Primary Explants Research Articles

Toxicity and Dermatokinetic Analysis of Ibrutinib in Human Skin Models

Background/Objectives: Ibrutinib (IBR) is a tyrosine kinase inhibitor under investigation in preclinical and clinical settings as an alternative treatment for melanoma. Nevertheless, the limited oral bioavailability of IBR and the need for high doses of the drug to kill melanoma cells are major drawbacks for this purpose. Considering that melanoma is restricted to the skin at early stages, the topical application of IBR might constitute an effective and safer administration route. In this study, we determined IBR’s toxicity and dermatokinetics using human primary cells and human organotypic skin explant cultures (hOSECs). Methods: After demonstrating that human primary fibroblasts and keratinocytes present IBR target genes, the cytotoxicity of the drug was determined using the MTT and annexin V/PI staining assays. IBR toxicity in the skin was assessed using the TTC assay, and the irritation potential was established using histological assessment. Finally, IBR cutaneous permeation was assessed ex vivo to determine the drug dermatokinetics. Results: Our findings reveal that IBR exerts dose-dependent toxicity towards skin cells, presenting an IC50 in the same range as melanoma cells. The topical application of the drug successfully reduced irritation and toxicity in the skin, and the drug was shown to successfully permeate the stratum corneum and reach the viable skin layers in therapeutic concentrations. Conclusions: Overall, our data encourage the topical application of IBR to treat melanoma, paving the way for future studies in this theme.

The Human Neural Cell Atlas of Zika Infection in developing human brain tissue: viral pathogenesis, innate immunity, and lineage reprogramming.

Zika virus (ZIKV) infection during pregnancy can lead to fetal brain infection and developmental anomalies collectively known as congenital Zika syndrome (CZS). To define the molecular features underlying CZS in a relevant human cell model, we evaluated ZIKV infection and neurodevelopment in primary fetal brain explants and induced pluripotent stem cell-derived mixed neural cultures at single cell resolution. We identified astrocytes as key innate immune sentinel cells detecting ZIKV and producing IFN-β. In contrast, neural progenitor cells displayed impaired innate immunity and supported high levels of viral replication. ZIKV infection of neurons suppressed differentiation and synaptic signaling networks and programmed a molecular switch from neurogenesis to astrogliogenesis. We identified a universal ZIKV-driven cellular stress response linked to intrinsic apoptosis and regulated by IFN-β. These findings reveal how innate immune signaling intersects with ZIKV-driven perturbations in cellular function to influence CZS outcomes including neuron developmental dysfunction and apoptotic cell death.

Omega-3 fatty acids protect cartilage from acute injurie by reducing the mechanical sensitivity of chondrocytes

Acute cartilage injuries, such as intra-articular fractures and blunt impacts, frequently result in chondrocyte death and extracellular matrix (ECM) degradation, significantly elevating the risk of post-traumatic osteoarthritis (PTOA). Despite advances in treatment, no effective therapies currently exist to fully cure PTOA or halt its progression. This study explores the protective effects of the dietary fatty acid eicosapentaenoic acid (EPA) on human primary chondrocytes (HPCs) and cartilage explants exposed to mechanical overload and blunt trauma. HPCs were isolated and subjected to mechanical stretching using BioFlex six-well culture plates, while cartilage explants were subjected to impact loading via a customized drop tower. EPA was incorporated into the culture medium, followed by assays to evaluate cell viability, calcium (Ca²⁺) influx, apoptosis, reactive oxygen species (ROS) levels, and collagen type II alpha (Col-2a) expression. EPA treatment markedly decreased chondrocyte mechanical sensitivity, as demonstrated by enhanced cell viability and reduced lactate dehydrogenase (LDH) release. Furthermore, EPA inhibited Piezo1 activation, leading to lower intracellular Ca²⁺ concentrations, decreased apoptosis, and diminished ROS levels. In cartilage explants, EPA improved chondrocyte viability, minimized structural damage, and sustained higher Col-2a expression compared to the blunt trauma group. These results indicate that EPA effectively shields chondrocytes and cartilage explants from mechanical overload-induced damage by inhibiting Piezo1 activation and mitigating Ca²⁺ influx, apoptosis, and oxidative stress. The findings suggest that EPA supplementation could offer a promising strategy for preventing PTOA progression following acute cartilage injuries. Further research is warranted to assess the clinical applications of EPA and confirm its efficacy in larger animal models and human trials.

