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Background: Peucedanum praeruptorum Dunn has typical stacked umbels and medicinal value; however, the lack of an effective tissue culture system for P. praeruptorum has limited the large-scale propagation of its seedlings. Results: We systematically established an in vitro regeneration system for P. praeruptorum using young leaves and stems as explants. Tissue culture plantlets were successfully obtained within 123 and 90 d of somatic embryogenesis and organogenesis, respectively. Combined plant growth regulators (PGRs) were optimized to promote efficient plant regeneration at each stage of the culture process. Specifically, embryogenic callus induction was superior in Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). For somatic embryonic development, the highest differentiation rates were achieved using BA, 2,4-D, and 6-furfuryl aminopurine (6-KT). Induction of organogenesis resulted in the highest differentiation rates and proliferation coefficients of buds in MS medium supplemented with BA and α-naphthaleneacetic acid (NAA). Moreover, regeneration of P. praeruptorum seedlings was achieved by adjusting the BA and indole-3-butyric acid (IBA) concentrations in 1/2 MS medium. Conclusion: Our results provide a technical system for the rapid propagation of P. praeruptorum, which can facilitate germplasm improvement, resource conservation, and further genetic transformation of Peucedanum species. [ABSTRACT FROM AUTHOR]

Tumor organoids, an in-vitro three-dimensional model, possess high potential for investigating tumor biology and treatment response and have been demonstrated more appropriate for drug assessment than two-dimensional cultures. Herein, we described two cases of invasive high-grade urothelial carcinoma who underwent radical cystectomy successfully following use of patient-derived organoids (PDOs) for drug screening to inform therapeutic decisions. In these two cases, the PDOs cultured by biopsy tissues were both sensitive to the combination of gemcitabine and cisplatin. After neoadjuvant chemotherapy (NAC) with gemcitabine and cisplatin, the patients responded well, and radical cystectomy was performed successfully. No recurrence or metastasis was observed within 6 months after surgery. This small case series suggests that the patient-derived urothelial carcinoma organoids contribute to optimizing NAC options to guide personalized treatment and improve the survival outcomes. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) is the pathogenic agent of pseudorabies, causing serious reproductive failure in swine. However, it is still unknown whether PRV uterine inoculation impairs blastocyst implantation. In the present study, a PRV infection mouse model was developed. Pregnant mice were inoculated with either 104 or 105 50% tissue culture infectious dose (TCID50) units of PRV on gestation day 2 (GD2). Viral DNA was detected in tissues by PCR and/or in situ hybridization. Histopathological change and expression of proinflammatory cytokines in uterus were analyzed by H.E. staining and qPCR, respectively. Apoptosis was also investigated by TUNEL assay, and the expression of apoptosis-related proteins including Bax and Bcl-2 was detected by Western blot. The results showed that intrauterine exposure of PRV on GD2 reduced the number of embryo implantation site. Abundant viral DNA was detected in spinal marrow and brain, and small amounts of PRV genomes were detected in embryo implantation site, ovary as well as thymus. Considerable inflammatory cells infiltrating in the endometrium, with high levels of pro-inflammatory cytokines of interleukin (IL)-6, IL-1β and tumor necrosis factor- α mRNA after infection. In addition, PRV infection promoted apoptosis in stroma and endothelium of the mouse endometrium. Collectively, intrauterine inoculation of PRV during early pregnancy causes cytokine release syndrome and apoptosis in endometrium, which impairs mouse embryo implantation. [ABSTRACT FROM AUTHOR]

