Plate Reader Research Articles

81 Effects of udder quarter location on colostrum yield, nutrients, and immunoglobulin G in beef heifers

Abstract We hypothesized that colostrum total nutrients and immunoglobulin G (IgG) would not be affected by side of the udder, but that they would be greater in the rear versus the front half of the udder due to difference in volume of colostrum produced. Complete colostrum yield was collected individually from each quarter pre-suckling [40.5 ± 21.0 min (SD)] from fall-calving crossbred beef heifers (body weight = 472 ± 34 kg; body condition score (= 5.3 ± 0.4) that were either individually-fed 100% (control; n = 12) or 70% (nutrient restricted; n = 12) of NASEM metabolizable energy and metabolizable protein requirements from d 160 of gestation to calving. Analysis of colostrum lactose, protein, triglycerides, and free glucose was performed using colorimetric assays on a UV-visible light microplate reader. Colostrum IgG was analyzed using a commercially available ELISA kit. Quarter yield, nutrient or IgG concentration, and total nutrients or IgG were analyzed using the mixed procedure of SAS with fixed effects of udder location, nutritional plane, and their interaction in the model. Nutritional plane was not part of the current objective, but was included to account for variation in colostrum yield due to nutrient restriction, as we have previously reported. Separate statistical comparisons were made between the front and rear quarters and between the right and left quarters of the udder, with each quarter considered a sampling unit within each experimental unit (half) for both analyses. The interaction of udder location × nutritional plane did not affect (P ≥ 0.11) any variable. Nutrient restriction decreased (P ≤ 0.03) colostrum yield, total lactose, and total free glucose compared with controls. Front quarters produced less volume (P < 0.001; 36.2% of total); total lactose, protein, and free glucose (P ≤ 0.02; 34.3 to 43.3%); and total IgG (P < 0.001; 33.7%) than rear quarters. Front quarters tended to produce less (P = 0.07; 43.3%) total triglycerides than rear quarters. Lactose concentration was 10.5% greater (P = 0.05) in front compared with rear quarters, but other nutrient or IgG concentrations were not affected (P ≥ 0.18). Udder side did not affect total volume (P = 0.78), total nutrients (P ≥ 0.36), or total IgG (P = 0.79). The left side represented 50.7% of the total volume and 50.1 to 53.4% of total nutrients and IgG produced. Concentration of nutrients and IgG also were not affected (P ≥ 0.31) by udder side. In conclusion, colostrum did not differ between udder sides, but, relative to front quarters, rear quarters produced greater nutrient and IgG yield due to greater colostrum volume with minimal differences in nutrient or IgG concentrations.

Abstract PO3-25-12: Tigecycline-Induced Metabolic Reprogramming and Cytokine Modulation Suppress the Characteristics of Breast Cancer Cells

