Biological Tissue Samples Research Articles

Hybrid dark-field and attenuation contrast retrieval for laboratory-based X-ray tomography

X-ray dark-field imaging highlights sample structures through contrast generated by sub-resolution features within the inspected volume. Quantifying dark-field signals generally involves multiple exposures for phase retrieval, separating contributions from scattering, refraction, and attenuation. Here, we introduce an approach for non-interferometric X-ray dark-field imaging that presents a single-parameter representation of the sample. This fuses attenuation and dark-field signals, enabling the reconstruction of a unified three-dimensional volume. Notably, our method can obtain dark-field contrast from a single exposure and employs conventional back projection algorithms for reconstruction. Our approach is based on the assumption of a macroscopically homogeneous material, which we validate through experiments on phantoms and on biological tissue samples. The methodology is implemented on a laboratory-based, rotating anode X-ray tube system without the need for coherent radiation or a high-resolution detector. Utilizing this system with streamlined data acquisition enables expedited scanning while maximizing dose efficiency. These attributes are crucial in time- and dose-sensitive medical imaging applications and unlock the ability of dark-field contrast with high-throughput lab-based tomography. We believe that the proposed approach can be extended across X-ray dark-field imaging implementations beyond tomography, spanning fast radiography, directional dark-field imaging, and compatibility with pulsed X-ray sources.

Large Dynamic Range Spectral Measurement in Terahertz Region Based on Frequency Up-Conversion Detection via OH1 Crystal.

Terahertz spectroscopy systems, which integrate terahertz sources and detectors, have important applications in many fields such as materials science and security checking. Based on highly sensitive frequency up-conversion detection, large dynamic range spectral measurements in a terahertz region are reported. Our system realized the detection sensitivity at a 10 aJ level with a 2-(3-(4-hydroxystyryl)-5,5-dime-thylcyclohex-2-enylidene) malononitrile (OH1) crystal and a dynamic range up to seven orders. Based on this system, we verified the validity of the spectral measurement with tests which were conducted on monohydrate glucose, anhydrous glucose and mixed tablet samples with a thickness of 0.8 mm in 1~3 THz, respectively. Also, a mini coppery elbow tube with an inner diameter of 1 mm was used for the transmission of a terahertz wave to simulate some strip biological tissue samples. By allowing terahertz to transmit through this tube filled with 0.5 g glucose powder, we successfully obtained the absorption spectrum with a minimum transmittance at the absorption peak in the order of 10-4.

A highly sensitive approach for the analysis of tyrosine phosphoproteome in primary T cells

Tyrosine phosphorylation, a common post-translational modification process for proteins, is involved in a variety of biological processes. However, the abundance of tyrosine-phosphorylated proteins is very low, making their identification by mass spectrometry (MS) is difficult; thus, milligrams of the starting material are often required for their enrichment. For example, tyrosine phosphorylation plays an important role in T cell signal transduction. However, the number of primary T cells derived from biological tissue samples is very small, and these cells are difficult to culture and expand; thus, the study of T cell signal transduction is usually carried out on immortalized cell lines, which can be greatly expanded. However, the data from immortalized cell lines cannot fully mimic the signal transduction processes observed in the real physiological state, and they usually lead to conclusions that are quite different from those of primary T cells. Therefore, a highly sensitive proteomic method was developed for studying tyrosine phosphorylation modification signals in primary T cells. To address the issue of the limited T cells numbers, a comprehensive protocol was first optimized for the isolation, activation, and expansion of primary T cells from mouse spleen. CD3+ primary T cells were successfully sorted; more than 91% of the T cells collected were well activated on day 2, and the number of T cells expanded to over 7-fold on day 4. Next, to address the low abundance of tyrosine-phosphorylated proteins, we used SH2-superbinder affinity enrichment and immobilized Ti4+affinity chromatography (Ti4+-IMAC) to enrich the tyrosine-phosphorylated polypeptides of primary T cells that were co-stimulated with anti-CD3 and anti-CD28. These polypeptides were resolved using nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Finally, 282 tyrosine phosphorylation sites were successfully identified in 1 mg of protein, including many tyrosine phosphorylation sites on the immunoreceptor tyrosine-based activation motif (ITAM) in the intracellular region of the T cell receptor membrane protein CD3, as well as the phosphotyrosine sites of ZAP70, LAT, VAV1, and other proteins related to signal transduction under costimulatory conditions. In summary, to solve the technical problems of the limited number of primary cells, low abundance of tyrosine-phosphorylated proteins, and difficulty of detection by MS, we developed a comprehensive proteomic method for the in-depth analysis of tyrosine phosphorylation modification signals in primary T cells. This protocol may be applied to map signal transduction networks that are closely related to physiological states.

