Analysis Of Biological Samples Research Articles

Synergistically biomimetic platform that enables droplets to be self-propelled

Droplet transport still faces numerous challenges, such as a limited transport distance, large volume loss, and liquid contamination. Inspired by the principle of ‘synergistic biomimetics’, we propose a design for a platform that enables droplets to be self-propelled. The orchid leaf-like three-dimensional driving structure provides driving forces for the liquid droplets, whereas the lotus leaf-like superhydrophobic surface prevents liquid adhesion, and the bamboo-like nodes enable long-distance transport. During droplet transport, no external energy input is required, no fluid adhesion or residue is induced, and no contamination or mass loss of the fluid is caused. We explore the influence of various types and parameters of wedge structures on droplet transportation, the deceleration of droplet speed at nodal points, and the distribution of internal pressure. The results indicate that the transport platform exhibits insensitivity to pH value and temperature. It allows droplets to be transported with varying curvatures in a spatial environment, making it applicable in tasks like target collection, as well as load, fused, anti-gravity, and long-distance transport. The maximum droplet transport speed reached (58 ± 5) mm·s−1, whereas the transport distance extended to (136 ± 4) mm. The developed platform holds significant application prospects in the fields of biomedicine and chemistry, such as high-throughput screening of drugs, genomic bioanalysis, microfluidic chip technology for drug delivery, and analysis of biological samples.

Optimizing and Predicting Antidepressant Efficacy in Patients with Major Depressive Disorder Using Multi-Omics Analysis and the Opade AI Prediction Tools

According to the World Health Organization (WHO), major depressive disorder (MDD) is the fourth leading cause of disability worldwide and the second most common disease after cardiovascular events. Approximately 280 million people live with MDD, with incidence varying by age and gender (female to male ratio of approximately 2:1). Although a variety of antidepressants are available for the different forms of MDD, there is still a high degree of individual variability in response and tolerability. Given the complexity and clinical heterogeneity of these disorders, a shift from “canonical treatment” to personalized medicine with improved patient stratification is needed. OPADE is a non-profit study that researches biomarkers in MDD to tailor personalized drug treatments, integrating genetics, epigenetics, microbiome, immune response, and clinical data for analysis. A total of 350 patients between 14 and 50 years will be recruited in 6 Countries (Italy, Colombia, Spain, The Netherlands, Turkey) for 24 months. Real-time electroencephalogram (EEG) and patient cognitive assessment will be correlated with biological sample analysis. A patient empowerment tool will be deployed to ensure patient commitment and to translate patient stories into data. The resulting data will be used to train the artificial intelligence/machine learning (AI/ML) predictive tool.

Deep learning-based virtual H& E staining from label-free autofluorescence lifetime images

Label-free autofluorescence lifetime is a unique feature of the inherent fluorescence signals emitted by natural fluorophores in biological samples. Fluorescence lifetime imaging microscopy (FLIM) can capture these signals enabling comprehensive analyses of biological samples. Despite the fundamental importance and wide application of FLIM in biomedical and clinical sciences, existing methods for analysing FLIM images often struggle to provide rapid and precise interpretations without reliable references, such as histology images, which are usually unavailable alongside FLIM images. To address this issue, we propose a deep learning (DL)-based approach for generating virtual Hematoxylin and Eosin (H&E) staining. By combining an advanced DL model with a contemporary image quality metric, we can generate clinical-grade virtual H&E-stained images from label-free FLIM images acquired on unstained tissue samples. Our experiments also show that the inclusion of lifetime information, an extra dimension beyond intensity, results in more accurate reconstructions of virtual staining when compared to using intensity-only images. This advancement allows for the instant and accurate interpretation of FLIM images at the cellular level without the complexities associated with co-registering FLIM and histology images. Consequently, we are able to identify distinct lifetime signatures of seven different cell types commonly found in the tumour microenvironment, opening up new opportunities towards biomarker-free tissue histology using FLIM across multiple cancer types.

