Multiple Passages Research Articles

SIRT5 -mediated desuccinylation of UQCRC2 attenuates osteogenic differentiation of aged BM-MSCs through impairing mitochondrial homeostasis.

The osteogenic differentiation potential of bone marrow mesenchymal stem cells (BM-MSCs) is critical for bone regeneration and repair. In recent years, the role of protein succinylation modification in regulating cellular metabolism has garnered increasing attention. However, its mechanism in osteogenic differentiation remains unclear. Oxygen consumption rate (OCR) and mitochondrial ROS (mtROS) were detected to assess mitochondrial function in BM-MSCs with successive passages. Alizarin red staining and western blot experiments were used to evaluate osteogenic differentiation capacity. .Succinylation modification omics and Co-IP detection were conducted to determine SIRT5-mediated desuccinylation of UQCRC2 . Bioinformatics analysis revealed that sirtuin 5 (SIRT5) expression is upregulated with multiple rounds of BM-MSCs' passages, and is associated with biological pathways such as oxidative phosphorylation (OXPHOS), cellular senescence, and inhibition of osteogenic differentiation. Experiments in vitro confirmed the up-regulation of SIRT5 and the suppression of osteogenic differentiation with the increased times of BM-MSCs' passages. Overexpression of SIRT5 enhanced OXPHOS and elevated mtROS levels, but reduced the expression of Runx2 and osteocalcin, and decreased calcified nodules, thereby inhibiting the osteogenic differentiation of BM-MSCs. SIRT5-mediated desuccinylation of ubiquinol-cytochrome C reductase core protein 2 (UQCRC2) at the site of K250 promoted UQCRC2 translocation from cytoplasm to mitochondria, which enhanced the activity of mitochondrial respiratory complex III. It further increased mtROS, accelerated cellular senescence and inhibited the osteogenic differentiation of BM-MSCs. SIRT5 reduces succinylation modification of UQCRC2, promotes mitochondrial respiration and mtROS, and thus reduces the osteogenic differentiation ability of BM-MSCs cells. SIRT5 might be a potential target to prevent the suppression of osteogenic differentiation of of BM-MSCs after multiple rounds passages.

Challenges in cellular agriculture: lessons from Pacific white shrimp, Litopenaeus vannamei.

The overall goal of this research was to develop an embryonic stem cell (ESC) line from the Pacific white shrimp, Litopenaeus vannamei, to support production of cell-based cultivated seafood products towards meeting a growing global demand for sustainable seafood. It was hypothesized that characteristics of ESCs, such as high proliferation and pluripotency, would facilitate development of a continuous cell line that could be triggered to differentiate into a muscle cell phenotype. The targeted approach was based on collection of ESCs from fertilized shrimp eggs at the blastomere stage. Various media, supplements, growth factors, and plate coatings were tested to achieve growth of the shrimp ESCs. Although successful in early culture, this manuscript describes substantial challenges encountered as cultures grew over time. The cell cultures were initially dominated by shrimp as indicated by 18S rDNA community analysis, but after multiple passages, thraustochytrids, a common contaminant of invertebrate cell culture, became the predominant cell type. Presence of shrimp cells was confirmed through species-specific primers for the cytochrome C oxidase subunit 1 gene. Presence of thraustochytrids was also confirmed using species-specific primers, morphological features, growth properties, and acriflavine staining. Unsuccessful attempts to eradicate thraustochytrid contamination prevented shrimp cells from thriving. The future of shrimp cell culture depends on eliminating culture contaminants while encouraging growth of shrimp ESCs.

Evaluating Countermeasures Against Marburg Virus Using a Mouse Model.

Animal models are key tools for understanding Marburg virus (MARV) pathogenesis and evaluating novel countermeasures. Rodents, in particular, are useful model systems because they are inexpensive and easy to house and handle in maximum containment laboratories. Unfortunately, wild-type MARV, like other filoviruses, does not cause disease in immune-competent rodents and must first be adapted to the rodent host, typically through serial passaging. In this way, mouse-adapted MARV (MA-MARV) variant Angola was generated through multiple passages in SCID mice. MA-MARV is uniformly lethal in BALB/c mice and produces many of the hallmarks associated with Marburg virus disease in humans and nonhuman primates, including robust virus replication, a dysregulated immune response, and extensive organ damage. In this chapter, we describe a protocol for inoculating mice with MA-MARV for the purpose of evaluating countermeasures.