Structural characterization of sphingomyelins from tissue using electron-induced dissociation.

Sphingomyelins (SMs) and resulting metabolic products serve important functional and cell signaling roles and can act as potential biomarkers and therapeutic targets in many pathological disorders. SMs each contain a sphingoid base, an amide-linked fatty acyl chain, and a phosphocholine headgroup. Despite these simple building blocks, variations and modifications of both the sphingoid base and the fatty acyl chain result in a diverse array of structurally complicated SM compounds. Conventional tandem mass spectrometry (MS/MS) using the collision-induced dissociation (CID) method only provides limited structural information, necessitating other tools to unravel the structural complexity of these lipids. We utilize electron-induced dissociation (EID) and sequential CID/EID approaches to elucidate detailed structural features of SMs. Integrating the CID/EID method into an imaging MS workflow enables accurate identification of SMs directly from kidney tissue. The application of EID enables identification of SMs at the molecular species level, identifying the sphingosine base and the amide-linked fatty acyl chains. Furthermore, removal of the phosphocholine headgroup via CID followed by sequential EID in an MS3 analysis (CID/EID) enhances the structural information obtained. CID/EID provides diagnostic fragmentation patterns revealing the hydroxylation site and double bond position in both the sphingosine base and amide-linked fatty acyl chains. Detailed structural information of SMs from synthetic standards and biological tissue samples is obtained using an alternative electron-based dissociation method. Accurate characterization of SMs promises to better inform studies of tissue biochemistry, lipid metabolism, and molecular pathology.

Visual detection of misfolded alpha-synuclein and prions via capillary-based quaking-induced conversion assay (Cap-QuIC)

Neurodegenerative protein misfolding diseases impact tens of millions of people worldwide, contributing to millions of deaths and economic hardships across multiple scales. The prevalence of neurodegenerative disease is predicted to greatly increase over the coming decades, yet effective diagnostics for such diseases are limited. Most diagnoses come from the observation of external symptoms in clinical settings, which typically manifest during relatively advanced stages of disease, thus limiting potential therapeutic applications. While progress is being made on biomarker testing, the underlying methods largely rely on fragile and expensive equipment that limits their point-of-care potential, especially in developing countries. Here we present Capillary-based Quaking Induced Conversion (Cap-QuIC) as a visual diagnostic assay based on simple capillary action for the detection of neurodegenerative disease without necessitating expensive and complex capital equipment. We demonstrate that Cap-QuIC has the potential to be a detection tool for a broad range of misfolded proteins by successfully distinguishing misfolded versus healthy proteins associated with Parkinson’s disease (α-synuclein) and Chronic Wasting Disease (prions). Additionally, we show that Cap-QuIC can accurately classify biological tissue samples from wild white-tailed deer infected with Chronic Wasting Disease. Our findings elucidate the underlying mechanism that enables the Cap-QuIC assay to distinguish misfolded protein, highlighting its potential as a diagnostic technology for neurodegenerative diseases.

A device for rapid calorimetric measurements on small biological tissue samples

A new calorimetric technique is described that allows high-throughput heat production rate measurements on small biological tissue samples. The technique is based on the widely used thermopile chip technology combined with an innovative method for precise transport and positioning of samples of different biological materials at the thermal power detector inside the calorimeter. The new transport and positioning technique is a combination of fluidic and mechanical transport, where the latter is realized by a magneto-motor drive. The transport facility ensures good diffusive oxygen penetration into the sample, which is essential for highly metabolically active materials. The proper functioning of the device is demonstrated by measuring the heat production of metabolically active brown adipose tissue, biopsied tegu lizard muscle, and live Drosophila larvae at different stages and temperatures.