Lipidomics Study of Type 1 Diabetic Rats Using Online Phase Transition Trapping-Supercritical Fluid Extraction-Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Chromatography-mass spectrometry-based lipidomics represents an essential tool for elucidating lipid dysfunction mechanisms and is extensively employed in investigating disease mechanisms and identifying biomarkers. However, the detection of low-abundance lipids in biological matrices, along with cumbersome operational procedures, complicates comprehensive lipidomic analyses, necessitating the development of highly sensitive, environmentally friendly, and automated methods. In this study, an online phase transition trapping-supercritical fluid extraction-chromatography-mass spectrometry (PTT-SFEC-MS/MS) method was developed and successfully applied to plasma lipidomics analysis in Type 1 diabetes (T1D) rats. The PTT strategy captured entire extracts at the column head by converting CO2 from a supercritical state to a gaseous state, thereby preventing peak spreading, enhancing peak shape for precise quantification, and boosting sensitivity without any sample loss. This method utilized only 5 μL of plasma and accomplished sample extraction, separation, and detection within 27 min. Ultimately, 77 differential lipids were identified, including glycerophospholipids, sphingolipids, and glycerolipids, in T1D rat plasma. The results indicated that the progression of the disease might be linked to alterations in glycerophospholipid and sphingolipid metabolism. Our findings demonstrated a green, highly efficient, and automated method for the lipidomics analysis of biological samples, providing a scientific foundation for understanding the pathogenesis and diagnosis of T1D.

Unbiased method for spectral analysis of cells with great diversity of autofluorescence spectra.

Autofluorescence is an intrinsic feature of cells, caused by the natural emission of light by photo-excitatory molecular content, which can complicate analysis of flow cytometry data. Different cell types have different autofluorescence spectra and, even within one cell type, heterogeneity of autofluorescence spectra can be present, for example, as a consequence of activation status or metabolic changes. By using full spectrum flow cytometry, the emission spectrum of a fluorochrome is captured by a set of photo detectors across a range of wavelengths, creating an unique signature for that fluorochrome. This signature is then used to identify, or unmix, that fluorochrome's unique spectrum from a multicolor sample containing different fluorescent molecules. Importantly, this means that this technology can also be used to identify intrinsic autofluorescence signal of an unstained sample, which can be used for unmixing purposes and to separate the autofluorescence signal from the fluorophore signals. However, this only works if the sample has a singular, relatively homogeneous and bright autofluorescence spectrum. To analyze samples with heterogeneous autofluorescence spectral profiles, we setup an unbiased workflow to more quickly identify differing autofluorescence spectra present in a sample to include as "autofluorescence signatures" during the unmixing of the full stained samples. First, clusters of cells with similar autofluorescence spectra are identified by unbiased dimensional reduction and clustering of unstained cells. Then, unique autofluorescence clusters are determined and are used to improve the unmixing accuracy of the full stained sample. Independent of the intensity of the autofluorescence and immunophenotyping of cell subsets, this unbiased method allows for the identification of most of the distinct autofluorescence spectra present in a sample, leading to less confounding autofluorescence spillover and spread into extrinsic phenotyping markers. Furthermore, this method is equally useful for spectral analysis of different biological samples, including tissue cell suspensions, peripheral blood mononuclear cells, and in vitro cultures of (primary) cells.

Intelligent near-infrared light-activatable DNA machine with DNA wire nano-scaffold-integrated fast domino-like driving amplification for high-performance imaging in live biological samples

While there is significant potential for DNA machine-built enzyme-free fluorescence biosensors in the imaging analysis of live biological samples, they persist certain shortcomings. These encompass a deficiency of signal enrichment within a singular interface, uncontrolled premature activation during bio-delivery, and a slow reaction rate due to free nucleic acid collisions. In this contribution, we are committed to resolving the above challenges. Firstly, a single-interface-integrated domino-like driving amplification is constructed. In this conception, a specific target acts as the domino promotor (namely the energy source), initiating a cascading chain reaction that grafts onto a singular interface. Next, an 808 nm near-infrared (NIR) light-excited up-converting luminescence-induced light-activatable biosensing technique is introduced. By locking the target-specific identification segment with a photo-cleavage connector, the up-converted ultraviolet emission can activate target binding in a completely controlled manner. Moreover, a fast reaction rate is achieved by confining nucleic acid collisions within the surface of a DNA wire nano-scaffold, leading to a substantial enhancement in local contact concentration (30.8-fold increase, alongside a 15 times elevation in rate). When a non-coding microRNA (miRNA-221) is positioned as the model low-abundance target for proof-of-concept validation, our intelligent DNA machine demonstrates ultra-high sensitivity (with a limit of detection down to 62.65 fM) and good specificity for this hepatic malignant tumor-associated biomarker in solution detection. Going further, it is worth highlighting that the biosensing system can be employed to carry out high-performance imaging analysis in live bio-samples (ranging from the cellular level to the nude mouse body), thereby propelling the field of DNA machines in disease diagnosis.

