT achieved higher tumor control and probable survival rates for patients with small peripheral lung and liver cancers. Four-dimensional radiotherapy (4DRT), which can reduce uncertainties in 3DRT due to organ motion by real-time (every 0.1-1 s) tumor-tracking and immediate (0.1-1 s) irradiation, have achieved reduced adverse effects for prostate and pancreatic tumors near the digestive tract and with similar or better tumor control. Particle beam therapy improved tumor control and probable survival for patients with large liver tumors. The clinical outcomes of locally advanced or multiple tumors located near serial-type organs can theoretically be improved further by integrating the 4DRT concept with particle beams.

Projected frontal area and its components during front crawl depend on lung volume.

We examined the influence of lung volume on the vertical body position, trunk inclination, and projected frontal area (PFA) during swimming and the inter-relationships among these factors. Twelve highly trained male swimmers performed a 15 m front crawl with sustained maximal inspiration (INSP), maximal expiration (EXP), and intermediate (MID) at a target velocity of 1.20 m·s-1 . Using our developed digital human model, which allows inverse kinematics calculations by fitting individual body shapes measured with a three-dimensional photonic image scanner to individually measured underwater motion capture data, vertical center of mass (CoM) position, trunk inclination, and PFA were calculated for each complete stroke cycle. In particular, the PFA was calculated by automatic processing of a series of parallel frontal images obtained from a reconstructed digital human model. The vertical CoM position was higher with a larger lung-volume level (p < 0.01). The trunk inclination was smaller in INSP and MID than in EXP (p < 0.01). PFA was smaller with a larger lung-volume level (p < 0.01). Additionally, there was a significant interaction of vertical CoM position and trunk inclination with PFA (p = 0.006). There was a negative association between PFA and vertical CoM position, and a positive association between PFA and trunk inclination less than the moderate vertical CoM position (each p < 0.05). These results obtained using our methodology indicate that PFA decreases with increasing lung volume due to an increase in vertical CoM position, and additionally due to a decrease in trunk inclination at low-to-moderate lung-volume levels.

Effect of Hospital Case-Volume on Mortality after Ovarian Cancer Surgery: A Population-Based Retrospective Cohort Study.

OBJECTIVES: The goal of ovarian cancer surgery has recently shifted from optimal cytoreduction to more complete resection. This study attempted to reassess and update the association between surgical case-volume and both in-hospital and long-term mortality after ovarian cancer surgery using recent data. DESIGN: This study is a population-based retrospective cohort study. Participants/Material: Data from all adult patients who underwent ovarian cancer surgery in Korea between 2005 and 2019 were obtained from the national database. A total of 24,620 patients underwent ovarian cancer surgery in 362 hospitals during the period. SETTING: In-hospital and 1-, 3-, 5-year mortality were set as primary and secondary outcomes. METHODS: Hospitals were categorized into high-volume (>90 cases/year), medium-volume (20-90 cases/year), and low-volume (<20 cases/year) centers considering overall distribution of case-volume. Postoperative in-hospital and long-term mortality were analyzed using logistic regression after adjusting for potential risk factors. RESULTS: Compared to high-volume centers (0.54%), in-hospital mortality was significantly higher in medium-volume (1.40%; adjusted odds ratio, 2.92; confidence interval, 1.82-3.73; p < 0.001) and low-volume (1.61%; adjusted odds ratio, 2.94; confidence interval, 2.07-4.17; p < 0.001) centers. In addition, 1-year mortality was 6.26%, 7.06%, and 7.94% for high-volume, medium-volume, and low-volume centers, respectively, and the differences among the groups were significant. However, case-volume effect was not apparent in 3- and 5-year mortality after ovarian cancer surgery. LIMITATIONS: Lacking clinical information such as staging or histologic diagnosis due to the nature of the administrative data should be considered in interpreting the data. CONCLUSIONS: Case-volume effect was observed for in-hospital and 1-year mortality after ovarian cancer surgery, while it was not clearly found in 3- or 5-year mortality. Dilution of the case-volume effect might be attributed to the high accessibility to care.

[Fiber direction estimation using constrained spherical deconvolution based on multi-model response function].