The ornamental plant Calathea ‘Network’, belonging to the Araceae and the Calathea, was the material for this study. We studied the effects of types and concentrations of plant growth regulators, inoculation methods on bud proliferation, different media formulations on rooting induction, and different cultivation medium on transplant survival rate. The results showed that the single bud inoculation method had a better effect. The culture medium MS+0. 1 mg / L NAA+0. 5 mg / L BA was suitable for the shoot proliferation in C. ‘Network’. After 30 days of cultivation, the induction rate of adventitious buds was 98. 89%, and proliferative buds was 3. 29. The optimal medium for rooting 1 / 2MS+ 0. 1 mg / L NAA+0. 5 g / L AC could reach transplanting standard in 15-25 days. The peat ∶ coir = 3 ∶ 1 substrate was suitable for transplanting the in vitro seedlings of C. ‘Network’ and the survival rate of transplanting was 97. 22%. This study successfully established an efficient in vitro propagation system for the industrialization of the plant, providing technical support for its industrial production. [ABSTRACT FROM AUTHOR]

Background : Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis. Results: Highly efficient calli were induced on MS medium added 1.5 mg·L− 1 6-benzylaminopurine (6-BA) and 0.02 mg·L− 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L− 1 6-BA and 0.1 mg·L− 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants. Conclusions: Here, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism. [ABSTRACT FROM AUTHOR]

Cell culture is a cornerstone of in vitro biological research. Whilst glassware was once commonplace in tissue culture facilities, in recent decades laboratories have moved towards a heavy reliance on single use plastics for routine procedures. Single use plastics allow for accessible, sterile, and often affordable equipment that comes at a high environmental cost. We developed a glassware preparation and cleaning process that allowed the comparison of "traditional" plastic-heavy, and adapted "sustainable," cell culture practices, to empirically compare the sterility, viability, and proliferative capacity of cells cultured with differing techniques, by observing IL-6 production, morphology, and proliferation rate of cultured human pulmonary fibroblast cells. During which, we calculated the carbon footprint of traditional versus sustainable methods. We additionally endeavored to provide a realistic overview of the steps required to transition to more sustainable cell culture practices and make suggestions to ease the cost, labor, and time required to uptake similar practices in other laboratories. Cells cultured using reusable glassware did not show signs of contamination or stress compared to cells grown solely with plasticware, and glassware baked at 180°C for 120 min was sufficiently decontaminated and depyrogenated for culturing these cells. An individual researcher adopting the same methodology could reduce their carbon footprint by 105.92 kg of Carbon dioxide equivalent (CO2e) whilst also saving money (£408.78) over a 10-year period. We predict that these benefits would be greater if more researchers were to uptake these adapted practices. We intend for this paper to reassure researchers that viable, sterile, and sustainable routine cell culture can be achieved with little upfront cost to the researcher, with the prospective benefit of greatly reducing the cost to the environment. We additionally hope that increased uptake, and thus demand of more sustainable practices, encourages suppliers, policy makers, and funding bodies to make sustainable practices more accessible to individual researchers and institutions worldwide. [ABSTRACT FROM AUTHOR]

Epigenetic changes accompany the dynamic changes in the cell wall composition during the development of callus cells. H3K4me3 is responsible for active gene expression and reaction to environmental cues. Chromatin immunoprecipitation (ChIP) is a powerful technique for studying the interplay between epigenetic modifications and the DNA regions of interest. In combination with sequencing, it can provide the genome-wide enrichment of the specific epigenetic mark, providing vital information on its involvement in the plethora of cellular processes. Here, we describe the genome-wide distribution of H3K4me3 in morphogenic and non-morphogenic callus of Fagopyrum tataricum. Levels of H3K4me3 were higher around the transcription start site, in agreement with the role of this mark in transcriptional activation. The global levels of methylation were higher in the non-morphogenic callus, which indicated increased gene activation compared to the morphogenic callus. We also employed ChIP to analyse the changes in the enrichment of this epigenetic mark on the cell wall-related genes in both calli types during the course of the passage. Enrichment of H3K4me3 on cell wall genes was specific for callus type, suggesting that the role of this mark in cell-wall remodelling is complex and involved in many processes related to dedifferentiation and redifferentiation. This intricacy of the cell wall composition was supported by the immunohistochemical analysis of the cell wall epitopes' distribution of pectins and extensins. Together, these data give a novel insight into the involvement of H3K4me3 in the regeneration processes in F. tataricum in vitro callus tissue culture. [ABSTRACT FROM AUTHOR]