CRIMINAL LIABILITY FOR ANALYSING GENOMIC DATA WITHOUT OWNER'S CONSENT: A COMPARATIVE STUDY

Background: In the rapidly evolving landscape of genomics and biotechnology, the United Arab Emirates (UAE) has launched the Emirates Genome Program to harness the potential of genomic technologies for advancing healthcare services. Central to this initiative is the informed and voluntary participation of citizens in genetic research aimed at contributing to national health objectives through genetic data utilisation. Notably, the enactment of UAE Decree-Law No. (49) of 2023 underscores the importance of safeguarding genomic privacy as a foundational element for data security and individual rights. This study addresses the challenge of striking a delicate balance between individual genetic rights and the pressing scientific and medical needs of genomic research. It aims to analyse the right to genomic privacy and examine crimes associated with the unauthorised analysis of biological samples. Methods: This research employed an analytical legal methodology and a comparative approach to explore the crime of disclosing genomic data. By analysing Decree-Law No. (49) of 2023 and other relevant Emirati legislation, we examined the legal framework governing genomic research and data protection in the UAE. Comparative legal analysis was then conducted between Emirati and French laws to identify similarities and differences in approaches to genomic data disclosure crimes. The study also considered international standards and ethical principles to provide a comprehensive, multidisciplinary understanding of the intersection of law, ethics, and science in genomic privacy. Results and conclusions: This study’s findings underscore the necessity of establishing a robust legal framework that safeguards individual rights and ensures the confidentiality of genetic data. Such measures are crucial for fostering public trust in genomic research and aligning the UAE's genomic endeavours with rigorous ethical and legal standards. Ultimately, Decree-Law No. (49) of 2023 exemplifies the state's commitment to promoting ethical and legal practices in genomic research, thereby facilitating sustainable advancements in medical science.

Innovative Solid-Phase Extraction Strategies for Improving the Advanced Chromatographic Determination of Drugs in Challenging Biological Samples.

In the past few decades, considerable scientific strides have been made in the subject of drug analysis in human biological samples. However, the risk caused by incorrect drug plasma levels in patients still remains an important concern. This review paper attempts to investigate the advances made over the last ten years in common sample preparation techniques (SPT) for biological samples based on solid sorbents, including solid-phase extraction (SPE) and solid-phase micro-extraction (SPME), and in particular in the field of molecularly imprinted polymers (MIPs), including non-stimuli-responsive and stimuli-responsive adsorbents. This class of materials is known as 'smart adsorbents', exhibiting tailored responses to various stimuli such as magnetic fields, pH, temperature, and light. Details are provided on how these advanced SPT are changing the landscape of modern drug analysis in their coupling with liquid chromatography-mass spectrometry (LC-MS) analytical techniques, a general term that includes high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), as well as any variation of MS, such as tandem (MS/MS), multiple-stage (MSn), and high-resolution (HRMS) mass spectrometry. Some notes are also provided on coupling with less-performing techniques, such as high-performance liquid chromatography with ultraviolet (HPLC-UV) and diode array detection (HPLC-DAD) detection. Finally, we provide a general review of the difficulties and benefits of the proposed approaches and the future prospects of this research area.

Polylactic acid micro/nanoplastic-induced hepatotoxicity: Investigating food and air sources via multi-omics

