Quantification Of Activity Research Articles

Marine Infrastructure Detection with Satellite Data—A Review

A rapid development of marine infrastructures can be observed along the global coasts. Offshore wind farms, oil and gas platforms, artificial islands, aquaculture, and more, are being constructed without a proper quantification of these human activities. Therefore, effective monitoring is required to maintain transparency towards environmental standards, marine resource management, inventorying objects, and global security. This study reviews remote sensing-based approaches to offshore infrastructure detection over the past 12 years. We analyzed 89 studies from over 30 scientific journals, highlighting spatial and temporal trends, methodological approaches, and regional and thematic research foci. Our results show a significant increase in research interest, especially since 2019. Asia, and especially China, is the predominant focus region in terms of first authorship, funding, and areas of investigation. Aquaculture is the most studied infrastructure, followed by platforms, offshore wind farms, and artificial islands. Gaofen, Sentinel, and Landsat are the most used satellite sensors for detection. The apparent shift towards automated detection methods, especially Deep Learning algorithms, reflects advances in computer vision. This study highlights the key role of earth observation in the field of off-shore infrastructure detection, which can contribute towards outlining effective monitoring practices for marine activities, as well as highlighting important knowledge gaps.

Nepeta cataria L. (catnip) can serve as a chassis for the engineering of secondary metabolic pathways

ObjectiveEvaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences.ResultsThe reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 μmol g−1 s−1, N. cataria cultivar ‘1000’, and the cultivar ‘Citriodora’ showed an activity of 37 ± 8 μmol g−1 s−1 and 18 ± 4 μmol g−1 s−1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides.ConclusionNepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.

Free radical scavenging, α-amylase, α-glucosidase, and lipase inhibitory activities of metabolites from strawberry kombucha: Molecular docking and in vitro studies

Obesity, a global issue, is linked to cardiometabolic syndrome. Dietary modification is one of the recommended modes for managing cardiometabolic syndrome. Strawberries, a functional food, and kombucha, a fermented tea beverage, have gained attention for their health benefits. This study investigated the bioactive components of strawberry kombucha drink (SKD) and their effects on antioxidant activities and improving metabolic disorder markers. An in vitro experiment was performed to determine the effect of SKD on enzymatic parameters: lipase, α-glucosidase, and α-amylase activities. In addition, antioxidant activity using the DPPH method and quantification of the radical scavenging activity were also measured. Furthermore, untargeted metabolomic profiling of SKD and molecular docking simulation were conducted. The findings suggest that SKD, rich in secondary metabolites, can inhibit lipase, α-glucosidase, and α-amylase activities. It demonstrated in vitro anti-obesity, anti-diabetic, and antioxidant properties, potentially reducing metabolic and inflammatory issues. Thus, SKD could be a therapeutic beverage to alter metabolic issues associated with obesity. Nevertheless, further preclinical study is warranted to determine SKD’s potential in vivo.

A novel method for accurate and real-time quantification of radiopharmaceutical activity in automated dose dispensers

In recent years, there has been a rapid growth in new technologies within nuclear medicine and PET diagnostics, particularly in the field of radiopharmaceutical dose preparation. These advancements are driven by the integration of automatic algorithms into the process. This evolution has brought about significant improvements in the efficiency and accuracy of radiopharmaceutical dose preparation. These automatic algorithms utilize state-of-the-art technologies to streamline the process and ensure precise and tailored doses for patients. The integration of these advanced technologies has revolutionized the field, allowing for more effective and personalized treatment options in nuclear medicine and PET diagnostics. By employing automatic radiopharmaceutical dose dispensers (ARDDs), radiation exposure to technologists is minimized while also mitigating the risk of potential infections for patients. Ionization chambers are currently the most used detectors to measure the activity of radiopharmaceuticals in ARDDs. However, they present certain limitations, including size constraints. This study is part of an international endeavor to develop a new approach for evaluating radiopharmaceutical activity in an ARDD named KARl 100. A new approach was used to develop a scintillation detector by linking a PVT detector to a SiPMs array consisting of four elements, each having an active surface of 6 × 6 mm with a bias voltage of 30 V. The detection system's amplified output was then fed to a single-channel analyzer called Enviro, developed by Tema Sinergie S.p.A in Italy. The counts per second (CPS) detected by the detector were studied using WinTAM software allowing for the radionuclide’s activity concentration to be read out at run-time. Three widely used medical radioisotopes, 18F, 11C and 68Ga, were used to evaluate the performance of the detector in terms of linearity and repeatability of the measurements. Half-life and linear response were evaluated over a wide range of activities with different decay schemes. Data analysis was conducted, resulting in half-lives of the 18F, 11C and 68Ga radioisotopes of 109.75 ± 0.35, 20.62 ± 0.34 and 67.82 ± 0.25 min, respectively, with calculated deviations of 0.0167%, 1.00% and 0.29% from expected values. The linear response of the detector over the entire range of desired activity concentrations was confirmed after data correction for the detector dead time. Calibration of the detector combined with the dispenser system was carried out in parallel investigations to provide the required device properties for reference applications in nuclear medicine. Furthermore, Monte Carlo simulations using FLUKA code were performed to investigate the contribution of beta particles in addition to gamma radiations to assess the influence of beta particles on the calibration factors. For better accuracy, a shielding was designed to prevent any beta particles from reaching the detector.