Establishment, characterization, and sensory characteristics (taste and flavor) of an immortalized muscle cell line from the seven-band grouper Epinephelus septemfasciatus: implications for cultured seafood applications.

Grouper muscle satellite cells (GMSCs) from the seven-band grouper (Epinephelus septemfasciatus) were isolated, and their growth conditions were optimized (10% fetal bovine serum, 24°C, 10ng/mL bFGF). The cells were immortalized at passage 14 and designated as grouper immortalized muscle satellite cells (GIMSCs). DNA barcoding confirmed the grouper origin of both GMSC and GIMSC lines. GIMSCs exhibited enhanced proliferation, accelerated differentiation, and robust myotube formation compared to pre-crisis GMSCs. Western blot analysis showed upregulation of key myogenic factors (Pax7, MyoD, MyoG) and structural proteins (Desmin) in GIMSC, indicating the differentiation potential. The immortalized GIMSC line maintained consistent morphology, growth rates, and viability across multiple passages. Biocompatibility studies showed GIMSCs were compatible with bio-inks (sodium alginate, gelatin, κ-carrageenan) at 250 to 10,000µg/mL, retaining ~ 80% viability at the highest concentration. Taste sensory analysis revealed GMSCs had the highest umami and lowest saltiness and sourness, contrasting with the muscle of the seven-band grouper, which had higher saltiness and sourness. Flavor analysis identified pronounced fishy, hot fat, and ethereal flavors in the cells at higher level than in the muscle. These findings suggest GMSCs and GIMSCs are promising for producing cultured meat with enhanced umami taste and flavors, advancing cellular agriculture and sustainable food production.

Ameliorating salt stress in tomato by a top-down approach of acclimatizing the rhizosphere microbiome.

Soil salinization adversely impacts plant and soil health. While amendment with chemicals is not sustainable, the application of bioinoculants suffers from competition with indigenous microbes. Hence, microbiome-based rhizosphere engineering, focussing on acclimatization of rhizosphere microbiome under selection pressure to facilitate plant growth, exhibits promise. This study aimed to acclimatize a salt-susceptible tomato cultivar to high salt concentration through a microbiome-based top-down approach of rhizosphere engineering. Multiple passaging of the rhizosphere microbiome of the cultivar was performed for twelve plant growth cycles in the presence of increasing salt stress. The rhizosphere microbiome of the phenotypically best-grown plant under stress was transferred as inoculum to the next plant growth cycle. Plant growth attributes and stress marker levels were assessed, expression levels of plant salt stress-responsive genes were examined, and the bacterial community composition in the initial and final plant growth cycles was analysed. Rhizosphere microbiome inoculation promoted plant growth under increasing salt concentrations. Stress markers were reduced in plants inoculated with an acclimatized microbiome, while the root architecture was enhanced, indicating salt tolerance. The salt stress-responsive genes were downregulated in salt-treated plants, whereas upregulation of these genes was observed upon microbiome inoculation. The relative abundance of Exiguobacterium, Arthrobacter, and Lysobacter was higher in microbiome-treated plants under salt stress compared to the salt-treated plants without microbiome inoculation. The strategy of acclimatizing the rhizosphere microbiome of a salt-susceptible tomato cultivar was successfully implemented for stress amelioration and plant growth promotion, thereby offering a sustainable means with immense potential for application in other crops.

Double Deletion of EP402R and EP153R in the Attenuated Lv17/WB/Rie1 African Swine Fever Virus (ASFV) Enhances Safety, Provides DIVA Compatibility, and Confers Complete Protection Against a Genotype II Virulent Strain.