Mammary epithelial cells, the only milk-producing cell type in the mammary gland, undergo dynamic proliferation and differentiation during pregnancy, culminating in lactation postpartum. The East FriEsian sheep ranks among the world's most prolific dairy breeds, while the Sewa sheep, a unique dual-purpose breed autochthonous to the Qinghai-Tibet Plateau, exhibits significantly lower milk production. Employing tissue culture methods, we successfully established mammary epithelial cell lines from both breeds. Morphological assessment of mammary epithelial cells and immunofluorescence identification of Cytokeratin 7 and Cytokeratin 8 confirmed the epithelial identity of the isolated cells. Subsequent RNA-seq analysis of these in vitro epithelial cell lines revealed 1813 differentially expressed genes (DEGs). Among these, 1108 were significantly up-regulated and 705 were down-regulated in Sewa epithelial sheep cells compared to East FriEsian epithelial cells. KEGG enrichment analysis identified cellular processes, environmental information processing, human diseases, metabolism, and organismal systems as the primary functional categories associated with DEGs. Gene ontology (GO) terms annotation, categorized into molecular function, biological processes, and cellular component, yielded "binding and catalytic activity," "molecular function regulator activity," and "cellular process," "biological regulation," and "regulation of biological process" as the top three terms within each domain, respectively. Clusters of Orthologous Groups of proteins (KOG) classification further revealed that "signal transduction mechanisms" accounted for the largest proportion of DEGs among all KOG categories. Finally, based on these analyses, ATF3 and MPP7 were identified as promising candidate genes for regulating lactation. [ABSTRACT FROM AUTHOR]

Introduction: The mechanisms of the SARS-CoV-2-triggered complex alterations in immune cell activation and production of cytokines in lung tissue remain poorly understood, in part because of the limited use of adequate tissue models that simulate the structure and cell composition of the lung in vivo. We developed a novel ex vivo model of SARS-CoV-2 infection of lung explants, that maintains the intact tissue composition and the viral load for up to 7-10 days. Using this model, we studied cytokine production during SARS-CoV-2 infection. Materials and methods: Lung tissue was monitored for viability and cell composition using flow cytometry and histological analysis. SARS-CoV-2 infection was verified immunohistochemically, viral loads in tissue and culture medium were monitored by qPCR. A panel of 41 cytokines was measured in culture medium using xMAP technology. Results: The explant lung tissue was viable and maintained viral infection that influenced the cytokine production. Elevated concentrations of G-CSF, GM-CSF, GRO-a, IFN-g, IL-6, IL-8, IP-10, MCP-3, MIP-1a, PDGF-AA, and VEGF, and decreased IL-1RA concentration were observed in infected tissue compared to non-infected tissue. Discussion: Our results generally reflect the data obtained in COVID-19 patients. GRO-a, IFN-g, IL-6, IL-8, MCP-1, MCP-3, and RANTES correlated with the viral load, forming a distinct pro-inflammatory cluster. Thus, our lung ex vivo model faithfully reproduces some aspects of cytokine alterations in COVID-19 patients at an early disease stage, making the investigation of SARS-CoV-2 infection mechanisms more accessible and providing a potential platform for antiviral drug testing. [ABSTRACT FROM AUTHOR]

1-deoxynojirimycin (DNJ) is a well-known α-glucosidase inhibitor. A series of phenyltriazole-deoxynojirimycin hybrids containing C4 and C6 (4 and 6 methylenes, respectively) linkers were synthesized. These novel compounds were assessed for preliminary glucosidase inhibition and cytotoxicity tests in vitro. Among them, compounds 12–14 and 16–20 (IC50: 105 ± 9–11 ± 1 μM) were more active than deoxynojirimycin (DNJ, IC50 = 155 ± 15 μM). The kinetics of enzyme inhibition measured by using Lineweaver–Burk plots indicated that compounds 18 and 19 were competitive inhibitors. In addition, a molecular docking study of α-glucosidase revealed that the interaction modes and the orientations of compound 18 and DNJ were clearly different. Furthermore, in tissue culture, HL60 cell compounds showed no cytotoxicity at low concentrations. When the concentration reached 50 µM, only compound 20 exhibited cytotoxicity. The structure–activity relationships exhibit that the length of the linker and the nature of 4-position substituents on the phenyl have a significant effect on the inhibitory potency of glucosidases and cytotoxicity. [ABSTRACT FROM AUTHOR]