Abstract Objective: Patients with advanced breast cancer are prone to recurrence and metastasis. Hence, there is still a need to find new potential drugs or adjuvants to improve prognosis. The Warburg effect, which is highly dependent on glycolysis, has been found to be present in the energy metabolism of tumors, including breast cancer. The effect also promotes the production of cytokines, to adapt the inflammatory microenvironment of tumor. However, drugs targeting Warburg effect and inflammatory environment are rarely reported in the treatment of breast cancer. Tigecycline (Tige) is a commonly used glycylcycline antibiotic. Previous research has indicated it impede mitochondrial oxidative phosphorylation (OXPHOS) in various solid tumors. Based on the team's previous research, our study aimed to investigate whether Tige could inhibit breast cancer development by interfering with the Warburg effect and the inflammatory environment. Methods: MB231, MB468 and MCF7, T47D, which represent triple-negative and estrogen receptor positive breast cancers, were treated with Tige at different concentrations for different periods of time. CCK8, colony formation and sphere formation assays were used to evaluate the effects on proliferation and tumor stemness in vitro, respectively. The effects on migration and invasion were evaluated by wound healing assay and Transwell. RNA sequencing was used to explore the genes altered and analyze the pathways involved. After labeling with probes, the changes of reactive oxygen species (ROS) were detected by fluorescence microscope, microplate reader, and flow cytometry. Cell cycle and apoptosis were detected by flow cytometry. Liquid cytokine chip was used to analyze the secretion of cytokines. Electron microscopy was performed to observe the changes in mitochondrial morphology. RT-PCR was used to detect the expression of rate-limiting enzymes in glycolysis pathway. Glucose consumption, lactate and pyruvate production were measured using glucose, lactate and pyruvate detection kits, respectively. Western blot was used to detect the changes of respiratory chain protein complex and other related pathway proteins in OXPHOS. Results: Four breast cancer cells showed a significant decrease in cell proliferation, invasion and migratory ability after Tige treatment. Sphere-forming assays also showed that Tige had an effect on the stemness of breast cancer. RNA-sequencing showed that glycolysis, cell cycle, and immune-related pathways were inhibited after Tige treatment, and ROS production analysis revealed a decrease in ROS production. Flow cytometry also revealed that the cell cycle was arrested in S phase without significant apoptosis. Cytokine liquid chip analysis showed that the secretion of many cytokines, mainly interleukin (such as IL-1B), was inhibited after Tige intervention. RT-PCR results showed that the expression of several rate-limiting enzymes of the glycolytic pathway decreased. Glucose, lactate and pyruvate assay results showed that Tige reduced glucose uptake and lactate, pyruvate production. Electron microscopy results revealed that mitochondria in Tige treated cells showed significant contractures. WB results also showed a decrease in the expression of OXPHOS-related proteins. However, due to the heterogeneity of the four breast cancer cells, the degree of change in these capacities was not entirely consistent. Conclusions: Tige not only inhibits mitochondrial OXPHOS, but also inhibits the glycolysis pathway, which results in the inhibition of glucose uptake and the production of pyruvate and lactate. The secretion of early pro-inflammatory cytokines also affected. Therefore, this study suggests that Tige may change the glucose metabolism and energy metabolism, and also change the inflammatory environment of breast tumors, which provides new evidence for the anti-tumor effect of Tige in breast cancer. Citation Format: Haochen Yu, Lingfeng Tang, Shengchun Liu. Tigecycline-Induced Metabolic Reprogramming and Cytokine Modulation Suppress the Characteristics of Breast Cancer Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-12.

Abstract PO4-28-04: Curcumin inhibits breast cancer cell proliferation by regulating ciRS-7/miR-7-5p/CKS2 axis