Bioactive compounds, Antioxidant and Antimicrobial Attributes of Cordia Sinensis Lam

The aim of this study is to discover the efficacy of various parts of Cordia sinensis plant against some particular pathogens that are recognized to cause diseases and to check antioxidant and bioactive compounds from different parts of plant C. sinensis. Plant extracts were examined by quantification of phytochemical compounds and antimicrobial activity. Phytochemicals and several secondary metabolites were quantified and also qualitatively analyzed in C. sinensis extracts such as phenolic compounds, flavonoids, alkaloids, tannins, steroids, glycosides, and saponins. Similarly, biochemical primary metabolites like protein, total sugar, and reducing sugar were also estimated in different parts of C. sinensis and these results correlated with antimicrobial activity. This study reveals that acetone extracts of stems, leaves, and roots of the C. sinensis showed excellent antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumonia and also against fungal species Aspergillus niger, Candida albicans, and Penicillium notatum.

Analysis of Brain Regions Activated by Afferent Renal Nerves in DOCA-Salt Hypertensive Mice

We have reported that the neurogenic component of DOCA-salt HTN is dependent on afferent renal nerves (ARNs) that modulate central autonomic networks, however, the central pathways are poorly understood. Targeted recombination in active populations (TRAP2) mice provide a model for global visualization and quantification of neuronal activity via the expression of the fluorescent reporter protein tdTomato which is expressed under the immediate-early gene, c-Fos, a neuronal activation marker, via tamoxifen-inducible Cre. In combination with the deoxycorticosterone-acetate (DOCA)-salt model of hypertension (HTN), neuronal activation in central autonomic centers can be evaluated in TRAP2 mice during DOCA-salt-induced HTN to identify the role of ARNs in the control of blood pressure. We evaluated the expression of tdTomato in TRAP2 mice as a measure of c-Fos activation following 7 days of DOCA-salt HTN and compared it to immunolabeled c-Fos at day 21 of DOCA-salt HTN in brain regions known to be involved in blood pressure regulation. We hypothesized that DOCA will increase the expression of tdTomato and c-Fos in mice with intact renal nerves and that afferent renal denervation (ARDN) will attenuate this DOCA-induced neuronal activity. Three groups of TRAP2-DOCA mice were studied: normotensive (Vehicle-Sham), hypertensive (DOCA-Sham), hypertensive with ARDN (DOCA-ARDN). Mice received a nephrectomy, sham or ARDN, implant of 50mg DOCA and 0.9% saline drinking water for the length of the study (drug-free pellet and diH2O for vehicle). TRAP2-DOCA-salt mice received an injection of 5mg/mL 4-hydroxytamoxifen (4-OHT), a short acting tamoxifen metabolite, on day 7 to induce Cre recombination and expression of tdTomato during the development of HTN. On day 21, mice were anesthetized and perfused with 4% paraformaldehyde. Brains were immunolabeled for c-Fos expression indicating day 21 neuronal activation and compared to endogenous Cre-induced tdTomato expression from day 7 4-OHT. Preliminary data indicate increased neuronal activity in TRAP2 DOCA-Sham mice compared to Vehicle-Sham in the paraventricular nucleus of the hypothalamus, lateral parabrachial nucleus, and nucleus of the solitary tract. This activation pattern was reduced in TRAP2 DOCA-ARDN compared to TRAP2 DOCA-Sham mice. A future study will evaluate c-Fos expression in wild-type DOCA-salt mice at day 7 and 21 of DOCA-salt HTN development to validate the observations in the TRAP2-DOCA model. This experiment will also validate TRAPing as a method for measuring neuronal activation at two time points within the same animal. These preliminary results establish an anatomical foundation for future investigations into the physiological role of ARN input to central autonomic control centers that regulate blood pressure in a salt-induced hypertensive model. NIH R01 HL116476. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Predicting classroom activity index through multi-scale head posture classification network