The article discusses the importance of trithiocarbonates in various applications, including organic synthesis, manufacturing, and pharmaceuticals. The focus is on developing cleaner and greener methods for synthesizing asymmetrical trithiocarbonates with minimal use of reagents. The synthesized compounds showed significant anticancer activity against different human cancer cell lines. The study highlights the eco-friendly approach and efficient use of resources in the preparation of these biologically potent molecules. [Extracted from the article]

Cryopreservation allows for the long-term storage of biological materials, ensuring their viability for future use and preserving biodiversity. This study aimed to develop an efficient protocol for the long-term in vitro conservation of fegra fig. In vitro precultured shoot tips of fegra fig with 0.3 M sucrose showed the highest regrowth percentage (100%) before plunging into liquid nitrogen. After plunging into liquid nitrogen, the regrowth percentage was 43.33%. Following cryopreservation, the recovered shoots were rooted and acclimatized to ex vitro conditions. The genetic fidelity of the acclimatized 8-week-old fegra fig plantlets after cryopreservation with their mother plant was tested using randomly amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) molecular markers. Genetic similarity between the stored plantlets with their mother plant was 100% ensuring uniformity and true-to-type regenerated plants following cryopreservation. This study presents, for the first time, an efficient protocol for the genetic conservation of fegra fig. [ABSTRACT FROM AUTHOR]

Background: The incidence of non-tuberculous mycobacterium infection has shown a gradual increasing trend in recent years, among which cutaneous manifestations as an important aspect. This study aimed to describe the clinical features and microbiological findings in 6 cases of primary cutaneous nontuberculous mycobacterium infection. Methods: In this retrospective study from June 2021 to June 2022, the clinical data and microbiological results of six cases diagnosed with primary cutaneous non-tuberculous mycobacterium infection in department of dermatology, Hangzhou Third People's Hospital were analyzed. Results: All six cases were primary cutaneous non-tuberculous mycobacterium infections, four of which had a history of trauma or exposure, and two had an underlying disease that could lead to compromised immunity. All patients presented with erythema nodular skin lesions, four on the upper or lower extremities, one on the face, and one on the right hip. The histopathological findings of five patients who underwent biopsy were granulomatous inflammatory changes with mixed infiltration. Laboratory cultures using tissue or tissue fluid were all successful, including four Mycobacterium marinum, one Mycobacterium abscessus, and one Mycobacterium avium. Metagenomics next-generation sequencing detected results consistent with culture colonies in only two cases. With the exception of case 4, all patients responded well to oral medication, with a course of treatment ranging from 4 months to 1 year, and the prognosis was good. Conclusions: The clinical features of primary cutaneous non-tuberculous mycobacterium infection are often lacking in specificity, and the identification of related strains is difficult for a variety of reasons. Although the results of metagenomics next-generation sequencing are useful for pathogen spectrum identification, its diagnostic value should be carefully reevaluated under certain circumstances. Patients with suspected triggers who do not respond well to conventional treatments should be suspected as atypical infection and potential immunosuppression. If diagnosed and treated promptly, the prognosis of primary cutaneous non-tuberculous mycobacterium infection is generally good. [ABSTRACT FROM AUTHOR]