Abstract Background Breast cancer (BC) is the most common cancer in women worldwide. Curcumin is a polyphenolic turmeric-derived compound that has anti-proliferative and anti-tumor properties in different cancer types by acting on multiple molecules. Circular RNAs (circRNAs) are non-coding, single-stranded, covalently closed RNA molecules that act as regulators of the microRNA (miRNA) activity. Recent studies show that circRNAs are potential contributors to the onset and progression of many cancer types. CircRNA ciRS-7 acts as an oncogene and accelerates tumor progression by competitively suppressing miR-7-5p in BC. However, whether curcumin can regulate ciRS-7 to inhibit BC progression is still unclear. Method Breast Cancer cell lines (MCF-7 and T47D) and normal epithelial cell line (MCF-10A) were cultivated and treated with Curcumin (5μM, 10 μM, 20 μM) or DMSO as a control. Also, the cell lines were transfected with ciRS-7 siRNA, miR-7-5p mimic, and their non-targeted controls. The cell viability was detected by Cell Viability Detection Kit-8 (CVDK-8) using an ELISA plate reader. To detect cell migration, scratch assay was performed after 24hs of transfection and a phase contrast microscope was used to acquire images. The effect on apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit using flow cytometry. Potential target genes of miR-7-5p were identified by searching for the overlapping genes in miRNet and miRTarBase v8 with the overexpressed genes in BC patient tissue samples in the TCGA database. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of ciRS-7, miR-7-5p, and the selected genes. qRT-PCR experiments were performed in duplicate and the 2−ΔΔCt method was used for relative quantitation analysis. Results Curcumin treatment, depending on the increased doses, decreased cell proliferation through inducing apoptosis in MCF-7 and T47D cancer cells. Curcumin's anti-proliferative effect was shown to be quite restricted on normal MCF-10A cells, as compared to MCF-7 and T47D cancer cells. In MCF-7 and T47D cells treated with curcumin, cell migration was dramatically inhibited. Curcumin has very little influence on the migration of MCF-10A cells. In both cancer cell group that was transfected with ciRS-7 siRNA and miR-7-5p mimic, it was observed that apoptosis was increased, proliferation was suppressed, and migration was decreased compared to its control groups. Moreover, the expression of ciRS-7 was found to be significantly decreased in the curcumin-treated group, while miR-7-5p was shown to be significantly higher. CKS2 gene one of the possible target genes of miR-7-5p that was identified by using in silico approaches, was also downregulated in curcumin-treated BC cell lines compared to its control group. ciRS-7 and CKS2 expression levels were found to be downregulated, whereas miR-7-5p expression level was found to be elevated in MCF-7 and T47D cells that were transfected with ciRS-7 siRNA. Additionally, CKS2 gene expression was found to be downregulated in miR-7-5p mimic transfected cells. Conclusion Curcumin is derived from the turmeric plant (Curcuma longa) and has been used since 3000 B.C. as a food additive. Nowadays, curcumin is one of the most essential anti-cancer substances that was examined. However, investigations on the influence of curcumin on the circRNA-miRNA-mRNA axis are scarce. Curcumin has been demonstrated to inhibit proliferation and induce apoptosis of BC cells via the ciRS-7/miR-7-5p/CKS2 axis in the present study. Keywords Breast Cancer, Curcumin, ciRS-7, miR-7-5p, CKS2 Citation Format: Asmaa Abuaisha, Murat Kaya, Ilknur Suer, Selman Emiroglu, Fahrunnisa Abanoz, Mustafa Tukenmez, Neslihan Cabıoğlu, Mahmut Muslumanoglu, Kivanc Cefle, Sukru Palanduz, Sukru Ozturk. Curcumin inhibits breast cancer cell proliferation by regulating ciRS-7/miR-7-5p/CKS2 axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-04.

Lithium acetate mediated paper-based assay for absorbance analysis of E. coli concentrations

Paper-based assays have revolutionized POC diagnostics, offering significant values across diverse sectors due to their simplicity, cost-effectiveness, and portability. Detecting E. coli is critical for public health but faces challenges in traditional methods, prompting ongoing exploration of alternative approaches. To address the need for heightened sensitivity, this study introduces lithium acetate as a novel element in a paper-based E. coli detection assay, accompanying absorbance analysis using microplate readers. Utilization of lithium acetate as a lysis reagent and carrying out absorbance measurements in paper microtiter plates provide a significant stride in improving sensitivity and assay efficiency. The assay substantially reduced the analysis time to 4hours with a lower quantification limit of 103 cfu/ml for detecting E. coli in pure cultures. The results suggest that the assay could determine 10 cfu/ml of E. coli in a contaminated milk sample with 4hours of enrichment time. Thus, the study contributes towards improving E. coli detection with lower quantification limits and shorter analysis times. It is anticipated that the diagnostic methodology would circumvent the challenges in conventional E. coli detection methods, placing emphasis on absorbance analysis to achieve a streamlined, accurate, and rapid paper-based detection technique.

A reproducible and affordable method of conducting Luciferase assay

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.

DNA tetrahedron-based dual-signal fluorescence detection of apoE4 gene sites on a microplate reader.

As a neurodegenerative disorder, Alzheimer's disease (AD) is characterized by cognitive dysfunction and behavioral impairment. Among the various genetic risk factors for AD, apoE4 gene plays a pivotal role in the onset and progression of AD, and detection of apoE4 gene holds significance for prevention and early diagnosis of AD. Herein, dual-signal fluorescence detection of fragments associated with apoE ε4 allele near codon 112 (Tc1) and codon 158 (Tc2) was achieved using DNA tetrahedron nanostructure (DTN). The Förster resonance energy transfer (FRET) process in the DTN was initiated in which the nucleic acid intercalating dye thiazole orange (TO) served as the donor and the cyanine dyes of cyanine3 (Cy3) and cyanine5 (Cy5) at the two vertices of DTN served as the acceptors. In the presence of Tc1 and Tc2, the FRET process between TO and the cyanine dyes was hindered by the enzymatic cleavage reaction, which ensures the dual-signal fluorescence assay of apoE4 gene sites. The limit of detection for Tc1 and Tc2 was estimated to be 0.82nM and 0.77nM, respectively, and the whole assay was accomplished within 1h on a microplate reader. The proposed method thus possesses the advantages of easy operation, short detection time, and high-throughput capability.