Constrained spherical deconvolution can quantify white matter fiber orientation distribution information from diffusion magnetic resonance imaging data. But this method is only applicable to single shell diffusion magnetic resonance imaging data and will provide wrong fiber orientation information in white matter tissue which contains isotropic diffusion signals. To solve these problems, this paper proposes a constrained spherical deconvolution method based on multi-model response function. Multi-shell data can improve the stability of fiber orientation, and multi-model response function can attenuate isotropic diffusion signals in white matter, providing more accurate fiber orientation information. Synthetic data and real brain data from public database were used to verify the effectiveness of this algorithm. The results demonstrate that the proposed algorithm can attenuate isotropic diffusion signals in white matter and overcome the influence of partial volume effect on fiber direction estimation, thus estimate fiber direction more accurately. The reconstructed fiber direction distribution is stable, the false peaks are less, and the recognition ability of cross fiber is stronger, which lays a foundation for the further research of fiber bundle tracking technology.

Bioinspired in vitro microenvironments to control cell fate: focus on macromolecular crowding.

The development of therapeutic regenerative medicine and accurate drug discovery cell-based products requires effective, with respect to obtaining sufficient numbers of viable, proliferative, and functional cell populations, cell expansion ex vivo. Unfortunately, traditional cell culture systems fail to recapitulate the multifaceted tissue milieu in vitro, resulting in cell phenotypic drift, loss of functionality, senescence, and apoptosis. Substrate-, environment-, and media-induced approaches are under intense investigation as a means to maintain cell phenotype and function while in culture. In this context, herein, the potential of macromolecular crowding, a biophysical phenomenon with considerable biological consequences, is discussed.

Partial volume correction in arterial spin labeling perfusion MRI: A method to disentangle anatomy from physiology or an analysis step too far?

The mismatch in the spatial resolution of Arterial Spin Labeling (ASL) MRI perfusion images and the anatomy of functionally distinct tissues in the brain leads to a partial volume effect (PVE), which in turn confounds the estimation of perfusion into a specific tissue of interest such as gray or white matter. This confound occurs because the image voxels contain a mixture of tissues with disparate perfusion properties, leading to estimated perfusion values that reflect primarily the volume proportions of tissues in the voxel rather than the perfusion of any particular tissue of interest within that volume. It is already recognized that PVE influences studies of brain perfusion, and that its effect might be even more evident in studies where changes in perfusion are co-incident with alterations in brain structure, such as studies involving a comparison between an atrophic patient population vs control subjects, or studies comparing subjects over a wide range of ages. However, the application of PVE correction (PVEc) is currently limited and the employed methodologies remain inconsistent. In this article, we outline the influence of PVE in ASL measurements of perfusion, explain the main principles of PVEc, and provide a critique of the current state of the art for the use of such methods. Furthermore, we examine the current use of PVEc in perfusion studies and whether there is evidence to support its wider adoption. We conclude that there is sound theoretical motivation for the use of PVEc alongside conventional, 'uncorrected', images, and encourage such combined reporting. Methods for PVEc are now available within standard neuroimaging toolboxes, which makes our recommendation straightforward to implement. However, there is still more work to be done to establish the value of PVEc as well as the efficacy and robustness of existing PVEc methods.

Analysis of right ventricular mass from magnetic resonance imaging data: a simple post-processing algorithm for correction of partial-volume effects.

Magnetic resonance imaging (MRI) of the right ventricle (RV) offers important diagnostic information, but the accuracy of this information is hampered by the complex geometry of the RV. Here, we propose a novel postprocessing algorithm that corrects for partial-volume effects in the analysis of standard MRI cine images of RV mass (RVm) and evaluate the method in clinical and preclinical data. Self-corrected RVm measurement was compared with conventionally measured RVm in 16 patients who showed different clinical indications for cardiac MRI and in 17 Wistar rats with different degrees of pulmonary congestion. The rats were studied under isoflurane anaesthesia. To evaluate the reliability of the proposed method, the measured end-systolic and end-diastolic RVm were compared. Accuracy was evaluated by comparing preclinical RVm to ex vivo RV weight (RVw). We found that use of the self-correcting algorithm improved reliability compared with conventional segmentation. For clinical data, the limits of agreement (LOAs) were -1.8 ± 8.6g (self-correcting) vs. 5.8 ± 7.8g (conventional), and coefficients of variation (CoVs) were 7.0% (self-correcting) vs. 14.3% (conventional). For preclinical data, LOAs were 21 ± 46 mg (self-correcting) vs. 64 ± 89 mg (conventional), and CoVs were 9.0% (self-correcting) and 17.4% (conventional). Self-corrected RVm also showed better correspondence with the ex vivo RVw: LOAs were -5 ± 80 mg (self-correcting) vs. 94 ± 116 mg (conventional) in end-diastole and -26 ± 74 mg (self-correcting) vs. 31 ± 98 mg (conventional) in end-systole. The new self-correcting algorithm improves the reliability and accuracy of RVm measurements in both clinical and preclinical MRI. It is simple and easy to implement and does not require any additional MRI data.NEW & NOTEWORTHY Magnetic resonance imaging (MRI) of the right ventricle (RV) offers important diagnostic information, but the accuracy of this information is hampered by the complex geometry of the RV. In particular, the crescent shape of the RV renders it particularly vulnerable to partial-volume effects. We present a new, simple, self-correcting algorithm that can be applied to correct partial-volume effects in MRI-based RV mass estimation. The self-correcting algorithm offers improved reliability and accuracy compared with the conventional approach.