Ultraviolet radiation (UVR) has been recognized as a potential trigger for the transformation of benign melanocytic nevi into melanoma. However, the mechanisms governing the formation and progression of melanocytic nevi remain poorly understood. This lack of understanding is partly due to the difficulty in isolating and culturing nevus tissues in vitro, resulting in a dearth of robust ex vivo models for nevi. Therefore, the establishment of a reliable melanocytic nevus model is imperative. Such a model is essential for elucidating nevus pathogenesis and facilitating the development of effective therapeutic interventions. Therefore, we have sought to establish an ex vivo nevus explant model to study UVR stimulation. And the structural integrity and tissue activity of the ex vivo nevi explant model was evaluated. We then observed melanogenesis and proliferation activity of the explants after UVR stimulation. There was less blister formation after Day 3 in nevi explants under our modified medium conditions. The nevi explant was able to maintain almost the same morphological structure and tissue activity as in vivo tissue within 24 h. Following UVR stimulation, we observed increased melanogenesis and proliferation activity in nevi explants. Nevi explants could serve as an ex vivo model for UVR‐induced nevi stimulation research. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus, a pathogen to ruminants, camelids, and humans, is an emerging mosquito-borne bunyavirus currently endemic to Africa and the Arabian Peninsula. Although animals are primarily infected via mosquito bites, humans mainly become infected following contact with infected tissues or fluids of infected animals. There is an urgent need for adequate countermeasures, especially for humans, because effective therapeutics or vaccines are not yet available. Here we assessed the safety, tolerability, and immunogenicity of a next-generation, four-segmented, live-attenuated vaccine candidate, referred to as hRVFV-4s, in humans. A first-in-human, single-centre, randomised, double-blind, placebo-controlled trial was done in Belgium in which a single dose of hRVFV-4s was administered to healthy volunteers aged 18–45 years. Participants were randomly assigned using an interactive web response system. The study population encompassed 75 participants naive to Rift Valley fever virus infection, divided over three dosage groups (cohorts) of 25 participants each. All participants were followed up until 6 months. Using a staggered dose escalating approach, 20 individuals of each cohort were injected in the deltoid muscle of the non-dominant arm with either 104 (low dose), 105 (medium dose), or 106 (high dose) of 50% tissue culture infectious dose of hRVFV-4s as based on animal data, and five individuals per cohort received formulation buffer as a placebo. Primary outcome measures in the intention-to-treat population were adverse events and tolerability. Secondary outcome measures were vaccine-induced viraemia, vaccine virus shedding, Rift Valley fever virus nucleocapsid antibody responses (with ELISA), and neutralising antibody titres. Furthermore, exploratory objectives included the assessment of cellular immune responses by ELISpot. The trial was registered with the EU Clinical Trials Register, 2022-501460-17-00. Between August and December, 2022, all 75 participants were vaccinated. No serious adverse events or vaccine-related severe adverse events were reported. Pain at the injection site (51 [85%] of 60 participants) was most frequently reported as solicited local adverse event, and headache (28 [47%] of 60) and fatigue (28 [47%] of 60) as solicited systemic adverse events in the active group. No vaccine virus RNA was detected in any of the blood, saliva, urine, or semen samples. Rift Valley fever virus nucleocapsid antibody responses were detected in most participants who were vaccinated with hRVFV-4s (43 [72%] of 60 on day 14) irrespective of the administered dose. In contrast, a clear dose–response relationship was observed for neutralising antibodies on day 28 with four (20%) of 20 participants responding in the low-dose group, 13 (65%) of 20 responding in the medium-dose group, and all participants (20 [100%] of 20) responding in the high-dose group. Consistent with the antibody responses, cellular immune responses against the nucleocapsid protein were detected in all dose groups, whereas a more dose-dependent response was observed for the Gn and Gc surface glycoproteins. Neutralising antibody titres declined over time, whereas nucleocapsid antibody responses remained relatively stable for at least 6 months. The hRVFV-4s vaccine showed a high safety profile and excellent tolerability across all tested dose regimens, eliciting robust immune responses, particularly with the high-dose administration. The findings strongly support further clinical development of this candidate vaccine for human use. The Coalition for Epidemic Preparedness Innovations with support from the EU Horizon 2020 programme. [ABSTRACT FROM AUTHOR]