DTT protein equalization and Tryptophan protein quantification as a powerful tool in analytical proteomics.

Assessing total protein levels in biological samples is a common procedure in biochemistry and molecular biology. In this study, we compare tryptophan fluorescence (WF) with Bradford and BCA assays to determine total protein in serum samples. Our results indicate that tryptophan fluorescence spectrometry is an efficient, sensitive, and straightforward technique for quantifying proteins in serum. We observed minimal variation between the three methods: BCA de one with the lowers LOD and LOQ. The tryptophan method offers the possibility of reusing the intact sample that does not need colourimetric reagents for quantification. Consequently, free tryptophan serves as a reliable universal standard. This assay can be performed using a conventional fluorescence spectrometer with cuvettes or in a 96-well plate format with a plate reader. The method was successfully used as proof of concept, using serum from patients diagnosed with myeloma and serum from healthy donors.

Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2μg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

P2X7 receptor-dependent increase in endocannabinoid 2-arachidonoyl glycerol production by neuronal cells in culture: Dynamics and mechanism.

Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall. 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRABeCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. 2-AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant-GRABeCB2.0. ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase β activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/β-hydrolase domain containing 6 (ABHD6). Considering that P2X7R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.

Development and evaluation of an artificial intelligence for bacterial growth monitoring in clinical bacteriology.

In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.

Controllable detection threshold achieved through the toehold switch system in a mercury ion whole-cell biosensor

Due to the toxicity of mercury and its harmful effects on human health, it is essential to establish a low-cost, highly sensitive and highly specific monitoring method with a wide detection range, ideally with a simple visual readout. In this study, a whole-cell biosensor with adjustable detection limits was developed for the detection of mercury ions in water samples, allowing controllable threshold detection with an expanded detection range. Gene circuits were constructed by combining the toehold switch system with lactose operon, mercury-ion-specific operon, and inducible red fluorescent protein gene. Using MATLAB for design and selection, a total of eleven dual-input single-output sensing logic circuits were obtained based on the basic logic of gene circuit construction. Then, biosensor DTS-3 was selected based on its fluorescence response at different isopropyl β-D-Thiogalactoside (IPTG) concentrations, exhibiting the controllable detection threshold. At 5-20 μM IPTG, DTS-3 can achieve variable threshold detection in the range of 0.005-0.0075, 0.06-0.08, 1-2, and 4-6 μM mercury ion concentrations, respectively. Specificity experiments demonstrated that DTS-3 exhibits good specificity, not showing fluorescence response changes compared with other metal ions. Furthermore spiked sample experiments demonstrated its good resistance to interference, allowing it to distinguish mercury ion concentrations as low as 7.5 nM by the naked eye and 5 nM using a microplate reader. This study confirms the feasibility and performance of biosensor with controllable detection threshold, providing a new detection method and new ideas for expanding the detection range of biosensors while ensuring rapid and convenient measurements without compromising sensitivity.

Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation

Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput in vitro assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA2 inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of C. rhodostoma and N. mossambica, which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.

Some Acute Phase Proteins and Biochemistry Parameters of Female Dogs Affected with Pyometra