Tea (Camellia sinensis L. O. Kuntze) callus development based on morphological and phytochemical traits applied by variations in plant growth regulators (PGRs)

A variation of the clonal propagation system for tea has been established through in vitro methods, utilizing shoots and leaves derived from primary explants of mature field-grown plants. The research aimed to assess the diversity of tea calluses based on morphological and phytochemical traits derived from leaf and shoot explants on MS media induced by the application of plant growth regulators, including auxin and cytokinin. The research was conducted at the Seed Technology Unit Tissue Culture Laboratory, Faculty of Agriculture, Jatinangor, from April 2022 to February 2023. The experiment employed MS-modified multiplication media and PGR, with BAP at 9 mg/L, TDZ at 1.0 mg/L, Zeatin at 0.1 mg/L, NAA at 0.01 mg/L, and 1 mg/L. Results indicated that applying PGR, auxin, and cytokinin with MS media influenced morphological and phytochemical traits of tea's callus derived from leaves and shoots. Specifically, the treatment of 1 mg/L NAA + 1 mg/L BAP showed the best performance on callus weight and total phenolic and flavonoid contents compared to other treatments., i.e. 9.038% % and 0.05% %, respectively. The success of clonal propagation in tea through in vitro methods is expected to increase by carefully selecting the appropriate source and type of explant, planting media, PGR, and addressing potential contaminants.

A First Approach for the In Vitro Cultivation, Storage, and DNA Barcoding of the Endangered Endemic Species Euonymus koopmannii.

Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies.

Indications and Outcomes of Intraocular Lens Explantation in a Tertiary Eyecare Center in Hungary between 2006 and 2020.

Our study aimed to evaluate the indications and outcomes of intraocular lens (IOL) explantation surgeries in a tertiary eyecare center in Hungary. This retrospective study included all IOL explantation surgeries performed between 2006 and 2020 at the Department of Ophthalmology of Semmelweis University, Budapest, Hungary. There were no exclusion criteria for this study. For each patient, the demographics, clinical history, preoperative status, indications for IOL explantation, and operative and postoperative details were reviewed. Primary outcomes included explantation indications and the type of secondary implanted IOL. A total of 161 eyes from 153 patients were included (96 males; 62.7%); age at the time of the IOL explantation was 65.0 ± 17.4 years. The mean time between primary cataract surgery and IOL explantation was 8.5 ± 7.7 years. In total, 139 (86.3%) PCIOLs and 22 (13.7%) ACIOLs were explanted. The main indications for IOL explantation were dislocation (n = 133; 95.7%) and refractive cause (n = 2; 1.4%) in the PCIOL group. Among ACIOL explantations, the main reasons were pseudophakic bullous keratopathy (n = 14; 63.6%), dislocation (n = 4; 18.2%), and refractive cause (n = 2; 9.1%). In the PCIOL group, 115 (82.7%) primary IOLs were implanted in the capsular bag, 16 (11.5%) were sulcus fixated, and 8 (5.8%) were scleral fixated. The most frequent ocular comorbidities were previous vitrectomy (n = 50, 31.1%), previous ocular trauma (n = 45, 28.0%), glaucoma (n = 16, 9.9%), pseudoexfoliation syndrome (n = 15, 9.3%), and high axial myopia (n = 14, 8.7%). The most commonly used secondary IOL implant was the prepupillary iris-claw IOL (n = 115, 73.7%), followed by the retropupillary iris-claw IOL (n = 32, 20.5%). Uncorrected visual acuity (UCVA) was significantly better following IOL exchange in the entire sample (1.57 ± 0.61 (range: 2.40-0.05) vs. 0.77 ± 0.56 (range: 2.40-0.00); p < 0.001). Best-corrected visual acuity (BCVA) was maintained or improved in 80.7% of cases after IOL explantation. The most common indication for IOL explantation at a tertiary eyecare center in Hungary is IOL dislocation, followed by pseudophakic bullous keratopathy. Prepupillary and retropupillary iris-claw IOL are the most frequently used secondary implants and their use resulted in a significant UCVA improvement following IOL exchange.

In Vitro Shoot Multiplication and Rooting of 'Kashan' and 'Hervy Azerbaijan' Damask Rose (Rosa damascena Mill.) Genotypes for Cosmetic and Ornamental Applications.