The quantification of the interplay between student behavior data and classroom teaching effectiveness using quantitative metrics has perennially posed a challenge in the evaluation of classroom instruction. Classroom activity serves as a reflection of student engagement, emotional ambiance, and other pertinent aspects during the pedagogical process. This article presents a methodology for quantifying student head posture during classroom instruction utilizing AI-driven video analysis technology, notably the Classroom Activity Index (CAI). A Classroom Activity Analysis System (CAAS) was designed and developed, integrating a multi-scale classification network based on ECA-ResNet50 and ECA-ResNet18. This network discerns and categorizes various head regions of students situated in both the frontal and real rows of a lecture-style classroom, irrespective of their dimensions. The classification network attains exceptional performance, boasting F1 score of 0.91 and 0.92 for student head-up and head-nodding. Drawing on the live classroom instruction at a higher vocational college in Wuhan, Hubei Province, China, a comparative experiment was executed. The findings revealed that three factors: teacher-student verbal interaction, teacher body language, and utilization of digital resource, all exert an influence on CAI. Simultaneously, the degree of classroom activity as gauged by FIAS and manual analysis fundamentally aligns with the CAI indicators quantified by CAAS, validating the efficacy of CAI in the quantification of classroom activity. Consequently, the incorporation of CAAS in teaching, research, and oversight scenarios can augment the precision and scientific rigor of classroom teaching assessment.

A Deep-Learning-Based Partial-Volume Correction Method for Quantitative 177Lu SPECT/CT Imaging.

With the development of new radiopharmaceutical therapies, quantitative SPECT/CT has progressively emerged as a crucial tool for dosimetry. One major obstacle of SPECT is its poor resolution, which results in blurring of the activity distribution. Especially for small objects, this so-called partial-volume effect limits the accuracy of activity quantification. Numerous methods for partial-volume correction (PVC) have been proposed, but most methods have the disadvantage of assuming a spatially invariant resolution of the imaging system, which does not hold for SPECT. Furthermore, most methods require a segmentation based on anatomic information. Methods: We introduce DL-PVC, a methodology for PVC of 177Lu SPECT/CT imaging using deep learning (DL). Training was based on a dataset of 10,000 random activity distributions placed in extended cardiac-torso body phantoms. Realistic SPECT acquisitions were created using the SIMIND Monte Carlo simulation program. SPECT reconstructions without and with resolution modeling were performed using the CASToR and STIR reconstruction software, respectively. The pairs of ground-truth activity distributions and simulated SPECT images were used for training various U-Nets. Quantitative analysis of the performance of these U-Nets was based on metrics such as the structural similarity index measure or normalized root-mean-square error, but also on volume activity accuracy, a new metric that describes the fraction of voxels in which the determined activity concentration deviates from the true activity concentration by less than a certain margin. On the basis of this analysis, the optimal parameters for normalization, input size, and network architecture were identified. Results: Our simulation-based analysis revealed that DL-PVC (0.95/7.8%/35.8% for structural similarity index measure/normalized root-mean-square error/volume activity accuracy) outperforms SPECT without PVC (0.89/10.4%/12.1%) and after iterative Yang PVC (0.94/8.6%/15.1%). Additionally, we validated DL-PVC on 177Lu SPECT/CT measurements of 3-dimensionally printed phantoms of different geometries. Although DL-PVC showed activity recovery similar to that of the iterative Yang method, no segmentation was required. In addition, DL-PVC was able to correct other image artifacts such as Gibbs ringing, making it clearly superior at the voxel level. Conclusion: In this work, we demonstrate the added value of DL-PVC for quantitative 177Lu SPECT/CT. Our analysis validates the functionality of DL-PVC and paves the way for future deployment on clinical image data.