Background/Objectives: African swine fever virus (ASFV) is a devastating disease affecting domestic and wild suids and causing significant economic losses in the global pig industry. Attenuated modified live virus (MLV) vaccines are the most promising approaches for vaccine development. This study aimed to evaluate the safety and efficacy of four recombinant ASFV genotype II strains, derived from the non-hemadsorbing (non-HAD) attenuated isolate Lv17/WB/Rie1, through the single or simultaneous deletion of virulence-associated genes. Methods: Recombinant viruses were engineered by deleting the UK, EP402R, and EP153R genes, either individually or in combination. Four recombinant strains were evaluated for safety and efficacy in domestic pigs vaccinated intramuscularly with 102 TCID₅₀. Clinical signs, viremia, virus shedding, and antibody responses were monitored. Protection efficacy was assessed by challenging vaccinated pigs with the virulent genotype II Armenia07 strain. Additionally, a reversion-to-virulence study involving an overdose of the vaccine candidate was conducted to evaluate its stability through serial immunizations. Results: Deletion of the UK gene alone increased virulence, whereas the double deletion of EP402R and EP153R (Lv17/WB/Rie1-ΔCD) significantly enhanced safety while maintaining full protective efficacy. Vaccinated pigs exhibited reduced viremia, no virus shedding, and robust virus-specific antibody responses, achieving complete protection against Armenia07. The reversion-to-virulence study revealed potential but limited pathogenicity after multiple passages, indicating areas for improvement in vaccine stability. Conclusions: The Lv17/WB/Rie1-ΔCD strain demonstrates excellent safety and efficacy, along with potential DIVA (differentiating infected from vaccinated animals) compatibility, positioning it as a strong candidate for an ASFV MLV vaccine. Further research is needed to refine the vaccine and address the potential risks of reversion to virulence.

Remodeling of cytoskeleton, chromatin, and gene expression during mechanical rejuvenation of aged human dermal fibroblasts.

Aging is associated with a progressive decline in cellular function. To reset the aged cellular phenotype, various reprogramming approaches, including mechanical routes, have been explored. However, the epigenetic mechanisms underlying cellular rejuvenation are poorly understood. Here, we studied the cytoskeletal, genome-wide chromatin and transcriptional changes in young, aged, and mechanically rejuvenated fibroblasts using immunofluorescence, RNA sequencing, and Hi-C experiments. The mechanically rejuvenated aged fibroblasts, that had partially reset their transcription to a younger cell state, showed a local reorganization of the interchromosomal contacts and lamina-associated domains. Interestingly, the observed chromatin reorganization correlated with the transcriptional changes. Immunofluorescence experiments in the rejuvenated state confirmed increased actomyosin contractility like younger fibroblasts. In addition, the rejuvenated contractile properties were maintained over multiple cell passages. Overall, our results give an overview of how changes in the cytoskeleton, chromatin, and gene activity are connected to aging and rejuvenation.

Screening Algal and Cyanobacterial Extracts to Identify Potential Substitutes for Fetal Bovine Serum in Cellular Meat Cultivation.

Cultured meat technology is a form of cellular agriculture where meat is produced from animal cells grown in a lab, instead of raising and slaughtering animals. This technology relies heavily on fetal bovine serum (FBS) in cell media; hence, production is costly and contributes significantly to ammonia and greenhouse gas emissions. Achieving the successful commercialization of cell-cultured food requires the critical resolution of manufacturing cost and safety concerns. Hence, our research efforts are focused on identifying commercially viable and ecologically sustainable alternatives to FBS. In this study, we evaluated the potential of twenty-six water-based algal and cyanobacterial extracts to stimulate cell growth for meat cultivation under 90% reduced serum conditions. The extracts were compared in viability, proliferation, and Trypan blue exclusion assays. In the first screening phase, the extracts were evaluated in a ZEM2S (zebrafish) cell culture in a 1% FBS regimen. Based on their ability to exhibit protein tolerance or promote cell proliferation, ten extracts were selected and further assayed in a QM7 cell culture. The QM7 cell line (myoblasts from Japanese quail) is highly relevant for meat cultivation because of its ability to differentiate into muscle fibers. Extracts derived from two microalgae species, Arthrospira platensis (Spirulina) and Dunaliella tertiolecta, demonstrated the highest tolerance in cell culture, above 10 μg/mL (expressed as total protein concentration). Tolerance at a 100 μg/mL concentration was demonstrated exclusively using an extract of blue spirulina (commercially purified Spirulina), which supported cell growth through multiple passages.

TMIC-67. EMERGING ROLE OF EXTRACELLULAR MATRIX STIFFNESS AS A HALLMARK OF GLIOBLASTOMA: INSIGHT TO MECHANICAL MEMORY FORMATION AND PROLONGED INVASION IN THE TUMOR PERIPHERY

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

Numerical investigation of soil arching and integral abutment bridge in a pile-supported railway embankment

ABSTRACT Rail transport plays a crucial role in promoting sustainable mobility and tackling environmental challenges. However, the construction of a railway track on soft soil poses significant challenges due to the inherent instability and potential for excessive settlement. To address these issues, this study numerically assessed a pile-supported embankment and an integral abutment bridge. Two different numerical models are simulated in two-dimensional (2D) plane strain conditions. Findings from numerical analyses of the pile-supported embankment revealed that soil arching is significantly influenced by embankment height and pile spacing. The maximum settlement of the embankment decreases with an increase in embankment modulus and friction angle. Furthermore, a correlation among soil arching indicators was established. Conversely, the analysis of integral abutment railway bridges (IARBs) indicates that the performance of the transition zone is significantly influenced by factors such as approach slab geometry and multiple axle passages.