The damask rose (Rosa damascena Mill.) is an ornamental-medicinal plant from the Rosaceae family, and its aromatic compounds and essential oils are applied globally in the food, cosmetic, and pharmaceutical industries. Due to its economic value, this research aimed to establish a protocol for an efficient, rapid, and cost-effective method for in vitro shoot multiplication and rooting of the R. damascena 'Kashan' and 'Hervy Azerbaijan' genotypes. Nodal segments (as primary explants) were cultured on the Murashige and Skoog (MS) medium with combinations of various plant growth regulators (PGRs) such as gibberellic acid (GA3), 6-benzylaminopurine (BAP), and indole-3-butyric acid (IBA), as well as a PGR-like substance, phloroglucinol (PG), vitamins such as ascorbic acid (AA), and activated carbon in the form of active charcoal (AC). For the establishment stage, 0.1 mg·L-1 PG, 0.2 mg·L-1 GA3, and 1 mg·L-1 BAP were added to the media. Secondary explants (nodal segments containing axillary buds produced from primary explants) were obtained after 30 days of in vitro culture and transferred to the proliferation media supplemented with different concentrations of BAP (0, 0.5, 1, 1.5, 2, and 2.5 mg·L-1) and GA3 (0, 0.1, 0.2, 0.4, 0.8, and 1 mg·L-1) together with 0.1 mg·L-1 PG and 20 mg·L-1 of AA. The rooting media were augmented with different concentrations of BAP and GA3 with 0.1 mg·L-1 of IBA, PG and 20 mg·L-1 of AA and AC. The results showed that the highest regeneration coefficient (4.29 and 4.28) and the largest number of leaves (23.33-24.33) were obtained in the explants grown on the medium supplemented with 2 mg·L-1 BAP and 0.4 mg·L-1 GA3 for the 'Kashan' and 'Hervy Azerbaijan' genotypes, respectively. Likewise, this PGR combination provided the shortest time until bud break (approximately 6.5 days) and root emergence (approximately 10 days) in both genotypes. The highest number of shoots (4.78 per explant) and roots (3.96) was achieved in this medium in the 'Kashan' rose. Stem and root lengths, as well as stem and root fresh and dry weights, were also analyzed. In most measured traits, the lowest values were found in the PGRs-free control medium. Rooted plantlets were transferred to pots filled with perlite and peat moss in a 2:1 proportion and were acclimatized to ambient greenhouse conditions with a mean 90.12% survival rate. This research contributes significantly to our understanding of Damask rose propagation and has practical implications for the cosmetic and ornamental plant industries. By offering insights into the manipulation of regeneration processes, our study opens up new possibilities for the effective production of high-quality plant material.

Different contractility modes control cell escape from multicellular spheroids and tumor explants.

Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by mesenchymal cells to pull themselves into the extracellular matrix (ECM). Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.