Metabolically Tailored Selection of Ornamental Rose Cultivars through Polyamine Profiling, Osmolyte Quantification and Evaluation of Antioxidant Activities

Roses (genus Rosa), renowned for their economic significance and aesthetic appeal, face multifaceted challenges in cultivation due to biotic and abiotic stressors. To address these challenges, this study explores the role of osmolytes, particularly polyamines, proline and glycine betaine, as well as antioxidant capacities and condensed tannins, in enhancing stress tolerance in roses. Despite the genetic diversity inherent in roses, the metabolic aspect of stress tolerance has been underexplored in breeding programs. This paper investigates the intraspecific variability among 22 rose cultivars, focusing on osmolyte content (proline and glycine betaine), individual polyamines (putrescine, spermine and spermidine), as well as antioxidant activities, measuring radical scavenging capacity against 2,2′-azinobis(3-ethylbenzothiozoline-6-sulfonic acid (ABTS•+) and NO• radicals. Employing a targeted metabolomic approach, we quantified the levels of individual polyamines in both the petals and leaves of rose cultivars. This was achieved through high-performance liquid chromatography coupled with fluorescent detection following a derivatization pretreatment process. Within the evaluated cultivars, “Unique Aroma”, “Andre Rieu”, “Aroma 3”, “Frayla Marija” and “Trendy Fashion” stood out for their significantly elevated levels of total foliar polyamines. The predominant polyamine detected at both petal and leaf levels was putrescine, with concentrations ranging from 335.81 (“Zora Frayla”) to 2063.81 nmol g−1 DW (“Unique Aroma”) at the leaf level. Following putrescine, foliar spermidine levels varied from 245.08 (“Olivera Frayla”) to 1527.16 nmol g−1 DW (“Andre Rieu”). Regarding antioxidant capacity, the leaf extracts of rose cultivars “Zora Frayla” and “Natalija Frayla” were prominent by showing 68.08 and 59.24 mmol Trolox equivalents (TE) g−1 DW, respectively. The results highlight the intricate biochemical variability across rose cultivars and show that osmolytes, such as glycine betaine, proline and polyamines, and other biochemical markers can be used as reliable criteria for the selection of rose cultivars that are more resilient to biotic stress factors, especially powdery and downy mildew. Bridging fundamental research with practical applications, this study aims to contribute to the development of stress-tolerant rose cultivars adaptable to dynamic environmental conditions.

Efficient extraction methods and callus culture of quercetin from Indian white radish: yield analysis via HPLC quantification and assessment of cytotoxic activity

SummaryIndian white radish Co. 1 (Raphanus sativus L.) contains medicinal quercetin, and its commercial value supports large‐scale quercetin isolation. Various extraction methods, including ultra‐assisted extraction (UAE), maceration extraction (ME), Soxhlet extraction (SE), conventional extraction (CSE) and serial maceration extraction (SME), were employed to extract quercetin from radish leaf sections. RP‐HPLC‐optimising key experimental parameters yielded a linear response for the individual target compounds in the concentration range, with a limit of quantification (LOQ) of 2.2 μg mL−1 (r2 = 0.9995), a limit of detection (LOD) of 2.2 μg per 1.0 g of crushed leaves and repeatability within the relative standard deviation (RSD) of 0.23–0.37%. For in vitro callus cultures, 2,4‐dichlorophenoxyacetic acid (2,4‐D), 6‐benzylaminopurine (BAP) and zeatin were used for callus induction from in vitro leaf segments of radish. MS medium enriched with 2,4‐D (0.5 mg) induced the highest percentage of callus at 4 weeks. Among the extraction methods, the SME method yielded the highest recovery of quercetin (96.9%; 1.92 mg g−1 DW), phenolic (26.07 mg g−1 DW) and flavonoid (34.03 mg g−1 DW), compared to other methods and solvents. The callus extract included 0.5 mg of 2,4‐D, 32.07 mg of phenolic, 42.03 mg of flavonoid and 6.46 mg of quercetin. The presence of phytocompounds and quercetin was confirmed by GC–MS/MS and RP‐HPLC. The isolated quercetin (57.9 μg) also showed lethal effects on HT‐29 human colon cancer cells. This method may be effective for evaluating quercetin in radish extracts.