Micro/nanoplastics (MNPs) are detected in human liver, and pose significant risks to human health. Oral exposure to MNPs derived from non-biodegradable plastics can induce toxicity in mouse liver. Similarly, nasal exposure to non-biodegradable plastics can cause airway dysbiosis in mice. However, the hepatotoxicity induced by foodborne and airborne biodegradable MNPs remains poorly understood. Here we show the hepatotoxic effects of biodegradable polylactic acid (PLA) MNPs through multi-omics analysis of various biological samples from mice, including gut, fecal, nasal, lung, liver, and blood samples. Our results show that both foodborne and airborne PLA MNPs compromise liver function, disrupt serum antioxidant activity, and cause liver pathology. Specifically, foodborne MNPs lead to gut microbial dysbiosis, metabolic alterations in the gut and serum, and liver transcriptomic changes. Airborne MNPs affect nasal and lung microbiota, alter lung and serum metabolites, and disrupt liver transcriptomics. The gut Lachnospiraceae_NK4A136_group is a potential biomarker for foodborne PLA MNP exposure, while nasal unclassified_Muribaculaceae and lung Klebsiella are potential biomarkers for airborne PLA MNP exposure. The relevant results suggest that foodborne PLA MNPs could affect the “gut microbiota-gut-liver” axis and induce hepatoxicity, while airborne PLA MNPs could disrupt the “airway microbiota-lung-liver” axis and cause hepatoxicity. These findings have implications for diagnosing PLA MNPs-induced hepatotoxicity and managing biodegradable materials in the environment. Our current study could be a starting point for biodegradable MNPs-induced hepatotoxicity. More research is needed to verify and inhibit the pathways that are crucial to MNPs-induced hepatotoxicity.

Triple Sensing Modes for Triggered β-Galactosidase Activity Assays Based on Kaempferol-Deduced Silicon Nanoparticles and Biological Imaging of MCF-7 Breast Cancer Cells.

β-Galactosidase (β-Gala) is an essential biomarker enzyme for early detection of breast tumors and cellular senescence. Creating an accurate way to monitor β-Gala activity is critical for biological research and early cancer detection. This work used fluorometric, colorimetric, and paper-based color sensing approaches to determine β-Gala activity effectively. Via the sensing performance, the catalytic activity of β-Gala resulted in silicon nanoparticles (SiNPs), fluorescent indicators obtained via a one-pot hydrothermal process. As a standard enzymatic hydrolysis product of the substrate, kaempferol 3-O-β-d-galactopyranoside (KOβDG) caused the fluorometric signal to be attenuated on kaempferol-silicon nanoparticles (K-SiNPs). The sensing methods demonstrated a satisfactory linear response in sensing β-Gala and a low detection limit. The findings showed the low limit of detection (LOD) as 0.00057 and 0.098 U/mL for fluorometric and colorimetric, respectively. The designed probe was then used to evaluate the catalytic activity of β-Gala in yogurt and human serum, with recoveries ranging from 98.33 to 107.9%. The designed sensing approach was also applied to biological sample analysis. In contrast, breast cancer cells (MCF-7) were used as a model to test the in vitro toxicity and molecular fluorescence imaging potential of K-SiNPs. Hence, our fluorescent K-SiNPs can be used in the clinic to diagnose breast cellular carcinoma, since they can accurately measure the presence of invasive ductal carcinoma in serologic tests.

Development and Validation of an LC-MS/MS Method for the Determination of Plasma and Red Blood Cell Omega Fatty Acids: A Useful Diagnostic Tool

Development and Validation of an LC-MS/MS Method for the Determination of Plasma and Red Blood Cell Omega Fatty Acids: A Useful Diagnostic Tool

Incorporation of Three Different Optical Trains into the IR-MALDESI Mass Spectrometry Imaging Platform to Characterize Artemisia annua.

Artemisinin is the leading medication for the treatment of malaria and is only produced naturally in Artemisia annua. The localization of artemisinin in both the glandular and non-glandular trichomes of the plant makes it an ideal candidate for mass spectrometry imaging (MSI) as a model system for method development. Infrared matrix-assisted laser desorption electrospray ionization MSI (IR-MALDESI-MSI) has the capability to detect hundreds to thousands of analytes simultaneously, providing abundance information in conjunction with species localization throughout a sample. The development of several new optical trains and their application to the IR-MALDESI-MSI platform has improved data quality in previous proof-of-concept experiments but has not yet been applied to analysis of native biological samples, especially the MSI analysis of plants. This study aimed to develop a workflow and optimize MSI parameters, specifically the laser optical train, for the analysis of Artemisia annua with the NextGen IR-MALDESI platform coupled to an Orbitrap Exploris 240 mass spectrometer. Two laser optics were compared to the conventional set up, of which include a Schwarzschild-like reflective objective and a diffractive optical element (DOE). These optics, respectively, enhance the spatial resolution of imaging experiments or create a square spot shape for top-hat imaging. Ultimately, we incorporated and characterized three different optical trains into our analysis of Artemisia annua to study metabolites in the artemisinin pathway. These improvements in our workflow, resulted in high spatial resolution and improved ion abundance from previous work, which will allow us to address many different questions in plant biology beyond this model system.