Graph theory analysis of the dopamine D2 receptor network in Parkinson's disease patients with cognitive decline.

Cognitive decline in Parkinson's disease (PD) is a common sequela of the disorder that has a large impact on patient well-being. Its physiological etiology, however, remains elusive. Our study used graph theory analysis to investigate the large-scale topological patterns of the extrastriatal dopamine D2 receptor network. We used positron emission tomography with [11 C]FLB-457 to measure the binding potential of cortical dopamine D2 receptors in two networks: the meso-cortical dopamine network and the meso-limbic dopamine network. We also investigated the application of partial volume effect correction (PVEC) in conjunction with graph theory analysis. Three groups were investigated in this study divided according to their cognitive status as measured by the Montreal Cognitive Assessment score, with a score ≤25 considered cognitively impaired: (a) healthy controls (n = 13, 11 female), (b) cognitively unimpaired PD patients (PD-CU, n = 13, 5 female), and (c) PD patients with mild cognitive impairment (PD-MCI, n = 17, 4 female). In the meso-cortical network, we observed increased small-worldness, normalized clustering, and local efficiency in the PD-CU group compared to the PD-MCI group, as well as a hub shift in the PD-MCI group. Compensatory reorganization of the meso-cortical dopamine D2 receptor network may be responsible for some of the cognitive preservation observed in PD-CU. These results were found without PVEC applied and PVEC proved detrimental to the graph theory analysis. Overall, our findings demonstrate how graph theory analysis can be used to detect subtle changes in the brain that would otherwise be missed by regional comparisons of receptor density.

Cortical parameters predict bone strength at the tibial diaphysis, but are underestimated by HR-pQCT and µCT compared to histomorphometry.

Cortical bone and its microstructure are crucial for bone strength, especially at the long bone diaphysis. However, it is still not well-defined how imaging procedures can be used as predictive tools for mechanical bone properties. This study evaluated the capability of several high-resolution imaging techniques to capture cortical bone morphology and assessed the correlation with the bone's mechanical properties. The microstructural properties (cortical thickness [Ct.Th], porosity [Ct.Po], area [Ct.Ar]) of 11 female tibial diaphysis (40-90 years) were evaluated by dual-energy X-ray absorptiometry (DXA), high-resolution peripheral-quantitative-computed-tomography (HR-pQCT), micro-CT (µCT) and histomorphometry. Stiffness and maximal torque to failure were determined by mechanical testing. T-Scores determined by DXA ranged from 0.6 to -5.6 and a lower T-Score was associated with a decrease in Ct.Th (p ≤ 0.001) while the Ct.Po (p ≤ 0.007) increased, and this relationship was independent of the imaging method. With decreasing T-Score, histology showed an increase in Ct.Po from the endosteal to the periosteal side (p = 0.001) and an exponential increase in the ratio of osteons at rest to those after remodelling. However, compared to histomorphometry, HR-pQCT and µCT underestimated Ct.Po and Ct.Th. A lower T-Score was also associated with significantly reduced stiffness (p = 0.031) and maximal torque (p = 0.006). Improving the accuracy of Ct.Po and Ct.Th did not improve prediction of the mechanical properties, which was most closely related to geometry (Ct.Ar). The ex-vivo evaluation of mechanical properties correlated with all imaging modalities, with Ct.Th and Ct.Po highly correlated with the T-Score of the tibial diaphysis. Cortical microstructural changes were underestimated with the lower resolution of HR-pQCT and µCT compared to the histological 'gold standard'. The increased accuracy did not result in an improved prediction for local bone strength in this study, which however might be related to the limited number of specimens and thus needs to be evaluated in a larger collective.

Molecular crowding accelerates aggregation of α-synuclein by altering its folding pathway.

Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.

Optimal molecular crowding accelerates group II intron folding and maximizes catalysis.

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an "optimum" [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.