3D polarization-interference holographic histology for wavelet-based differentiation of the polycrystalline component of biological tissues with different necrotic states. Forensic applications.

The interference-holographic method of phase scanning of fields of scattered laser radiation is proposed. The effectiveness of this method for the selection of variously dispersed components is demonstrated. This method made it possible to obtain polarization maps of biological tissues at a high level of depolarized background. The scale-selective analysis of such maps was used to determine necrotic changes in the optically anisotropic architectonics of biological tissues. Development and experimental approbation of layered phase polarimetry of repeatedly scattered fields in diffuse layers of biological tissues. Application of scale-selective processing of the found coordinate distributions of polarization states in various phase sections of object fields. Determination of criteria (markers) for histological differential diagnosis of the causes of necrotic changes in optical anisotropy of biological tissues. We used a synthesis of three instrumental and analytical methods. Polarization-interference registration of laser radiation scattered by a sample of biological tissue. Digital holographic reconstruction and layered phase scanning of distributions of complex amplitudes of the object field. Analytical determination of polarization maps of various phase cross-sections of repeatedly scattered radiation. Application of wavelet analysis of the distributions of polarization states in the phase plane of a single scattered component of an object field. Determination of criteria (markers) for differential diagnosis of necrotic changes in biological tissues with different morphological structure. Two cases are considered. The first case is the myocardium of those who died as a result of coronary heart disease and acute coronary insufficiency. The second case is lung tissue samples of deceased with bronchial asthma and fibrosis. A method of polarization-interference mapping of diffuse object fields of biological tissues has been developed and experimentally implemented. With the help of digital holographic reconstruction of the distributions of complex amplitudes, polarization maps in various phase sections of a diffuse object field are found. The wavelet analysis of azimuth and ellipticity distributions of polarization in the phase plane of a single scattered component of laser radiation is used. Scenarios for changing the amplitude of the wavelet coefficients for different scales of the scanning salt-like MHAT function are determined. Statistical moments of the first to fourth orders are determined for the distributions of the amplitudes of the wavelet coefficients of the azimuth maps and the ellipticity of polarization. As a result, diagnostic markers of necrotic changes in the myocardium and lung tissue were determined. The statistical criteria found are the basis for determining the accuracy of their differential diagnosis of various necrotic states of biological tissues. Necrotic changes caused by "coronary artery disease-acute coronary insufficiency" and "asthma-pulmonary fibrosis" were demonstrated by the method of wavelet differentiation with polarization interference with excellent accuracy.

Study on anisotropy orientation due to well-ordered fibrous biological microstructures.

Most biological fibrous tissues have anisotropic optical characteristics, which originate from scattering by their fibrous microstructures and birefringence of biological macromolecules. The orientation-related anisotropic interpretation is of great value in biological tissue characterization and pathological diagnosis. We focus on intrinsic birefringence and form birefringence in biological tissue samples. By observing and comparing the forward Mueller matrix of typical samples, we can understand the interpretation ability of orientation-related polarization parameters and further distinguish the sources and trends of anisotropy in tissues. For glass fiber, silk fiber, skeletal muscle, and tendon, we construct a forward measuring device to obtain the Mueller matrix image and calculate the anisotropic parameters related to orientation. The statistical analysis method based on polar coordinates can effectively analyze the difference in anisotropic parameters. For those birefringent fibers, the statistical distribution of fast-axis values derived from Mueller matrix polar decomposition was found to exhibit bimodal characteristics, which is a key point in distinguishing the single-layer birefringent fiber sample from a layered, multioriented fibrous sample. The application conditions and interference factors of anisotropic orientation parameters are analyzed. Based on the parameters extracted from the orientation bimodal distribution, we can evaluate the relative change trend of intrinsic birefringence and form birefringence in anisotropic samples. The cross-vertical bimodal distribution of the fast axis of anisotropic fibers is beneficial to accurately analyze the anisotropic changes in biological tissues. The results imply the potential of anisotropic orientation analysis for applications in pathological diagnosis.