Abstract PO3-26-11: Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors

Abstract Background: Estrogen receptor alpha (ERα) mutations are common (30-40%) in metastatic endocrine therapy-resistant breast cancers (ETR-BC), enable resistance to endocrine therapies and are the molecular drivers of ETR-BC. We had previously shown that an oligobenzamide, ERX-41, could enhance endoplasmic reticulum stress in ETR-BCs driven by mutant (MT) ERα, resulting in cancer cell death in vitro and and in vivo. To enable clinical translation of ERX-41, we performed lead optimization, followed by preclinical and IND-enabling studies. Methods: Over 2000 oligo-benzamides were designed, synthesized and tested in multiple BC models including those that express WT-ERα (MCF7, and ZR75) and BC models with acquired resistance (MCF7-Tam, and MCF7-LTLT) and engineered models that express MT-ERα (MCF7-ERα-D538G, MCF7-ERα-Y537S, ZR75-ERα-D538G, ZR75-ERα-Y537S). For our lead compound, mechanistic validation studies were performed using CRISPR LIPA mutants, RT-qPCR and Western blotting. Explants, organoids, cell line-(CDX) and patient-derived (PDX) xenografts were used to test the ex vivo and in vivo effectiveness of our lead compound as a monotherapy and in combination with abemaciclib. Results: Testing of &amp;gt;2000 synthesized oligo-benzamides identified a lead compound, ERX-315, that had broad and potent activity (IC50 between 20-100 nM) against both WT and MT (mutant) ERα-driven BC cells in in vitro assays. CRISPR KO of LIPA (which encodes lysosomal acid lipase (LAL) in BC cells abrogated cytotoxic response to ERX-315, validating LIPA as the molecular target of ERX-315. Ultrastructural and molecular studies confirm that ERX-315 induces significant endoplasmic reticulum stress, leading to a shutdown of de novo protein synthesis and apoptotic cell death in BC. Importantly, ERX-315 does not induce endoplasmic reticulum stress or cell death in normal cells and is non-toxic in animal models. We have shown that this capacity of ERX-315 to induce endoplasmic reticulum stress is unique among drugs targeting ERα, including selective ERα modulators and degraders, such as GDC-0180, AZD-9496 and fulvestrant. ERX-315 has potent anti-proliferative activity against MT-ERα-driven BC, as seen in genetically modified cell lines both grown as monolayer or spheroids in vitro, patient derived explants (PDEs) ex vivo and cell line derived (CDX) and patient-derived (PDX) xenografts in vivo. The combination of ERX-315 and CDK4/6 inhibitor abemaciclib was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BC cells, in vitro, in xenograft models in vivo, and in primary patient-derived explants ex vivo. We are currently optimizing both the synthesis of ERX-315 using GMP process for kilogram scale production and validated methods for pharmacokinetic studies. We have developed formulations for both intravenous and oral administration with favorable pharmacokinetic parameters and proven utility of the formulated ERX-315 against CDX and PDX tumors in vivo. Toxicity studies in dogs and rodents have demonstrated &amp;gt;8-fold therapeutic to toxicity window. A phase I clinical trial is planned to be open to enrollment by Q1 2024. Conclusions: We have identified a lead compound ERX-315, which represents a novel class of agent that induce catastrophic levels of ER stress resulting in cancer cell death and that can effectively work against multiple forms of ETR-BC, including those driven by MT-ERα. Preclinical studies, GMP manufacturing, formulation and IND-enabling studies are being completed in time for the commencement of the phase I clinical trial by Q1 2024. Citation Format: Suryavathi Viswanadhapalli, Karla Parra, Tae-Kyung Lee, Rahul Gopalam, Kara Kassees, Tanner Reese, Gaurav Sharma, Xihui Liu, Xue Yang, ChiaYuan Chen, Carlos Roggero, Liping Chen, Sautam Bhattacharya, Uday Pratap, Russell Hayward, Sharron Gargosky, Jung-Mo Ahn, Ganesh V. Raj, Ratna Vadlamudi. Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors [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 PO3-26-11.

Abstract 384: SLC7A11 modulates sensitivity to the first-in-class mitochondrial peroxiredoxin 3 inhibitor thiostrepton (RSO-021) via a ferroptosis independent pathway

Abstract Mitochondrial peroxiredoxin 3 (PRX3) has been identified as an actionable cancer vulnerability that is currently being investigated in the first-in-human phase 1 clinical trial, MITOPE (NCT05278975). RSO-021 (thiostrepton or TS) is a redox-active drug that inhibits PRX3’s peroxidase activity via covalent adduction of its active site peroxidatic and resolving cysteine residues, forming an irreversible crosslink across the protein dimer. Covalent inhibition of PRX3 results in tumor cell death due to a diminished ability to remove high levels of mitochondrial reactive oxygen species (ROS) in cancer cells. To better understand the mechanism underpinning sensitivity and resistance, TS-tolerant human mesothelioma cell lines were generated under constant TS pressure and subjected to a drug-screen comprising chemotherapeutic agents, metabolic inhibitors, ROS modulating agents and ferroptosis-inducing compounds, to identify synergistic interactions compared with TS- sensitive cell lines. Erastin, an SLC7A11 inhibitor exhibited the greatest synergy when combined with TS, potentiated PRX3 covalent crosslinking and cellular ROS levels. Synergy with TS was enhanced by cystine depletion and could be phenocopied by siRNA knockdown of SLC7A11. SLC7A11 was up-regulated in TS-tolerant cells and resistance could be overcome with erastin. TS-tolerant cell lines have significantly reduced glutathione levels and slowed proliferation. Ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 did not inhibit TS or TS in combination with erastin. In summary, SLC7A11 up-regulation confers tolerance to TS through a mechanism involving a cysteine-dependent modulation of the redox state independent of canonical ferroptosis, that can be overcome by the addition of an SLC7A11 inhibitor. The correlation between SLC7A11 with response to TS in a 33-patient primary mesothelioma explant co-clinical trial, and patients enrolled into the MITOPE phase 1/2 trial will be presented. Citation Format: Terri Messier, Victoria Gibson, Aleksandra Bzura, Joanna Dzialo, Charlotte Poile, Stephanie Stead, Alexis Saaman, George N. Naumov, Dean A. Fennell, Brian Cunniff. SLC7A11 modulates sensitivity to the first-in-class mitochondrial peroxiredoxin 3 inhibitor thiostrepton (RSO-021) via a ferroptosis independent pathway [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 384.