Biomonitoring of volatile organic compounds and organophosphorus flame retardands in commercial aircrews after „fume and smell events“

Health risks to humans after „fume and smell events“, short-term incidents on aircrafts that are accompanied by unpleasant odour or visible smoke, remain a subject of controversy. We assessed exposure to volatile organic compounds (VOC) and organophosphorus compounds (OPC) by biomonitoring in 375 aircrew members after self-reported “fume and smell events” and in 88 persons of the general population. A total of 20 parameters were analysed in blood and urine by gas chromatography and mass spectrometry. Median levels of acetone in blood and urine and 2-propanol in blood were elevated in aircrews compared to controls (p < 0.0001). Additionally, elevated peak exposures, best estimated by the 95th percentiles, were observed in aircrews for n-heptane and n-octane in blood, and acetone, 2,5-hexanedione and o-cresol in urine. Only the maximum observed levels of 2,5-hexandione in urine (768 μg/L) and toluene in blood (77 μg/L) in aircrew members were higher than the current biological exposure indices (BEI® levels) (500 and 20 μg/L, respectively) of the American Conference of Governmental Industrial Hygienists (US-ACGIH) for workers occupationally exposed to n-hexane and toluene, two well-accepted human neurotoxicants. Low-level exposures to n-hexane and toluene could be also observed in controls. The majority of OPC parameters in urine, including those of neurotoxic ortho-isomers of tricresylphosphate, were below the limit of quantitation in both aircrews and controls. Our comparative VOC and OPC analyses in biological samples of a large number of aircrew members and controls suggest that exposures are similar in both groups and generally low.

Development of CO2-switchable deep eutectic solvent-based liquid-liquid microextraction approach: Application in urinary excretion and tissue distribution studies

BackgroundPharmacokinetic studies are pivotal in drug development, focusing on absorption, distribution, and excretion of active compounds. Effective sample preparation methods play a crucial role in these studies. Traditional techniques like protein precipitation and liquid-liquid extraction often involve toxic solvents and are time-consuming. Recently, deep eutectic solvent (DES) has emerged as an eco-friendly alternative due to its high efficiency, low cost, and low toxicity. This study introduces a novel sample pretreatment method using CO2-switchable DES in liquid-liquid microextraction (LLME) to enhance speed, accuracy, and sensitivity in complex biological samples analysis. ResultsA liquid-liquid microextraction sample pretreatment method based on switchable DES combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the analysis of urine and tissue samples. The method was optimized through systematic investigation of key parameters, including DES type, volume, molar ratio, pH, vortex time, gas purge time, and salt addition. The resulting procedure exhibited satisfying linearity (r2 ≥ 0.9958), good precision (RSD ≤6.01 %), desirable recovery (52.44%–98.12 %) and matrix effect (86.22%–119.30 %), and the accuracy and precision of stability were within the ±15 % limit. The proven methods were further applied to urinary excretion study and tissue distribution study of Nelumbinis plumula (NP) extract. The results indicated that the total cumulative excretion of liensinine, isoliensinine and neferine in urine within 240 h was 4.96 %, 0.66 % and 0.44 %, respectively. The tissue distribution study showed that alkaloids mainly distribute in liver, kidney, and spleen. SignificanceThis research introduces a groundbreaking technique distinguished by its simplicity, speed, cost-effectiveness, and environmental friendliness. This approach, utilizing CO2-switchable DES as an extraction solvent for LLME, integrates deproteinization and removal of interfering molecules into a single step. This integration showcases its efficiency and convenience, demonstrating significant promise for various applications in the analysis of biological samples. Additionally, this study provides the first report on urinary excretion and tissue distribution of alkaloids from NP using a DES-LLME method. These findings offer valuable insights into the in vivo behavior of herbal medicine, enhancing understanding of pharmacological actions and facilitating clinical rational administration.

Bridging biological samples to functional nucleic acid biosensor applications: current enzymatic-based strategies for single-stranded DNA generation.