Canine pyometra is a common reproductive disorder of intact diestrual bitches. The disease is associated with inflammation and infection of the uterus leading to generalized illness. The varied reactions of the host to infection, inflammation, or trauma are collectively known as the acute-phase response (APR) and encompass a wide range of pathophysiological responses. One of these systemic responses to disease is an increase in the production by the liver of a number of serum proteins, which are known collectively as the acute-phase proteins (APP). The most important APPs in dogs are C-reactive protein (CRP) and serum amyloid A (SAA). When diagnosed with pyometra, the preferred and most effective treatment is ovariohysterectomy, a procedure that in itself causes systemic inflammation due to the tissue trauma caused by surgery. The purpose of this study was to identify the effect pyometra and ovariohysterectomy have on acute phase proteins and routinely measured biochemistry variables in serum of affected bitches. We achieved this by evaluating concentrations of CRP and SAA, as well as of some biochemistry parameters in serum before and after ovariohysterectomy in bitches with pyometra. Blood samples were collected from the cephalic vein immediately prior to and 24h, 72h and 168 hours after initiation of surgery. The levels of CRP, SAA and some biochemistry parameters - total protein (TP), albumin (ALB), globulin (GLOB), as well as activity of some enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKLP) and lactate dehydrogenase (LDH) were determined
 Aims: The aim of this study was to investigate the impact of pyometra and its surgical treatment, ovariohysterectomy, on acute phase proteins, specifically C-reactive protein (CRP) and serum amyloid A (SAA), as well as on routine serum biochemistry variables in affected bitches. By evaluating the concentrations of CRP and SAA, alongside key biochemistry parameters before and after surgery, this research seeks to elucidate the physiological and biochemical responses to both the disease and its treatment, providing insight into the systemic effects of pyometra and the surgical stress response in dogs.
 Study Design:  The study design involved a cohort of twenty-seven female dogs, divided into two groups: seventeen clinically healthy intact female dogs serving as the control group, which were examined for routine health care or vaccinations, and ten female dogs diagnosed with pyometra at the Faculty of Veterinary Medicine, University of Sarajevo. The diagnosis of pyometra was made based on case history, clinical signs, ultrasonography, and confirmed by gross examination of the pus-filled uterus during and after ovariohysterectomy. The surgical procedure was performed using a standard mid-line approach under general inhalation anesthesia, with no post-operative complications reported.
 Place and Duration of Study: Faculty of Veterinary Medicine, University of Sarajevo, between June 2012 and September 2012.
 Methodology: Blood samples were collected from the distal cephalic vein into serum separating tubes (Vacutainer SST; Becton Dickinson, USA) for CRP and SAA measurement and serum biochemical evaluation just before the ovariohysterectomy (base values) as well as after 24 hours (day one), 72 hours (day three) and 168 hours (day seven). Blood samples from control dogs were collected only once. Serum samples were prepared by centrifugation (1.500 × g for 10 min) and stored in plain micro tubes (Eppendorf, Germany) at −20°C prior to analysis. Serum CRP concentrations in the dogs were measured with ELISA microplate reader (Anthos 2001 ELISA-reader, Anthos Mikrosysteme, Krefeld, Germany) using a commercial canine CRP enzyme-linked immunosorbent assay (ELISA) kit (Tridelta PhaseTM Range CRP Assay, Tridelta Development Limited, County Kildare, Ireland). For SAA, the analyses were performed using also a commercially available ELISA kit (Tridelta PhaseTM Range SAA Assay, Tridelta Development Limited, County Kildare, Ireland). Total protein (TP), albumin (ALB), globulin (GLOB) concentrations and alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALKP) and lactate dehydrogenase (LDH) activities were determined by using standard procedures and slides by Catalyst Dx Chemistry Analyzer, Idexx.
 Statistical analyses were performed using the SPSS software package (for Windows, Version 11.5, SPSS Inc, USA). All data were shown as mean ± standard deviation. The results were analyzed using the independent t-test. A P-value less than 0.05 were considered statistically significant.
 Results: Our results indicated significantly higher concentrations of CRP and SAA in dogs with pyometra throughout the study period compared to the control group, with CRP concentrations rising up to 15-fold and SAA up to 10-fold. Notably, these increases in CRP and SAA concentrations were mostly observed before surgery, with only limited additional increases post-surgery. This suggests that while ovariohysterectomy induces a proinflammatory response, it does not significantly escalate the levels of these proteins beyond the initial disease impact. The variability observed in CRP and SAA levels points to potential interindividual differences, impacting their diagnostic value. However, on the last postoperative day, the significantly elevated levels of both proteins in dogs with pyometra suggest a persistent, though less severe, inflammatory process, with a subsequent decline as homeostasis was gradually restored.
 Conclusion: The role of the acute phase response is to limit inflammation, remove the damaging factor and restore homeostasis. The systemic effects of pyometra are reflected by acute phase proteins and several biochemistry parameters.