MULTI-PARAMETER MUELLER-MATRIX TOMOGRAPHY OF HISTOLOGICAL SAMPLES OF BIOLOGICAL TISSUES AS AN ACCURATE AND EFFECTIVE METHOD FOR DETERMINING THE DEGREE OF BLOOD LOSS

Establishing the volume of blood loss is extremely important in the context of forensic practice, as it can indicate various circumstances of death and is important in solving criminal cases. Consideration of modern methods of determining this parameter in the article is relevant and aims to reveal new opportunities and perspectives in this field of forensic medical examination.
 Aim of the work. To develop a set of new forensic criteria for accurate determination of the volume of blood loss using the method of multichannel polarization Muller-matrix tomography of histological sections of parenchymal organs and human blood samples.
 Materials and methods. Samples of parenchymal organs and human blood were collected from 76 corpses of both sexes with varying degrees of blood loss from 0 mm3 to 2500 mm3. The research was carried out using the method of multichannel polarization Muller-matrix linear dichroism tomography of biological tissue samples.
 Results. For all studied biological samples, a decrease in the level of circular birefringence (CB) of formed blood elements against the background of a gradual necrotic decrease in distributions of linear birefringence (LB) of the optical anisotropy of parenchymal tissues and blood films was established for the process of blood loss. The range of sensitivity of the method of differential Mueller-matrix tomography with algorithmic reproduction of linear dichroism maps to changes in blood loss volume of the deceased, which is (0±2000) mm3, is determined.
 Conclusions. The accuracy of the method of differential Mueller matrix mapping with algorithmic reproduction of linear dichroism maps of biological preparations was determined, which is 86-92 % in the range of the level of blood loss ΔV = (0±2000) mm3.

Experimental study of the interstitial effect of a semiconductor laser with a wavelength of 445 nm on a biological model

Introduction. This study presents the results of interstitial exposure of a semiconductor laser with a wavelength of 445 nm to biological tissue samples at different pulsed wave power in a constant mode, with a contact method.Aim. To study the interstitial effect of a semiconductor laser with a wavelength of 445 nm on experimental tissue samples in a constant mode at different power.Materials and methods. As an experimental sample, we used biological tissue with a developed vascular structure in the form of pig liver. The source of laser radiation was a semiconductor laser with a wavelength of 445 nm, with a power range from 0.5 to 4 watts. When working with biological tissue samples, we evaluated their external and internal changes after laser exposure. The exposure time during interstitial exposure was 1 mm/sec with a 20 mm immersion depth of the laser fiber into the fabric. The results of the macro and microscopic picture were evaluated using histological examination and morphometry of the zones of destruction and coagulation necrosis, on a transverse section of the tissue.Results. The results of an experimental study indicate that interstitial laser exposure has a pronounced coagulation effect combined with a cutting effect. The optimal combination of coagulation and cutting effect of exposure, accompanied by visual contractility of the tissue, without excessive carbonation at a power of 3.0 watts.Conclusion. The use of interstitial exposure of a semiconductor laser with a wavelength of 445 nm on experimental tissue samples in a constant mode at different power showed the predominance of the coagulation effect in combination with the cutting effect with a pronounced reduction in tissue volume. Experiments have shown that the power of 3 W is the optimal power of laser exposure in the interstitial method, in which there is a pronounced reduction in the volume of the studied drug without excessive carbonation.

Two-Dimensional Correlation Spectroscopy (2D-COS) Analysis of Evolving Hyperspectral Images.