The escalating threat of emerging diseases, often stemming from contaminants and lethal pathogens, has precipitated a heightened demand for sophisticated diagnostic tools. Within this landscape, the functional nucleic acid (FNA) biosensor, harnessing the power of single-stranded DNA (ssDNA), has emerged as a preeminent choice for target analyte detection. However, the dependence on ssDNA has raised difficulties in realizing it in biological samples. Therefore, the production of high-quality ssDNA from biological samples is critical. This review aims to discuss strategies for generating ssDNA from biological samples for integration into biosensors. Several innovative strategies for ssDNA generation have been deployed, encompassing techniques, such as asymmetric PCR, Exonuclease-PCR, isothermal amplification, biotin-streptavidin PCR, transcription-reverse transcription, ssDNA overhang generation, and urea denaturation PAGE. These approaches have been seamlessly integrated with biosensors for biological sample analysis, ushering in a new era of disease detection and monitoring. This amalgamation of ssDNA generation techniques with biosensing applications holds significant promise, not only in improving the speed and accuracy of diagnostic processes but also in fortifying the global response to deadly diseases, thereby underlining the pivotal role of cutting-edge biotechnology in public health and disease prevention.

Preparation of a β-cyclodextrin grafted magnetic biochar for efficient extraction of four antiepileptic drugs in plasma samples

Simultaneous monitoring of plasma concentration levels of multiple antiepileptic drugs (AEDs) is essential for dose adjustment in comprehensive epilepsy treatment, necessitating a sensitive technique for accurate extraction and determination of AEDs. Herein, a magnetic solid-phase extraction (MSPE) technique on the basis of modified biochar (BC) is investigated to extract four AEDs from plasma, in conjunction with high performance liquid chromatography. BC derived from Zizyphus jujuba seed shells was activated by phosphoric acid (PBC) and magnetized via coprecipitation to produce MPBC. The MPBCCD obtained after modification with β-cyclodextrin (CD) was characterized and evaluated for adsorption. It exhibited fast adsorption kinetics based on second-order kinetics and satisfactory adsorption capacity for AEDs. Then it was employed as the MSPE adsorbent and the influencing parameters were optimized. The enrichment factor was 18.75. The validation analysis revealed a favorable linearity that ranged from 0.04 to 20 μg·mL−1 along with a low limit of detection of 6.85 to 10.19 ng·mL−1. The recovery of the AEDs ranged from 78.7 to 109.2 %, with relative standard deviations below 6.7 %. Using quantum chemistry theory calculations and experimental results analysis, the adsorption mechanism was investigated. It disclosed that the suggested strategy built upon MPBCCD was appropriate for the assessment of AEDs in plasma and expanded the usage of BC as the environmentally favorable matrix for the analysis of biological samples.

Determination of Δ9-tetrahydrocannabinol, 11-nor-carboxy-Δ9-tetrahydrocannabinol and cannabidiol in human plasma and urine after a commercial cannabidiol oil product intake.

Cannabidiol (CBD) products are widely used for pain relief, sleep improvement, management of seizures etc. Although the concentrations of Δ9-tetrahydrocannabinol (Δ9-THC) in these products are low (≤0.3% w/w), it is important to investigate if its presence and/or that of its metabolite 11-nor-carboxy-Δ9-THC, is traceable in plasma and urine samples of individuals who take CBD oil products. A sensitive GC/MS method for the determination of Δ9-THC, 11-nor-carboxy-Δ9-THC and CBD in plasma and urine samples was developed and validated. The sample preparation procedure included protein precipitation for plasma samples and hydrolysis for urine samples, solid-phase extraction and finally derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide) with 1% trimethylchlorosilane. For all analytes, the LOD and LOQ were 0.06 and 0.20ng/mL, respectively. The calibration curves were linear (R2 ≥ 0.992), and absolute recoveries were ≥91.7%. Accuracy and precision were within the accepted range. From the analysis of biologic samples of 10 human participants who were taking CBD oil, it was realized that Δ9-THC was not detected in urine, while 11-nor-carboxy-Δ9-THC (0.69-23.06ng/mL) and CBD (0.29-96.78ng/mL) were found in all urine samples. Regarding plasma samples, Δ9-THC (0.21-0.62ng/mL) was detected in 10, 11-nor-carboxy-Δ9-THC (0.20-2.44ng/mL) in 35, while CBD (0.20-1.58ng/mL) in 25 out of 38 samples, respectively. The results showed that Δ9-THC is likely to be found in plasma although at low concentrations. In addition, the detection of 11-nor-carboxy-Δ9-THC in both urine and plasma samples raises questions and concerns for the proper interpretation of toxicological results, especially considering Greece's zero tolerance law applied in DUID and workplace cases.