Comparison and evaluation of the methods for measuring hemolytic activity of <i>Stomolophus meleagris</i> jellyfish tentacle extract

Hemolytic activity assessment is a widely used method to evaluate the toxicity of marine organisms, including jellyfish. However, there are some challenges associated with testing hemolytic activity. In this study, four methods were employed to compare the hemolytic activity of jellyfish tentacle extract (TE). Firstly, a microplate reader was used to compare the mouse hemoglobin solution at three different wavelengths (415 nm, 541 nm, and 576 nm), and the most sensitive wavelength was selected for further experiments. Secondly, photomicrograph counting was used to determine the number of complete red blood cells in the field of view. Thirdly, a microplate reader was used to test hemolytic activity in a 96-well plate at 415 nm. Fourthly, a Bicinchoninic Acid (BCA) kit was used to test the concentration of hemoglobin in the solution. Finally, a UV-Vis Spectrophotometer was used to test hemolytic activity at 415 nm. Among the three wavelengths tested, the absorption value was most sensitive at 415 nm. The photomicrograph counting method was able to reflect changes in the shape of Red Blood Cells (RBCs). The microplate reader method may exhibit deviations when the solution concentration is high, while external factors could influence the BCA kit when the toxin concentration in the experimental group is low. The spectrophotometer method was found to be relatively accurate and sensitive to changes. When optimizing the method, it is important to consider the applicability of the Beer-Lambert law and the concentration of solutions.

In Vivo Fluorescent Labeling of Foam Cell-Derived Extracellular Vesicles as Circulating Biomarkers for In Vitro Detection of Atherosclerosis.

Real-time monitoring of the development of atherosclerosis (AS) is key to the management of cardiovascular disease (CVD). However, existing laboratory approaches lack sensitivity and specificity, mostly due to the dearth of reliable AS biomarkers. Herein, we developed an in vivo fluorescent labeling strategy that allows specific staining of the foam cell-derived extracellular vesicles (EVs) in atherosclerotic plaques, which are released into the blood as circulating biomarkers for in vitro detection of AS. This strategy relies on a self-assembled nanoprobe that could recognize foam cells specifically, where the probe is degraded by the intracellular HClO to produce a trifluoromethyl-bearing boron-dipyrromethene fluorophore (termed B-CF3), a lipophilic dye that can be transferred to the exosomal membranes. These circulating B-CF3-stained EVs can be detected directly on a fluorescence spectrometer or microplate reader without resorting to any sophisticated analytical method. This liquid-biopsy format enables early detection and real-time differentiation of lesion vulnerability during AS progression, facilitating effective CVD management.

Weissella sp. SNUL2 as potential probiotics with broad-spectrum antimicrobial activities

Probiotics have been applied to a wide range of bacteria, causing gastrointestinal and vaginal infections. However, probiotics generally possess limited antimicrobial spectra and are primarily utilized as dietary supplements. Recognizing the need for more versatile probiotics, this study focuses on isolating and characterizing strains suitable for antibiotic replacement. Among these strains, Weissella sp. SNUL2, derived from a traditional fermented food in Korea (i.e., Sikhae), emerged as a promising candidate. The correlation between optical density at 600 nm and colony-forming units was verified and applied in subsequent experiments. To assess the therapeutic potential of probiotics, antibacterial tests were conducted using a microplate reader to evaluate the inhibition of 60 bacterial strains (including common foodborne pathogens) induced by Weissella sp. SNUL2 cell-free supernatant (CFS). The results confirmed its broad-spectrum antibacterial properties compared to previously known probiotics. Furthermore, enzymatic treatment with proteinases (trypsin and pepsin) and a time-kill assay were conducted to elucidate the nature of the antibacterial substance in Weissella sp. SNUL2 CFS. Through sequential chromatography involving gel filtration and ion-exchange chromatography, specific fractions with enhanced antibacterial properties were identified. LC-MS/MS analysis of the secretome fraction revealed the presence of various proteins from the C39 family, peptidoglycan endopeptidases, and N-acetylmuramoyl-l-alanine amidase domain-containing protein precursors. Hence, the combined action of these proteins may contribute to Weissella sp. SNUL2's broad antimicrobial activity.