The evolutionary behavior is examined for heterogeneously distributed hyperspectral images of a simulated biological tissue sample comprising lipid-like and protein-like components during the aging process. Taking a simple planar average of a spectral image loses useful information about the spatially resolved nature of the data. In contrast, multivariate curve resolution (MCR) analysis of a spectral image at a given stage of aging produces a set of loadings of major component groups. Each loading represents the combined spectral contributions of a mixture of similar but not identical constituents (i.e., lipid-like and protein-like components). Temporal analysis of individual component groups using two-dimensional correlation spectroscopy (2D-COS) and MCR provides much-streamlined results without interferences from the overlapped contributions. Grouping of data into separate components also allows for the effective comparison of the parallel processes of lipid oxidation and protein denaturation involving a number of constituents using the heterocomponent 2D-COS analysis. The complex interplays of lipid constituents and protein secondary structures during the tissue aging process are unambiguously highlighted. The possibility of extending this approach to a much more general form of applications using a moving window analysis is also discussed.

Investigating the mechanistic impact of pork soft tissue preparation techniques on the classification precision of laser-induced breakdown spectroscopy.

The investigation of the mechanism underlying the impact of biological soft tissue sample preparation methods on laser-induced breakdown spectroscopy (LIBS) signals can enhance the stability of LIBS signals. Our study focused on four specific preparation methods applied to pork samples: rapid freezing, fresh slicing, drying, and pressing. The influence of various preparation techniques on the signal-to-noise ratio and fluctuation of Ca, Na, Mg, and CN bands within the sample spectra was assessed. The signal-to-noise ratios for samples that were dried and pressed notably improved. And the pressing method effectively mitigated the uneven distribution of pork tissue components, displaying superior spectral line stability. To explain this phenomenon, we used the Saha-Boltzmann diagram to estimate the plasma temperature. Remarkably, there was a significant reduction in plasma temperature fluctuations across four pressed samples, with a standard deviation of 108.53. Furthermore, we undertook a classification analysis employing support vector machine models to corroborate the generalization efficacy of the sample preparation technique. Dried and pressed samples demonstrated notably higher classification accuracy, precision, and recall (all >93%) compared to frozen and fresh samples, where these metrics remained below 86%. The performance of the SVM model was ultimately evaluated using Receiver Operating Characteristic (ROC) curves and the Area Under the Curve (AUC). The AUC for the frozen, fresh, dried, and pressed samples was 0.854, 0.907, 0.989, and 0.996, respectively. The findings revealed that the pressing method exhibited superior performance, followed by drying, fresh slicing, and freezing, in descending order of effectiveness.

Sample preparation and data collection for serial block face scanning electron microscopy of mammalian cell monolayers.

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focused ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe step-by-step protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events by light microscopy, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables.

Two Different Strategies for Stabilization of Brain Tissue and Extraction of Neuropeptides.

Neuropeptides are bioactive peptides that are synthesized and secreted by neurons in signaling pathways in the brain. Peptides and proteins are extremely vulnerable to proteolytic cleavage when their biological surrounding changes. This makes neuropeptidomics challenging due to the rapid alterations that occur to the peptidome after harvesting of brain tissue samples. For a successful neuropeptidomic study, the biological tissue sample analyzed should resemble the living state as much as possible. Heat stabilization has been proven to inhibit postmortem degradation by denaturing proteolytic enzymes, hence increasing identification rates of neuropeptides. Here, we describe two different stabilization protocols for rodent brain samples that increase the number of intact mature neuropeptides and minimize interference from degradation products of abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by sequentially using an aqueous and an organic extraction medium.

Single source CARS-based multimodal microscopy system for biological tissue imaging [Invited

A coherent anti-Stokes Raman scattering (CARS)-based multimodality microscopy system was developed using a single Ti:sapphire femtosecond laser source for biological imaging. It provides three complementary and co-registered imaging modalities: CARS, MPM (multiphoton microscopy), and RCM (reflectance confocal microscopy). The imaging speed is about 1 frame-per-second (fps) with a digital resolution of 1024 × 1024 pixels. This microscopy system can provide clear 2-dimensional and 3-dimensional images of ex-vivo biological tissue samples. Its spectral selection initiates vibrational excitation in lipid cells (approximately 2850 cm-1) using two filters on the pump and Stokes beam paths. The excitation can be tuned over a wide spectral range with adjustable spectral filters. The imaging capability of this CARS-based multimodal microscopy system was demonstrated using porcine fat, murine skin, and murine liver tissue samples.