Abstract 4071: The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis

Abstract Antibody-cytokine conjugates leverage orthogonal mechanisms of action (MoA) in one molecule to induce potent antitumor immune responses. PD-1-targeting conjugates are of particular interest since they preferentially target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME) while simultaneously blocking the PD-(L)1 pathway and inducing potent cytokine receptor stimulation in the same CD8+ T cell in cis (cis-signaling). Interleukin 18 (IL-18) is a proinflammatory cytokine able to integrate both innate and adaptive immunity resulting in profound anti-tumor immune responses mediated by T effector and NK cells. BPT567 has been generated via chemical conjugation of the anti-PD-1 Ab LipustobartTM to an enhanced human IL-18 variant of increased potency and reduced sensitivity to IL-18 binding protein (IL-18BP). BPT567 is designed to specifically target and activate a subset of tumor-infiltrating PD-1+ IL18R+ CD8+ T effector cells recently described to exhibit superior cytotoxic and proliferative activity (Codarri Deak et al., Nature 2022). Due to its ability to signal in cis, BPT567 shows enhanced potency as well as increased resistance to IL-18BP when analyzing IFNγ release in vitro in PD-1+ cells. Subsequent PD-1 receptor occupancy analyses showed that it is fully sufficient for BPT567 to engage a low number of PD-1 receptors to induce maximum IFNγ release in NK92 cells expressing human PD-1. Strikingly, despite addressing overlapping epitopes in PD-1, even a 200-fold excess of pembrolizumab has no impact on the in vitro potency of BPT567. In vivo, BPT567 exhibits strong anti-tumor efficacy at significantly lower IL-18 doses compared to the combination of an untargeted antibody-IL-18 conjugate and an anti-PD1 Ab further corroborating the importance of cis-signaling as a unique MoA of this PD1-IL18 conjugate. Detailed analyses of tumor-infiltrating immune leukocytes (TIL) revealed that BPT567 triggers a preferential expansion of CD8+ T effector memory cells within the TME. In line with this finding, activity in the MC38 tumor model is dependent on the presence of CD8+ T cells while depletion of other immune cell subsets (e.g. NK cells, CD4+ T cells, macrophages) had no impact on efficacy. Although not required for anti-tumor efficacy, CD4+ T cells are essential for proficient formation of an immunological memory. In an effort to confirm findings generated in murine models in relevant human ex vivo systems, we analyzed the release of cytokines and chemokines in dissociated cells isolated from primary human tumor explants. Also in this human model, BPT567 is able to induce significantly stronger IFNγ release compared to either single agents or the combination of untargeted IL-18 and an anti-PD1 Ab, thus confirming the unique MoA of the PD1-IL18 conjugate triggering superior TIL activation in cis. Citation Format: Kea Martin, Thuy Luu, Jean-Philippe Carralot, Lilian Gremlich, Petra Herzig, Caoimhe Herr, Roy Meoded, Philipp Moosmann, Arnaud Goepfert, Alfred Zippelius, Vijaya Pattabiraman, Bertolt Kreft. The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis [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 4071.