Development of Green and High Throughput Microwell Spectrophotometric Methods for Determination of Galidesivir in Bulk Drug and Dosage Forms Based on Simple Oxidimetric Reactions with Inorganic Agents.

Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form. The objective of this study was the investigation of oxidation reactions of GDV with five inorganic oxidizing reagents and the employment of the reactions in the development of five green microwell spectrophotometric methods (MW-SPMs) with simple procedure and high throughputs for determination of GDV in its bulk and dosage forms (capsules). The reactions were carried out in 96-well plates and the absorbances of reaction solutions were measured by an absorbance microplate reader. Variables influencing the reactions were carefully investigated and optimized. Under the refined optimum conditions, Beer's law with excellent correlation coefficients (0.9992-0.9997) was followed in GDV concentrations in a general range of 5-700 µg/mL, and the limits of detection were ≥1.8 µg/mL. All validation parameters of all methods were acceptable. The methods were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision; the recovery percentages were 98.6-101.2 ± 0.58-1.14%. The greenness of MW-SPMs was evaluated by three comprehensive metric tools, which demonstrated the adherence of MW-SPMs to the principles of the green analytical chemistry approach. The proposed MW-SPMs combined the advantages of microwell-based practice and the use of common laboratory reagents for the analysis. The advantages of microwell analysis were the high throughput, readily available for semi-automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of using common laboratory reagents were the availability, consistency, compatibility, safety, and cost-effectiveness. Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Abstract 4233: Rapid and robust generation of ex vivo tumoroid samples from fresh patient samples in a serum-free, conditioned medium-free system

Abstract Functional precision oncology describes a method in which an individual patient’s cancer cells are tested for response to various treatments ex vivo to aid in therapy selection. In many cases, these cultures take the form of tumoroids, which are 3D, patient-derived “mini tumors” that retain phenotypic and genotypic characteristics of the patient cancer. While interest in functional precision oncology is increasing due to strong correlation between patient responses and those experimentally observed using matched tumoroid models in the literature, a lack of standardization has precluded its widespread translation to clinical settings. To study the ability to generate short-term tumoroid cultures using a commercially available, serum-free, and conditioned medium-free system, we processed fresh tumor resection samples and performed culture in Gibco™ OncoPro™ Tumoroid Culture Medium. Tumor resection samples spanning a variety of indications were stored in supplemented Hibernate™-A Medium (including antibiotics) and shipped overnight on gel packs to a central tissue processing site. Samples were minced, enzymatically dissociated, and counted. Average yields of ~2000-12,000 cells per mg of tumor resection tissue were observed, with lower yields in lung, head and neck, and breast cancer samples and higher yields in endometrial and colorectal cancer samples. Donor-to-donor variability generated a wide range of yields within a given indication. Similarly, cell viability following dissociation ranged widely, reaching as low as 20% for some samples and exceeding 90% for others. Though yields and initial viability varied, dissociated cells that were plated in OncoPro Tumoroid Culture Medium formed tumoroids in >85% of samples within 7 days of tissue receipt. Both mutational profiles and gene expression patterns were highly conserved (>90%) between initial tumor samples and cells cultured in OncoPro for 7 days. Computational approaches designed to identify cell-type specific signatures from bulk RNA sequencing data indicated that immune cell population signatures were present after one week in OncoPro medium. Additionally, we have developed methods to analyze tumoroid response to compounds and candidate cell therapies by both plate reader- and imaging-based analyses using ~1.2e6 initial tumor cells per 96-well plate. Due to the high success rate of tumoroid formation from fresh tumor samples and retention of donor-specific characteristics in OncoPro Tumoroid Culture Medium, we believe that it will be a valuable tool for functional precision oncology of solid tumor samples. Citation Format: Colin Paul, Amber Bullock, Anthony Chatman, Brittany Balhouse, Chris Yankaskas, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Rapid and robust generation of ex vivo tumoroid samples from fresh patient samples in a serum-free, conditioned medium-free system [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 4233.