A Critical View on the Quantification of Model Catalyst Activity

The conversion of reactants, reaction rate referred to catalyst mass, and turnover frequency (TOF) are values typically employed to compare the activity of different catalysts. However, experimental parameters have to be chosen carefully when systems of different complexity are compared. In order to characterize UHV-based model systems, we use a highly sensitive sniffer setup which allows us to investigate the catalytic activity by combining three different measurement modes: temperature-programmed desorption, continuous flow, and pulsed-reactivity experiments. In this article, we explore the caveats of quantifying catalytic activity in UHV on the well-studied and highly defined reference system of CO oxidation on Pt(111), which we later compare to the same reaction on Pt19 clusters deposited on Fe3O4(001). We demonstrate that we can apply fast heating ramps for TOF quantification, thus inducing as little sintering as possible in the metastable clusters. By changing the reactant ratio, we find transient reactivity effects that influence the TOF, which should be kept in mind when comparing catalysts. In addition, the TOF also depends on the surface coverage that itself is a function of temperature and pressure. At a constant reactant ratio, in the absence of transient effects, however, the TOF scales linearly with total pressure over the entire measured temperature range from 200 to 700 K since the reaction rate is dependent on both reactant partial pressures with temperature-dependent reaction order. When comparing the maximum TOF at this particular reactant ratio, we find a 1.6 times higher maximum TOF for Pt19/Fe3O4(001) than for Pt(111). In addition, pulsed-reactivity measurements help identify purely reaction-limited regimes and allow for a more detailed investigation of limiting reactants over the whole temperature range.

Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology

Cellulose-degrading microorganisms hold immense significance in utilizing cellulose resources efficiently. The screening of natural cellulase bacteria and the optimization of fermentation conditions are the hot spots of research. This study meticulously screened cellulose-degrading bacteria from mixed soil samples adopting a multi-step approach, encompassing preliminary culture medium screening, Congo red medium-based re-screening, and quantification of cellulase activity across various strains. Particularly, three robust cellulase-producing strains were identified: A24 (MT740356.1 Brevibacillus borstelensis), A49 (MT740358.1 Bacillus cereus), and A61 (MT740357.1 Paenibacillus sp.). For subsequent cultivation experiments, the growth curves of the three obtained isolates were monitored diligently. Additionally, optimal CMCase production conditions were determined, keeping CMCase activity as a key metric, through a series of single-factor experiments: agitation speed, cultivation temperature, unit medium concentration, and inoculum volume. Maximum CMCase production was observed at 150 rpm/37 °C, doubling the unit medium addition, and a 5 mL inoculation volume. Further optimization was conducted using the selected isolate A49 employing response surface methodology. The software model recommended a 2.21fold unit medium addition, 36.11 °C temperature, and 4.91 mL inoculant volume for optimal CMCase production. Consequently, three parallel experiments were conducted based on predicted conditions consistently yielding an average CMCase production activity of 15.63 U/mL, closely aligning with the predicted value of 16.41 U/mL. These findings validated the reliability of the model and demonstrated the effectiveness of optimized CMCase production conditions for isolate A49.

Multifunctional Sr, Mg, Ag-substituted octacalcium phosphate/carboxymethyl chitosan scaffolds: Antibacterial activity and osteogenic differentiation of human mesenchymal stem cells