Abstract A069: High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants

Abstract Introduction: As high-grade serous ovarian cancer (HGSOC) rarely shows symptoms, most patients present with metastases. Tumor heterogeneity between metastases may impede a patient’s response to therapy and thus impact survival. We aim to decipher the heterobiology of HGSOC lesions using high-resolution imaging of patient-derived explants (PDEs), to determine the differential treatment response between primary and metastatic tumors. Methods: PAX8, CK7, RAD51, γH2AX, Ki67, cPARP, and CD3 were chosen to enumerate tumor and T cells, and their activity. Antibodies were optimized in monolayer and spheroid cultures of HGSOC cell lines, COV318 and OVCAR4. CD3 was optimized in FFPE human tonsillitis tissue. Primary and metastatic HGSOC explants were cultured for 48h followed by 24h fixation. Several immunostaining and optical clearing protocols were tested on PDEs, which were imaged using a fully automated confocal high-content screening system. Results: Immunostaining and automated image analysis facilitated the identification of individual nuclei within PDEs without Refractive Index (RI) matching or optical clearing. However, high background impeded further image analysis, preventing the quantification of certain antibodies. Glycerol-based RI matching and ScaleS tissue clearing were implemented to try and overcome this. However, neither generated sufficiently high-quality images for analysis. A novel Clearing-enhanced 3D (Ce3D) immunostaining system recently developed for high-quality multiplex fluorescence microscopy of large tissue volumes was tested. While individual nuclei were recognized, as well as RAD51 foci and CK7 cytoskeleton staining, image analysis was still impeded due to high background. Conclusions: Protocols without RI matching and optical clearing can stain large, complex structures such as PDEs. However, high background impedes image analysis suggesting the need for optical clearing and RI matching. The inclusion of various RI matching and/or optical clearing steps failed to overcome high background staining. Further work is ongoing to refine immunostaining, RI matching, optical clearing, and mounting of PDEs to allow the quantification of all antibodies. Citation Format: Adele E. Connor, Joanne Lysaght, Donal J. Brennan, Jeremy C. Simpson, Antoinette S. Perry. High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A069.

Establishing the &lt;i&gt;in vitro&lt;/i&gt; culture of and micropropagating edible honeysuckle

Edible honeysuckle is a popular fruit crop. Its therapeutic and health-promoting effects are attributed to a high content of bioactive compounds in the fruits. Unlike the traditional plant multiplication methods, the in vitro propagation allows scientists to obtain high-quality planting material of honeysuckle in a great quantity and within a short time. The research was carried out at the Laboratory of Breeding and Genetic Research on Field Crops of the Federal Scientific Center of Agricultural Biotechnology of the Far East named after A.K. Chaiki. Honeysuckle variety Podarok amurchanam created by the Far Eastern State Agrarian University was used as the research object. The research materials were sterilized according to the methodology of N.I. Vavilov All-Russian Institute of Plant Genetic Resources with some modifications. Several products were used as chemical agents for sterilization in the following sequence: a 5% solution of surfactants, fungicide Fundazol, EC (1 g/l), the bleaching agent ACE freshly diluted with distilled water in the proportion 1:9 (0.50% of NaOCl in the working solution), and 70% ethanol. The primary explants were cultured on an MS containing 20 g/l sucrose and 6 g/l agar (hereafter – MS) and supplemented with 6-benzylaminopurine (BA) at a concentration of 0.5 mg/l. The pH of the medium was adjusted to 5.7-5.8 using 1N КОН. The explants (microcuttings with one-two internodes) were subcultured on an MS supplemented with BA (0.5 mg/l). The morphometric parameters of the plants were measured on the 35th day of cultivation. The sterilization of the explants with Fundazol (1 g/l) and the ACE diluted with distilled water in the proportion 1:9 allowed us to obtain a high number of viable microclones (50%). The elimination of leaves from the honeysuckle microcuttings drastically decreased the survival rate and led to the death of the microclones in most cases (the mortality rate was 98.7 %). Subculturing the microcuttings on the MS supplemented with BA at a concentration of 0.5 mg/l facilitated the normal growth and development of the regenerated honeysuckle plants (the average reproduction rate was 4.65).

Размножение in vitro перспективных сортов картофеля для возделывания в условиях Республики Коми

The paper deals with the results of clonal micropropagation of promising potato varieties for cultivation in the conditions of the Komi Republic. The study material is three potato varieties (Zyryanets, Pechorsky, Vychegodsky), characterized by the high resistance to late blight and abiotic stress factors in the conditions of the Komi Republic. The primary explants were weakly germinated eyeholes, as well as cuttings and shoot tips isolated from 14- and 30-day-old potato plants which underwent surface sterilization and then were cultivated in the Murashige–Skoog nutrient medium containing various hormones. The Pechorsky variety was identified to have the high growth index and specific growth rate of shoots in vitro. This variety can be included in studies on cell and genetic engineering aimed at creating potato forms with the complex resistance.

AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

In vitro culture introduction and microclonal reproduction of triploid Aspen (Populus Tremula L.)

In response to global warming issues, the entire world community seeks to decarbonize its energy and economy. One of the ways to reduce concentrated greenhouse gases in the atmosphere is to enlarge forest areas and encourage forest regeneration after fires. The given paper describes sterilization methods of primary explants. Studies have shown that 0.1% diacid solution supplemented with Tween 20 is the most effective at producing aseptic viable cultures of triploid aspen (Populus tremula L.) in vitro. This method provides the necessary amount of viable sterile cultures for their subsequent micro-multiplication (clonal), sufficient for the further mass production of planting material. Murashige & Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) had a more intensive formation of adventive buds compared to the medium with kinetin. Higher BAP concentration resulted in more regenerants from one explant with their height shortened. Woody Plant Medium (WPM) with different concentrations of 6-benzylaminopurine and indolyl-acetic acid (IAA) is more effective for working with triploid aspen. Effective reproduction at triploid aspen cultivation was shown at 0.5 mg/l concentration of BAP. The research results can be successfully used in vitro culture introducing of triploid aspen, experiments on micro-reproduction of other tree crops to get high-quality planting material.

Type I collagen concentration affects neurite outgrowth of adult rat DRG explants by altering mechanical properties of hydrogels

Type I collagen is a predominant fibrous protein that makes up the extracellular matrix. Collagen enhances cell attachment and is commonly used in three-dimensional culture systems, to mimic the native extracellular environment, for primary sensory neurons such as dorsal root ganglia (DRG). However, the effects of collagen concentration on adult rat DRG neurite growth have not been assessed in a physiologically relevant, three-dimensional culture. This study focuses on the effects of type I collagen used in a methacrylated hyaluronic acid (MAHA)-laminin-collagen gel (triple gel) on primary adult rat DRG explants in vitro. DRGs were cultured in triple gels, and the neurite lengths and number of support cells were quantified. Increased collagen concentration significantly reduced neurite length but did not affect support cell counts. Mechanical properties, fiber diameter, diffusivity, and mesh size of the triple gels with varying collagen concentration were characterized to further understand the effects of type I collagen on hydrogel property that may affect adult rat DRG explants. Gel stiffness significantly increased as collagen concentration increased and is correlated to DRG neurite length. Collagen concentration also significantly impacted fiber diameter but there was no correlation with DRG neurite length. Increasing collagen concentration had no significant effect on mesh size and diffusivity of the hydrogel. These data suggest that increasing type I collagen minimizes adult rat DRG explant growth in vitro while raising gel stiffness. This knowledge can help develop more robust 3D culture platforms to study sensory neuron growth and design biomaterials for nerve regeneration applications.

5-Bromo-3,4-dihydroxybenzaldehyde stabilizes diabetic retinal neurovascular units by inhibiting the inflammatory microenvironment

BackgroundDiabetic retinopathy (DR) is a leading cause of blindness characterized by damage to the retinal neurovascular unit, which is caused by hyperglycemia-induced metabolic and inflammatory responses. 5-Bromo-3,4-dihydroxybenzaldehyde (BDB) is a compound derived from marine red algae and known for its anti-inflammatory effects. MethodsThis study aimed to investigate the potential protective effects of BDB on DR using primary human retinal vascular endothelial cells and retinal tissue explants. The analysis involved assessing vascular integrity, expression of tight junction protein, hyperglycemia-induced permeability, and retinal ganglion cell (RGC) apoptosis. The protective effect of BDB in maintaining the diabetic retinal neurovascular units was verified using type 1 diabetic mouse models. Additionally, the inhibitory effect of BDB on the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 were examined. ResultsIn vitro experiments revealed that BDB promoted vascular integrity, inhibited the transcription of pro-inflammatory factors, and alleviated hyperglycemia-induced permeability. BDB also protected RGC from hyperglycemia-induced apoptosis. In diabetic mice models, BDB treatment maintained the integrity of diabetic retinal neurovascular units and inhibited the secretion of TNF-α, IL-1β, and IL-6. ConclusionBDB demonstrated a protective effect on DR by inhibiting the secretion of inflammatory factors, suggesting its potential as a therapeutic agent for the treatment of DR. Further research is warranted to validate its safety and efficacy for clinical application.