Optical coherence tomography (OCT) imaging technology has significant advantages in in situ and noninvasive monitoring of biological tissues. However, it still faces the following challenges: including data processing speed, image quality, and improvements in three-dimensional (3D) visualization effects. OCT technology, especially functional imaging techniques like optical coherence tomography angiography (OCTA), requires a long acquisition time and a large data size. Despite the substantial increase in the acquisition speed of swept source optical coherence tomography (SS-OCT), it still poses significant challenges for data processing. Additionally, during in situ acquisition, image artifacts resulting from interface reflections or strong reflections from biological tissues and culturing containers present obstacles to data visualization and further analysis. Firstly, a customized frequency domain filter with anti-banding suppression parameters was designed to suppress artifact noises. Then, this study proposed a graphics processing unit (GPU)-based real-time data processing pipeline for SS-OCT, achieving a measured line-process rate of 800 kHz for 3D fast and high-quality data visualization. Furthermore, a GPU-based real-time data processing for CC-OCTA was integrated to acquire dynamic information. Moreover, a vascular-like network chip was prepared using extrusion-based 3D printing and sacrificial materials, with sacrificial material being printed at the desired vascular network locations and then removed to form the vascular-like network. OCTA imaging technology was used to monitor the progression of sacrificial material removal and vascular-like network formation. Therefore, GPU-based OCT enables real-time processing and visualization with artifact suppression, making it particularly suitable for in situ noninvasive longitudinal monitoring of 3D bioprinting tissue and vascular-like networks in microfluidic chips. [ABSTRACT FROM AUTHOR]

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic and adult skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of skin, including nucleated/enucleated epithelium, mesenchyme, and hair follicle, as well as the role of collagens in maintaining its integrity. Furthermore, quantitative analysis comparing stiffness distributions in live tissue samples with those in preserved tissues, including formalin-fixed and cryopreserved tissue samples, unveiled the distinct impacts of preservation processes on tissue stiffness patterns. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples to accurately capture the true spatiotemporal variations in mechanical properties. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. Stiffness, a simple mechanical parameter, has drawn attention in understanding the mechanobiological principles underlying the homeostasis and pathology of living tissues. To explore tissue-scale mechanobiology, we propose a technique integrating an upright structured illumination microscope and an atomic force microscope. This technique enables live measurements of stiffness distribution and fluorescent observation of thick living tissue slices. Experiments revealed the highly heterogeneous mechanical nature of mouse embryonic and adult skin in three dimensions and the previously unnoticed influences of preservation techniques on the mechanical properties of tissue at microscopic resolution. This study provides a new technical platform for live stiffness measurement and biomolecular observation of tissue-scale samples with micron-scale resolution, thus contributing to future studies of tissue- and organ-scale mechanobiology. [Display omitted] [ABSTRACT FROM AUTHOR]

Ruffled vegetables, having wrinkled leaves with a crispy and tender texture, are highly favored by consumers. However, the genetic resources of crinkled leaf vegetables in the Brassica genus are limited. In this study, distant hybridization was performed between 'Mottle-leaf Tai-cai' (Brassica campestris L. ssp. chinensis (L.) Makino var. tai-tsai Hort. AA, 2n = 20) and 'Big Yellow Flower Chinese Kale' (B. oleracea var. alboglabra Bailey. CC, 2n = 18) by hand emasculation followed by tissue culture. As a result, all obtained three embryos were identified as true hybrids (AC, 2n = 19) through morphology, cytology, and molecular markers. After chromosome doubling, an allotetraploid hybrid (AACC, 2n = 38) was obtained with a giant leaf, serrated leaf margin, and wrinkled leaf surface; therefore, its most agronomical characters were biased toward the female parent of 'Mottle-leaf Tai-cai'. In comparison with diploid hybrid, the pollen stainability of allotetraploids were significantly improved and similar to their parents. Additionally, all 5 pairs of SSR primers showed the authenticity of the hybrids. Furthermore, the contents of soluble sugar, vitamin C, and cellulose in the hybrids increased when compared with their parents. Briefly, the obtained hybrid is interspecific euhybrid, and has wrinkle-leaf characteristics, improved nutritional quality, so it is a novel Brassica wrinkle-leaf vegetable germplasm. [ABSTRACT FROM AUTHOR]