Abstract CT139: Immunological activity of oncolytic adenovirus encoding TNFα and IL-2 (TILT-123) in combination with pembrolizumab in platinum resistant or refractory ovarian cancer patients

Abstract Background TILT-123 (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2) is a serotype chimeric dual safety device oncolytic adenovirus armed with tumor necrosis factor alpha and interleukin-2. It was designed to improve efficacy of T-cell therapies such as immune checkpoint blockade or adoptive cell transfer. We present biological and immunological endpoints from an open-label phase 1 dose escalation study of TILT-123 in combination with anti-PD-1 pembrolizumab in platinum resistant or refractory ovarian cancer patients. Experimental procedures In this dose escalation trial (NCT05271318), patients were first injected with an intravenous (i.v.) dose of TILT-123 ranging from 3x1011 to 4x1012 virus particles (VP), followed by five intratumoural (i.t.) or intraperitoneal (i.p.) injections ranging from 1x1011 to 5x1011 VP. Patients also received intravenous injections of 200 mg pembrolizumab every three weeks starting 36 days after the first dose of TILT-123. The primary endpoint was safety, while efficacy by RECIST1.1 was a secondary endpoint. Collection of tumor biopsies, circulating immune cells, serum, saliva, urine and feces enabled multi-omic evaluation of mechanism of action and identification of predictive biomarkers. Summary of data As of January 2nd 2024, 15 patients received trial therapy. No dose-limiting toxicities were observed and the most frequent AEs associated with therapy included chills and nausea (&amp;gt;10% of patients). Stable disease by RECIST 1.1 was seen in 67% in 12 evaluable patients. TILT-123 replication was detected in both injected and non-injected tumors as assessed by qPCR and anti-hexon IHC. Multiplex immunofluorescence demonstrated induction of robust immunological activity in both injected and non-injected tumors, including significant increase in granzyme B+, PD-1+ CD4+ and CD8+ T cells as well as PD-1+ NK cells. Baseline intratumoral CD4+, FOXP3+ regulatory T cells were not associated with clinical response. Tertiary lymphoid structures and lymphoid aggregates were identified in patients showing long term disease control. Low to medium titers of neutralizing antibodies against 5/3 chimeric adenovirus were detected in 57% of patients at baseline and were not a predictive factor of disease control. No patients had high titers at baseline. Conclusion TILT-123 in combination with anti-PD-1 pembrolizumab was safe and resulted in disease control in both platinum resistant and refractory ovarian cancer patients. Analysis of biological samples revealed insights into mechanism of action, including an immune profile predictive of clinical response. These results, together with those from four other trials (NCT04695327, NCT04217473, NCT06125197 and NCT05222932) set the stage for continued clinical evaluation of TILT-123. Citation Format: James H. Clubb, Matthew S. Block, Johanna Mäenpää, Santeri Pakola, Lyna Haybout, Dafne C. Quixabeira, Tatiana Kudling, Mirte van der Heijden, Elise Jirovec, Tuomo Alanko, Daniel A. Adamo, Susan Ramadan, Jorma Sormunen, Juha Kononen, Julia W. Cohen, Nadena Singh, John Goldfinch, Suvi Sorsa, Riikka Havunen, Claudia Kistler, João M. Santos, Víctor Cervera-Carrascon, Akseli Hemminki. Immunological activity of oncolytic adenovirus encoding TNFα and IL-2 (TILT-123) in combination with pembrolizumab in platinum resistant or refractory ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT139.

Enhancing comparative T cell receptor repertoire analysis in small biological samples through pooling homologous cell samples from multiple mice

Accurate characterization and comparison of Tcell receptor (TCR) repertoires from small biological samples present significant challenges. The main challenge is the low material input, which compromises the quality of bulk sequencing and hinders the recovery of sufficient TCR sequences for robust analyses. We aimed to address this limitation by implementing a strategic approach to pool homologous biological samples. Our findings demonstrate that such pooling indeed enhances the TCR repertoire coverage, particularly for cell subsets of constrained sizes, and enables accurate comparisons of TCR repertoires at different levels of complexity across Tcell subsets with different sizes. This methodology holds promise for advancing our understanding of Tcell repertoires in scenarios where sample size constraints are a prevailing concern.

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. We found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.