The Burden of Revision Total Ankle Replacement Has Markedly Increased from 2010 to 2020

Category: Ankle Arthritis Introduction/Purpose: Total ankle replacement (TAR) surgery has steadily increased in recent decades. The aim of the current study was to investigate the evolving burden of revision surgery and risk factors and timing of revision or implant removal. We hypothesized that there has been an increase in revision TAR and explant surgeries in the past decade and that younger age and higher comorbidity burden are risk factors for revision and explant. Methods: Using the 2010-2020 PearlDiver M151Ortho dataset, this retrospective cohort study identified primary TAR, TAR revision, and TAR explant patients. This is a large national database containing billing claims information on over 151 million patients in the United States across all payer types and sites of care. Patient factors investigated included age, sex, and Elixhauser Comorbidity Index (ECI, a longitudinal measure of patient comorbidity burden generated using ICD-9 and ICD-10 diagnosis codes). Annual incidence for primary and revision TAR, and explant were recorded. Multivariate logistic regression analyses were performed to determine independent risk factors for revision TAR or explant relative to primary TAR. For explants, the eventual intervention by two years was analyzed. Ten-year timing and survival to revision or explant surgery following patient-matched unilateral TAR were characterized. Results: Over the study period, there were 10,531 primary, 1,218 revision, and 1,735 explant TARs (increasing by 310%, 398%, and 9.6%, respectively). Independent predictors of revision TAR included: younger age (odds ratio [OR] 1.29 per decade decrease) and higher ECI (OR 1.23 per 2-point increase) (p &lt; 0.0001 for both). Independent predictors of explant surgery included: younger age (OR 1.80 per decade decrease, p&lt; 0.0001), female sex (OR 1.17, p=0.0011), and higher ECI (OR 1.35 per 2-point increase, p&lt; 0.0001). Explanted patients went on to arthrodesis (37.7%), amputation (26.1%), revision TAR (14.7%), or no identified procedure/other procedures (21.5%) within the subsequent two years. The 10-year implant survival rate following primary TAR was 91.8%. 73% and 83% of revisions and explants occurred in the first three years following index TAR, respectively. Conclusion: The burden of TAR revision has grown substantially over the past decade. Younger age, female sex, and higher patient comorbidity burden were associated with increased risk of revision or explant surgery. If explant is pursued, most went on to arthrodesis or amputation. These results suggest that care should be taken in patient selection for TAR to minimize the occurrence of revision and explant surgery. Treatment algorithms should be modified to optimize outcomes among patients who undergo explant or revision surgery.

Фенотипическая и генетико-селекционная оценка березы повислой (Betu-la pendula ROTH) и березы пушистой (Betula pubescens EHRH.) ex situ и in vitro

The results of a study of birch test cultures obtained by different methods of ex situ pollination and introduced into culture in vitro are presented. To identify the phenotypic, cultural and cytological characteristics of two birch species naturally growing in the Central Black Earth Region - silver birch (diploid) and downy birch (tetraploid), test cultures of seed origin F1 and F2 were specially created. The article presents data on phenotypic (growth in height in the juvenile and reproductive periods of ontogeny), genetic selection (seed propagation system) variability and some characteristics of the introduction of F1 seed progeny of breeding forms of local birch species into in vitro culture. To predict the growth characteristics of seed progeny of breeding forms of local birch species obtained by different methods of pollination - self-pollination and open pollination at different stages of ontogeny (at the age of 2 and 10 years), a statistical processing of the feature of growth in height of families was carried out using the rank Spearman's correlation coefficient ρ. Species specificity was shown in response to different pollination methods, morphogenic activity of self-fertile and self-sterile forms of these birch species on a nutrient medium ½ MC + 6-benzylaminopurine 1 mg/l, primary shoot formation of explants and regenerative capacity in these species, the nature of morphogenesis (formation of the main escape). For the polyploid species (downy birch), a higher level of self-fertility was established, as well as a more intensive growth of shoots in height than for drooping birch; primary explants in the former had a different character of morphogenesis (there was a joint initiation of the main shoot with adventitious ones) than in silver birch (formation of the main shoot), which indicates a greater adaptive potential for polyploid species and their greater opportunities for inclusion in genetic breeding experiments and obtaining valuable breeding forms.