The emergence of hybrid scaffolds, blending biomaterials with diverse properties, offers promise in musculoskeletal tissue engineering. However, a need for in vitro platforms investigating biological behavior and the interplay of different load‐bearing and colonizable synthetic bone substitute materials remains. Herein, we present a novel, in‐house producible, and scalable clamp culture system designed for facile in vitro analysis of interactions between biomaterials, hydrogels, and cells. The system, constructed here from an exemplary 3D‐printable polymer and photopolymerizable hydrogel using a widely available benchtop 3D printer, ensures mechanical stability and protection for the embedded hydrogel via its double‐clamp structure, facilitating various analytical methods while preserving culture integrity. Hybrid clamp cultures were additively manufactured from polylactic acid, filled with a bone precursor cell‐laden methacrylate gelatin hydrogel, cultured for 14 days, and analyzed for cell viability, mineralization, and osseous differentiation. Results indicate no adverse effects on osteogenic differentiation or mineralization compared to conventional droplet cultures, with enhanced cell viability and simplified handling and downstream analysis. This system demonstrates the potential for robust experimentation in tissue engineering and is adaptable to various plate formats, and thus highly suitable for the investigation of biomaterial‐cell interactions and the development of implants for musculoskeletal tissue defects. [ABSTRACT FROM AUTHOR]

Ferocactus peninsulae (F.A.C. Weber) Britton & Rose, commonly known as the barrel cactus, is a distinctive and visually striking succulent belonging to the Cactaceae family. It is renowned for its impressive size and robust, barrel-shaped structure. F. peninsulae faces potential threats from habitat destruction and illegal collection. This study investigates the response of transverse Thin Cell Layer (tTCL) explants of F. peninsulae to various concentrations (0.5 to 2.0 mg L−1) of two cytokinins, 6-benzylaminopurine (BAP) and 6-furfuryl amino purine (Kinetin/Kin) to induce multiple shoots in vitro. The tTCL explants were inoculated on Murashige and Skoog’s medium supplemented with 1.0 mg L−1 BAP to induce fresh shoots (98.6% response). The shoots were proliferated (19.3 shoots per tTCL explant with 3.5 cm length) on a combination of BAP (0.75 mg L−1) and indole-3-acetic acid (IAA; 0.15 mg L−1). The results demonstrated the differential impact of cytokinins and auxins combinations on shoot multiplication, with BAP and IAA proving optimal for robust shoot growth while the higher concentrations led to abnormal morphogenesis and callus formation. The freshly generated shoots were rooted on 0.75 mg L−1 indole-3 butyric acid (95.8% rooting response) and hardened in the greenhouse, and the plantlets were successfully shifted to the field with a 100% survival rate. The study could help in the large-scale production of the conservation-prioritized cactus species using the tTCL culture technique.Key message: The areoles on transverse thin cell layer explants of Ferocactus peninsulae were activated using a combination of cytokinin and auxin, leading to successful shoot regeneration, rooting, and hardening of the plantlets. [ABSTRACT FROM AUTHOR]