Imaging of N-Linked Glycans in Biological Tissue Sections Using Nanospray Desorption Electrospray Ionization (nano-DESI) Mass Spectrometry.

N-linked glycans are complex biomolecules vital to cellular functions that have been linked to a wide range of pathological conditions. Mass spectrometry imaging (MSI) has been used to study the localization of N-linked glycans in cells and tissues. However, their structural diversity presents a challenge for MSI techniques, which stimulates the development of new approaches. In this study, we demonstrate for the first time spatial mapping of N-linked glycans in biological tissues using nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI). Nano-DESI MSI is an ambient ionization technique that has been previously used for imaging of metabolites, lipids, and proteins in biological tissue samples without special sample pretreatment. N-linked glycans are released from glycoproteins using an established enzymatic digestion with peptide N-glycosidase F, and their spatial localization is examined using nano-DESI MSI. We demonstrate imaging of N-linked glycans in formalin-fixed paraffin-embedded human hepatocellular carcinoma and human prostate tissues in both positive and negative ionization modes. We examine the localization of 38 N-linked glycans consisting of high mannose, hybrid fucosylated, and sialyated glycans. We demonstrate that negative mode nano-DESI MSI is well-suited for imaging of underivatized sialylated N-linked glycans. On-tissue MS/MS of different adducts of N-linked glycans proves advantageous for elucidation of the glycan sequence. This study demonstrates the applicability of liquid extraction techniques for spatial mapping of N-linked glycans in biological samples, providing an additional tool for glycobiology research.

Wrapped phase aberration compensation using deep learning in digital holographic microscopy

In digital holographic microscopy (DHM), phase aberration compensation is a general problem for improving the accuracy of quantitative phase measurement. Current phase aberration compensation methods mainly focus on the continuous phase map after performing the phase filtering and unwrapping to the wrapped phase map. However, for the wrapped phase map, when larger phase aberrations make the fringes too dense or make the noise frequency features indistinct, either spatial-domain or frequency-domain based filtering methods might be less effective, resulting in phase unwrapping anomalies and inaccurate aberration compensation. In order to solve this problem, we propose and design a strategy to advance the phase aberration compensation to the wrapped phase map with deep learning. As the phase aberration in DHM can be characterized by the Zernike coefficients, CNN (Convolutional Neural Network) is trained by using massive simulated wrapped phase maps as network inputs and their corresponding Zernike coefficients as labels. Then the trained CNN is used to directly extract the Zernike coefficients and compensate the phase aberration of the wrapped phase before phase filtering and unwrapping. The simulation results of different phase aberrations and noise levels and measurement results of MEMS chip and biological tissue samples show that, compared with current algorithms that perform phase aberration compensation after phase unwrapping, the proposed method can extract the Zernike coefficients more accurately, improve the phase data quality of the consequent phase filtering greatly, and achieve more accurate and reliable sample profile reconstruction. This phase aberration compensation strategy for the wrapped phase will have great potential in the applications of DHM quantitative phase imaging.

Mechanical properties of atherosclerotic plaques, caps and walls of arterial vessels: a mobile test bench experiments

Within the framework of this study, a methodology and a prototype of a test bench for conducting experiments on compression of biological tissue samples have been developed. The test bench for the implementation of the technique consists of high–precision scales (measuring accuracy up to 0.01 g, maximum weight - 5 kg), an electronic caliper (measuring accuracy up to 0.01 mm) with 3D-printer pads that are attached to grips, as well as video cameras. Thanks to such a mobile stand, it was possible to conduct a series of experiments (a total of 99 tests) to determine the Young's modulus of atherosclerotic plaques and areas of vascular walls removed from the body no later than a few hours. This made it possible to collect a database of the mechanical characteristics of the plaques as close as possible to the real properties. In addition, regression dependences were constructed between the Hounsfield numbers corresponding to dense atherosclerotic deposits discernible on a CT scan and the Young modules obtained during experiments. Such dependencies will further allow determining the mechanical properties of plaques in vivo based on computed tomography data. The technique was verified by mechanical experiments on the universal testing machine Instron 3342 and on a mobile test bench on samples of hard (with pronounced calcification) and soft plaques. 7 experiments were conducted for each type of samples. The results differed by no more than 4.3 % for soft plaques and no more than 9.5 % for hard plaques. To test the inter-expert reliability of the methodology, a number of experiments were conducted with the involvement of two independent participants. Each of the three mobile stand operators performed 5 tests on soft rubber samples. The study of inter-expert reliability allowed us to show the independence of the methodology from the skills and qualifications of the operator.