Numerous studies indicate that octacalcium phosphate (OCP) possesses promising bone regenerative properties. To further improve the regenerative properties of OCP, several ionic substitutions have been proposed. However, knowledge regarding the synergic effect of multiple ions substituted in OCP crystals and fabricated scaffolds is lacking. In this work, plate-like OCP crystals substituted with 1 and 2.5 mol.% of Mg2+, Sr2+ and Ag+ were prepared by the wet precipitation method. Rietveld refinement analysis indicated a successful substitution of Ca2+ by Mg2+, Sr2+and Ag+ ions. The prepared substituted OCP powders showed a high antibacterial effect towards Gram-negative and Gram-positive bacteria. Further, composite scaffolds based on substituted OCP and carboxymethyl chitosan were prepared by the freeze-crosslinking method. The scaffolds had a highly porous structure, with interconnected pores and homogeneously dispersed OCP within the polymer matrix, and high stability in simulated physiological conditions during 28 days. Good viability and proliferation of human bone marrow-derived mesenchymal stem/stromal cells were confirmed by Live/Dead and CyQUANT assays. Additionally, successful osteogenic differentiation was confirmed by quantification of alkaline phosphatase activity and immunocytochemical staining for type I collagen. Results indicate that ionic substitutions with 1.0 mol.% have the most beneficial effect on stem cell differentiation towards osteoblast phenotype.

Activity quantification and dosimetry in radiopharmaceutical therapy with reference to 177Lutetium.

Radiopharmaceutical therapy has been widely adopted owing primarily to the development of novel radiopharmaceuticals. To fully utilize the potential of these RPTs in the era of precision medicine, therapy must be optimized to the patient's tumor characteristics. The vastly disparate dosimetry methodologies need to be harmonized as the first step towards this. Multiple factors play a crucial role in the shift from empirical activity administration to patient-specific dosimetry-based administrations from RPT. Factors such as variable responses seen in patients with presumably similar clinical characteristics underscore the need to standardize and validate dosimetry calculations. These efforts combined with ongoing initiatives to streamline the dosimetry process facilitate the implementation of radiomolecular precision oncology. However, various challenges hinder the widespread adoption of personalized dosimetry-based activity administration, particularly when compared to the more convenient and resource-efficient approach of empiric activity administration. This review outlines the fundamental principles, procedures, and methodologies related to image activity quantification and dosimetry with a specific focus on 177Lutetium-based radiopharmaceuticals.

Design and validation of a reporter mouse to study the dynamic regulation of TFEB and TFE3 activity through in vivo imaging techniques

ABSTRACT TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor’s validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism. Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

Mitotic activity: A systematic literature review of the assessment methodology and prognostic value in feline tumors.

Increased proliferation is a driver of tumorigenesis, and quantification of mitotic activity is a standard task for prognostication. This systematic review is an analysis of all available references on mitotic activity in feline tumors to provide an overview of the assessment methods and prognostic value. A systematic literature search in PubMed and Scopus and a nonsystematic search in Google Scholar were conducted. All articles on feline tumors that correlated mitotic activity with patient outcome were identified. Data analysis revealed that of the 42 eligible articles, mitotic count (MC, mitotic figures/tumor area) was evaluated in 39 studies, and mitotic index (MI, mitotic figures/tumor cells) in 3 studies. The risk of bias was considered high for most studies (26/42, 62%) based on small study populations, insufficient details of the MC/MI methods, and lack of statistical measures for diagnostic accuracy or effect on outcome. The MC/MI methods varied between studies. A significant association of MC with survival was determined in 20 of 28 (71%) studies (10 studies evaluated other outcome metrics or provided individual patient data), while 1 study found an inverse effect. Three tumor types had at least 4 studies, and a prognostic association with survival was found in 5 of 6 studies on mast cell tumors, 5 of 5 on mammary tumors, and 3 of 4 on soft-tissue sarcomas. MI was shown to correlate with survival for mammary tumors by 2 research groups; however, comparisons to MC were not conducted. Further studies with standardized mitotic activity methods and appropriate statistical analysis for discriminant ability of patient outcome are needed to infer the prognostic value of MC and MI.