Molecular characterization and phylogenetic analysis of infectious laryngotracheitis virus isolates from commercial chicken flocks in Turkey.

Infectious laryngotracheitis virus (ILTV) causes an acute and highly contagious respiratory disease in poultry. Live-attenuated vaccines are generally used to control and prevent infectious laryngotracheitis (ILT). However, these vaccines can revert to a virulent form due to multiple passages and thereby become an ILT source. Hence, monitoring of ILTV in the field through molecular characterization is critically important for controlling infection and differentiating circulating isolates. In this study, we genotypically characterized and phylogenetically analyzed eight ILTV isolates from chicken flocks located in four different cities of Turkey between 2019 and 2022. For all isolates, we analyzed two regions of the infected cell protein 4 gene (ICP4-1 and ICP4-2) and the thymidine kinase (TK) gene. The isolates were 100%, 100%, and 99.8-100% identical to each other in the ICP4-1 and ICP4-2 gene fragments and the TK gene, respectively. None of the ICP4 sequences had a deletion at nt 272-283, confirming that they were field isolates. None of the isolates were predicted to have a T252M mutation in the thymidine kinase, suggesting that they have low virulence. The isolates were 100%, 99.36%, and 99.91% identical to Turkish ILTV isolates in their ICP4-1, ICP4-2, and TK gene region, respectively. Phylogenetic analysis based on the ICP4-1 and TK genes confirmed that the ILTV isolates are closely related to Turkish ILTV isolates. This suggests that these ILTVs were endemic isolates, which in turn suggests that the ILTV isolates circulating in Turkey were evolutionarily close, originated from the field, and had low virulence.

Quantum-Inspired Fusion for Open-Domain Question Answering

Open-domain question-answering systems need models capable of referencing multiple passages simultaneously to generate accurate answers. The Rational Fusion-in-Decoder (RFiD) model focuses on differentiating between causal relationships and spurious features by utilizing the encoders of the Fusion-in-Decoder model. However, RFiD reliance on partial token information limits its ability to determine whether the corresponding passage is a rationale for the question, potentially leading to inappropriate answers. To address this issue, we propose a Quantum-Inspired Fusion-in-Decoder (QFiD) model. Our approach introduces a Quantum Fusion Module (QFM) that maps single-dimensional into multi-dimensional hidden states, enabling the model to capture more comprehensive token information. Then, the classical mixture method from quantum information theory is used to fuse all information. Based on the fused information, the model can accurately predict the relationship between the question and passage. Experimental results on two prominent ODQA datasets, Natural Questions and TriviaQA, demonstrate that QFiD outperforms the strong baselines in automatic evaluations.

Use of terrestrial laser scanning point cloud in the inventory of Mechowo Caves

An inventory is a set of technical operations for obtaining reliable information about a site in order to prepare technical and descriptive documentation, presenting the current state of objects. One of the measurement technologies allowing for the acquisition of reliable and comprehensive information about a site is terrestrial laser scanning. A point cloud from terrestrial laser scanning generates both 2D surveys and 3D models of various types of objects. The scope of research work included the application of terrestrial laser scanning (TLS) technology in the inventory of the Mechowo Caves – a cave in the village of Mechowo. The survey of the area in front of the cave and its part accessible to visitors was carried out using a Leica P40 laser scanner. Due to the highly varied shape of the cave ( multiple low and narrow passages) and its unique character, the measurement had to be performed in a non-standard way – with the use of numerous measuring stations with different combinations of measuring instrument settings and variable scanning parameters. As a result of the work, a point cloud was generated, based on which cross-sections presenting the spatial layout of the Mechowo Caves were created, as well as a 3D model of the area covered by the survey.