Neuro-Urology and Biobanking: An Integrated Approach for Advancing Research and Improving Patient Care.

Understanding the molecular mechanisms underlying neuro-urological disorders is crucial for the development of targeted therapeutic interventions. Through the establishment of comprehensive biobanks, researchers can collect and store various biological specimens, including urine, blood, tissue, and DNA samples, to study these mechanisms. In the context of neuro-urology, biobanking facilitates the identification of genetic variations, epigenetic modifications, and gene expression patterns associated with neurogenic lower urinary tract dysfunction. These conditions often present as symptoms of neurological diseases such as Alzheimer's disease, multiple sclerosis, Parkinson's disease, spinal cord injury, and many others. Biobanking of tissue specimens from such patients is essential to understand why these diseases cause the respective symptoms and what can be done to alleviate them. The utilization of high-throughput technologies, such as next-generation sequencing and gene expression profiling, enables researchers to explore the molecular landscape of these conditions in an unprecedented manner. The development of specific and reliable biomarkers resulting from these efforts may help in early detection, accurate diagnosis, and effective monitoring of neuro-urological conditions, leading to improved patient care and management. Furthermore, these biomarkers could potentially facilitate the monitoring of novel therapies currently under investigation in neuro-urological clinical trials. This comprehensive review explores the synergistic integration of neuro-urology and biobanking, with particular emphasis on the translation of biobanking approaches in molecular research in neuro-urology. We discuss the advantages of biobanking in neuro-urological studies, the types of specimens collected and their applications in translational research. Furthermore, we highlight the importance of standardization and quality assurance when collecting samples and discuss challenges that may compromise sample quality and impose limitations on their subsequent utilization. Finally, we give recommendations for sampling in multicenter studies, examine sustainability issues associated with biobanking, and provide future directions for this dynamic field.

Prognostic impact of molecular profiles and molecular signatures in clear cell ovarian cancer

ObjectiveOvarian Clear cell carcinomas (OCCC) are characterized by low response to chemotherapy and a poor prognosis in advanced stages. Several studies have demonstrated that OCCC are heterogenous entities. We have earlier identified four molecular profiles based on the mutational status of ARID1A and PIK3CA. In this study we aimed to examine the association between molecular profiles, Tumor Mutational Burden (TMB), and molecular signatures with the clinical outcome in OCCC MethodsWe identified 55 OCCC cases with corresponding data and biological tissue samples in the Danish Gynecological Cancer Database during 2005-2016. Mutational profiling and TMB were performed using the Oncomine Tumor Mutational Load Assay. Chi-square and Cox regression analyses were used. P-values < 0.05 were considered statistically significant. ResultsMutations in the PIK3CA gene (p=0.04) and low TMB (p=0.05) were associated with disease progression. In multivariate analyses adjusted for stage, patients with tumor mutations in the ARID1A and/or PIK3CA genes had a significantly impaired Progression Free Survival (PFS) and Overall Survival (OS) compared to patients who were wildtype ARID1A and PIK3CA (undetermined subgroup) (HR= 5.42 and HR= 2.77, respectively). High TMB status was associated with an improved PFS (HR= 0.36) and OS (HR= 0.46). A trend towards an improved PFS in patients with APOBEC enrichment was observed (HR 0.45). ConclusionTMB-High was associated with decreased risk of progression and with an improved PFS and OS. Furthermore, OCCC with mutations in either ARID1A and/or PIK3CA genes had a significantly impaired prognosis compared to the undetermined subgroup in stage adjusted analyses.