Abstract 7619: Tissue-derived peripheral biomarkers that reflect activity of T-cells, macrophages, and neutrophils in patients with solid tumors

Abstract Background: A toolbox of tissue-derived peripheral biomarkers that reflect the activity of T-cells, macrophages and neutrophils is a medical need for immuno-oncology drug development allowing non-invasive quantification of immune cell activity before - and during - treatment, both for predictive and pharmacodynamic purposes. By identifying and quantifying specific extracellular matrix fragments with neo-epitopes that are generated by proteases specific for T-cells, macrophages, and neutrophils, respectively, it is possible to develop peripheral biomarkers that reflect the activity of these immune cells. Such biomarkers have previously shown associations with outcome of patients treated with immune checkpoint inhibitors. Here we investigated the distribution of 3 such biomarkers in different cancer indications. Moreover, these data were compared to data from melanoma patients showing associations with response to cancer immunotherapy. Methods: Tissue-derived biomarkers related to activity of T-cells (C4G: Collagen-4 degraded by Granzyme B), Neutrophils (CPA9-HNE: Calprotectin degraded by neutrophil elastase), Macrophages (VICM: citrullinated and MMP-degraded vimentin) were measured by validated ELISA/ECLIA in serum from patients with various solid tumor types (bladder-, breast- colorectal-, head and neck-, kidney-, lung-, ovarian-, pancreatic, stomach-, prostate- cancer, and melanoma, n=220) and compared to age-matched healthy controls (n=33) by ANOVA. The biomarker levels were compared to historical data. Results: Median biomarker levels of CPA9HNE (neutrophil activity/NETosis) and VICM (macrophage activity) were significantly increased in all solid tumor types (p<0.001), respectively and with a ~5-fold inter-patient variation as well. Median C4G levels was only slightly elevated in a few indications (p<0.05) and with a 5-10-fold inter-patient variation across tumor types. All biomarker levels were independent of disease stage. Similar biomarkers levels were seen to those found to be associated with objective response rate and overall survival of melanoma patients treated with cancer immunotherapy. Conclusion: Tissue-derived peripheral biomarkers that reflect the activity of T-cells (C4G), macrophages (VICM) and neutrophils (CPA9HNE) has biomarker potential in solid tumors and may serve as prognostic, predictive and pharmacodynamic biomarkers in clinical trials investigating cancer immunotherapy. Citation Format: Nicholas Willumsen, Neel I. Nissen, Morten A. Karsdal. Tissue-derived peripheral biomarkers that reflect activity of T-cells, macrophages, and neutrophils in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7619.

ClonoScreen3D – A Novel 3-Dimensional Clonogenic Screening Platform for Identification of Radiosensitizers for Glioblastoma

PurposeGlioblastoma (GBM) is a lethal brain tumor. Standard of care treatment comprising surgery, radiation and chemotherapy results in median survival rates of 12-15 months. Molecular targeted agents identified using conventional two-dimensional (2D) in vitro models of GBM have failed to improve outcome in patients, rendering such models inadequate for therapeutic target identification. A previously developed 3D GBM in vitro model that recapitulates key GBM clinical features and responses to molecular therapies was investigated for utility for screening novel radiation-drug combinations using gold-standard clonogenic survival as readout. ResultsPatient-derived GBM cell lines were optimized for inclusion in a 96-well plate 3D clonogenic screening platform, ClonoScreen3D. Radiation responses of GBM cells in this system were highly reproducible and comparable to those observed in low-throughout 3D assays. The screen methodology provided quantification of candidate drug single agent activity (EC50) and the interaction between drug and radiation (radiation interaction ratio, RIR). The PARP inhibitors talazoparib, rucaparib and olaparib, each showed a significant interaction with radiation by ClonoScreen3D and were subsequently confirmed as true radiosensitizers by full clonogenic assay. Screening a panel of DNA damage response inhibitors revealed the expected propensity of these compounds to interact significantly with radiation (13/15 compounds). A second screen assessed a panel of compounds targeting pathways identified by transcriptomic analysis and demonstrated single agent activity and a previously unreported interaction with radiation of dinaciclib and cytarabine (RIR 1.28 and 1.90, respectively). These compounds were validated as radiosensitizers in full clonogenic assays (sensitizer enhancement ratio 1.47 and 1.35, respectively). ConclusionsThe ClonoScreen3D platform was demonstrated to be a robust method to screen for single agent and radiation-drug combination activity. Using gold-standard clonogenicity, this assay is a tool for identification of radiosensitizers. We anticipate this technology will accelerate identification of novel radiation-drug combinations with genuine translational value.

RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring

BackgroundRibulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity.ResultsWe simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5–7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3− to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited ~ 3.4-fold slower reaction rate (0.37 min−1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation.ConclusionsOverall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.