Design and tests of filtering actions for an AI-based RSOs detection and tracking algorithm

Space debris represent a huge threat for the actual and future space traffic. The continuous monitoring of the space environment around the Earth and the build-up, maintenance of Resident Space Objects (RSOs) catalogue are essential to enhance the space segment safety. In the last few years, trend towards Space Based Space Surveillance (SBSS) missions was relevant. In particular, the monitoring of space debris through on-board Star Trackers (STs) coupled with Artificial Intelligence (AI) based algorithms is being studied, modeling RSOs as clear streaks on a dark background. This work presents the advances in the development of an AI-based algorithm for streaks-like RSOs Detection & Tracking for on-board optical sensors applications like STs. The initial design of the RSOs monitoring algorithm (Mastrofini et al., 2022) is extended and tested for what concerns the tracking part with new filtering actions. The goal is the improvement of the tracking outputs quality and reliability when multiple RSOs passages occur, with different configurations. Tests both on real and simulated images from STs are shown and discussed.

On the Dynamical Evolution of the Asteroid Belt in a Massive Star–Neutron Star Binary

Some fast radio bursts (FRBs) exhibit repetitive behaviors, and their origins remain enigmatic. It has been argued that repeating FRBs could be produced by the interaction between a neutron star and an asteroid belt. Here, we consider the systems in which an asteroid belt dwells around a massive star, while a neutron star, as a companion of the massive star, interacts with the belt through gravitational force. Various orbital configurations are assumed for the system. Direct N-body simulations are performed to investigate the dynamical evolution of the asteroids' belt. It is found that a larger orbital eccentricity of the neutron star will destroy the belt more quickly, with a large number of asteroids being scattered out of the system. A low inclination not only suppresses the collisions but also inhibits the ejection rate at early stages. However, highly inclined systems may undergo strong oscillations, resulting in the Kozai–Lidov instabilities. Among the various configurations, a clear periodicity is observed in the collision events for the case with an orbital eccentricity of 0.7 and mutual inclination of 0◦. It is found that such a periodicity can be sustained for at least eight neutron star orbital periods, supporting this mechanism as a possible explanation for periodically repeating FRBs. Our studies also suggest that the active stage of these kinds of FRB sources should be limited, since the asteroid belt would finally be destroyed by the neutron star after multiple passages.

The Three Hundred: The existence of massive dark matter-deficient satellite galaxies in cosmological simulations

The observation of a massive galaxy with an extremely low dark matter content (i.e. NGC 1277) has posed questions about how such objects form and evolve in a hierarchical universe. We here report on the finding of several massive, dark matter-deficient galaxies in a set of 324 galaxy clusters theoretically modelled by means of full-physics hydrodynamical simulations. We first focus on two example galaxies selected amongst the most massive and dark matter-deficient ones. By tracing the evolution of these galaxies, we find that their lack of dark matter is a result of multiple pericentre passages. While orbiting their host halo, tidal interactions gradually strip away dark matter while preserving the stellar component. A statistical analysis of all massive satellite galaxies in the simulated clusters shows that the stellar-to-total mass ratio today is strongly influenced by the number of orbits and the distance at pericentres. Galaxies with more orbits and closer pericentres are more dark matter-deficient. Additionally, we find that massive, dark matter-deficient galaxies at the present day are either the remnants of very massive galaxies at infall or former central galaxies of infalling groups. We conclude that such massive yet dark matter-deficient galaxies exist and are natural by-products of typical cluster galaxy evolution, with no specific requirement for an exotic formation scenario.

Delineation of global, absolutely essential and conditionally essential pangenomes of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative, anaerobic oral pathobiont, an etiological agent of periodontitis and the most commonly studied periodontal bacterium. Multiple low passage clinical isolates were sequenced, and their genomes compared to several laboratory strains. Phylogenetic distances were mapped, a gene absence-presence matrix generated, and core (present in all genomes) and accessory (absent in one or more genomes) genes delineated. Subsequently, a second pangenome delineating the prevalence of inherently essential genes was generated. The prevalence of genes conditionally essential for surviving tobacco exposure, abscess formation and epithelial invasion was also determined, in addition to genes encoding key proteolytic enzymes containing putative signal peptides. While the absolutely essential pangenome was highly conserved, significant differences in the complete and conditionally essential pangenomes were apparent. Thus, genetic plasticity appears to lie primarily in gene sets facilitating adaptation to variant disease-related environments. Those genes that are highly pervasive in the P. gingivalis absolutely essential pangenome or are highly prevalent and essential for fitness in disease-relevant models, may represent particularly attractive therapeutic targets worthy of further investigation. As mutations in absolutely essential genes are expected to be lethal, the data provided herein should also facilitate improved planning for P. gingivalis gene mutation strategies.

Design and Testing of a Closed Multi-Channel Air-Blowing Seedling Pick-Up Device for an Automatic Vegetable Transplanter

In this study, a closed multi-channel air-blowing plug seedling pick-up device and a combined plug tray were designed to address the issues of complex structure, high seedling damage rates and low pick-up efficiency in fully automated vegetable transplanter systems. The device operates by sealing the plug seedlings in a seedling cup, where compressed air is channeled into the sealed cavity through multiple passages during the seedling pick-up process. The upper surface of the seedling plug is subjected to uniform force, overcoming the friction and adhesion between the plug seedlings and the tray. This process presses the seedlings into the guide tube, completing the pick-up operation. A mechanical model for the plug seedlings was developed, and the kinetics of the pick-up process were analyzed. The multi-channel high-pressure airflow was simulated and evaluated, identifying three key parameters affecting seedling pick-up performance: water content of the seedling plug, air pressure during pick-up, and air-blowing duration. Using these factors as variables, and with seedling pick-up rate and substrate loss rate as evaluation indicators, single-factor experiments and a three-factor, three-level orthogonal experiment were conducted. The experiments’ results showed that the best seedling pick-up performance was achieved when the water content of the plug was 20%, the air pressure was 0.3 MPa, and the air-blowing time was 30 ms. Under these conditions, the seedling pick-up success rate was 97.22%, and the substrate loss rate was 10.46%.

RESTORATION OF VIRULENCE OF B.MELITENSIS 16 M STRAINS AND BRUCELLA ABORTUS 544

When testing anti-brucellosis vaccines, an important role is played by studying the infecting dose of control strains. To restore the virulent properties of the control strains Brucella melitensis 16 M and Brucella abortus 544, it is necessary to carry out multiple successive passages in different doses (starting from 1,000,000 colony-forming units, CFU/cm3 to 5 CFU/cm3), through the body of sensitive laboratory animals. In this case, for experimental work, we took 54 heads of guinea pigs (males) weighing 350-380 g, from which we created 9 groups of 6 heads each. The article reflects the progress and results of research into determining the minimum fixing infectious dose of the control reference strains Brucella melitensis 16 M and Brucella abortus 544.Based on the results of the studies, the minimum infectious dose (5 CFU) of Brucella after the ninth passage, which causes the generalized form of brucellosis, was determined. Spent virulent fixing doses (5 CFU/cm3) strains of Brucella melitensis 16 M and Brucella abortus 544 can be used for control infection when setting up various experiments to determine and evaluate the immunogenicity of anti-brucellosis vaccines and chemoprophylaxis.

166 Development of non-model organoids in animal research: A paradigm shift in in vitro models

Abstract For decades, two-dimensional (2D) cell cultures have been used as the gold standard in vitro models for basic research, drug discovery and preclinical screening applications. However, since 2009, the field has quickly shifted towards the use of three-dimensional (3D) stem cell-derived organoids. Organoids offer a variety of advantages compared with conventional 2D models, including being self-organized, retaining their cellular polarity, and being simplified versions of organs in vitro. Adult stem cells can self-renew, differentiate into multiple cell types, and are genomically stable over multiple passage lines, providing a variety of technical benefits and offering the closest recapitulation of the in vivo environment. Our laboratory has focused on expanding, characterizing, and utilizing animal-derived 3D organoid models, specifically focusing on canines; but also including other animals such as swine, turtles, snakes, and squirrels to study their unique adaptations. Practically, we can use 3D canine colonoids for the screening of anti-inflammatory candidates using our tumor necrosis factor (TNF)-α stimulation model. Additionally, we have grown and used swine lung organoids as a model for studying H1N1 viral uptake. Furthermore, we have grown and bio-banked tumor organoids/spheroids from a variety of canine cancers, including urothelial carcinoma (derived from urine and biopsies) and nasal carcinoma, among others, with future applications in precision veterinary medicine and comparative oncology. To accomplish this goal, we have carried out in vitro screening of a variety of chemotherapies and combination therapies at different concentrations across patients to predict in vivo responses and demonstrate the predictive ability of the organoid model. Finally, our laboratory has derived and characterized a variety of healthy organoid lines from canines including the pancreas, lung, liver, bladder, colon, and pituitary to act as healthy controls in mechanistic studies looking at disease pathogenesis and for uses in basic biology. In summary, in vitro organoid models from various animal species and tissues can be used in a variety of basic and applied research fields, including virology, toxicology, and oncology, with the potential to broaden our scientific understanding of disease and overall improve human and veterinary medicine.