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Obtaining virus-free plants is a crucial step in disease management that enables reliable and profitable fruit farming. The present study applied various in vitro virus elimination protocols, including apical shoot culture, chemotherapy, thermotherapy, cryotherapy, and their combination, to eliminate black mulberry Idaeovirus (BMIV) from sour black mulberry. First, a shoot tip (0.5–2 mm) culture protocol was optimized, and four ribavirin concentrations (0, 10, 20, and 30 mg/L) were investigated over five weeks as a form of chemotherapy (ch). For the first thermotherapy treatment (Ch + Th1st), chemotherapy treatment was followed by a gradual increase in the temperature (24–33 °C). In another experiment (Th2nd + Ch), in vitro shoots were incubated in the dark for two weeks at two different temperatures (35 ± 1 °C and 37 ± 1 °C, for one week each). Subsequently, the shoot tips were incubated with various ribavirin doses. Finally, cryotherapy (Cr) was used with or without immersing the shoot tips in liquid nitrogen. A two-step RT-PCR was performed to assess the presence of the virus in 7–8-week-old in vitro plants. Th2nd + Ch significantly increased the shoot tip burst and plant survival/morphogenesis compared to the other treatments. Except for the application of cryotherapy, the protocols eliminated BMIV in different proportions, and the highest virus elimination rate (50%) was obtained by applying 30 mg/L ribavirin during the Ch + Th1st treatment. These findings are essential in preventing the dissemination of the virus and enabling the safe movement of germplasm around the world. [ABSTRACT FROM AUTHOR]

Individual cells grown in culture exhibit remarkable differences in their growth, with some cells capable of forming large clusters, while others are limited or fail to grow at all. While these differences have been observed across cell lines and human samples, the growth dynamics and associated cell states remain poorly understood. In this study, we performed clonal tracing through imaging and cellular barcoding of an in vitro model of esophageal epithelial cells (EPC2-hTERT). We found that about 10% of clones grow exponentially, while the remaining have cells that become non-proliferative leading to a halt in the growth rate. Using mathematical models, we demonstrate two distinct growth behaviors: exponential and logistic. Further, we discovered that the propensity to grow exponentially is largely heritable through four doublings and that the less proliferative clones can become highly proliferative through increasing plating density. Combining barcoding with single-cell RNA-sequencing (scRNA-seq), we identified the cellular states associated with the highly proliferative clones, which include genes in the WNT and PI3K pathways. Finally, we identified an enrichment of cells resembling the highly proliferative cell state in the proliferating healthy human esophageal epithelium. Author summary: Single cells derived from tissues and cell lines can have variable abilities to grow in culture. Understanding the molecular differences that lead to this variability has the potential to reveal regulators of growth and to improve in vitro culture for tissue regeneration. In this study, we investigated differences in growth between esophageal epithelial cells and found that these differences are non-genetic in origin. Through quantitative analysis of clonal growth at different time points, we show that the heterogeneity in growth can be modeled as two separate populations, one that is highly proliferative and grows exponentially, and another that is less proliferative and is able to transition into a non-proliferative state. Tracking clones through cell barcoding with scRNA-seq, we define the molecular differences between these populations and their associated signaling pathways. Together, this work highlights the contributions of non-genetic heterogeneity in proliferation, which has applications for tissue regeneration and stem cell biology. [ABSTRACT FROM AUTHOR]

Objective Materials and Methods Results Conclusion To evaluate the relationship between renal pelvis pressure and infection after ureteroscopy, using a live swine model.In anaesthetised pigs, a 1‐h ureteroscopy was performed using a pressure‐sensing guidewire, with renal pelvis pressure maintained at either 37 mmHg or 75 mmHg for the entire procedure and infusion with saline alone or with a standardised concentration of uropathogenic Escherichia coli strain CFT073 (1.5 × 107 colony‐forming units [CFU]/mL). Venous blood sampling was performed during and after the procedure. Vital signs, inflammatory biomarkers, and renal tissue and blood cultures were assessed.In 21 pig kidneys, study groups were: 37 mmHg with saline irrigation (n = 3); 75 mmHg with saline irrigation (n = 4); 37 mmHg with saline irrigation with 1.5 × 107 CFU/mL E. coli (n = 7); and 75 mmHg with saline irrigation with 1.5 × 107 CFU/mL E. coli (n = 7). Statistically significant changes in inflammatory biomarkers were most pronounced in the group with 75 mmHg saline irrigation + E. coli and were significantly elevated compared with the control group and the group receiving E. coli irrigation at 37 mmHg. Positive blood cultures were noted in 5/7 animals treated with E. coli at 75 mmHg; no others developed bacteraemia.In this swine model of ureteroscopy, irrigation with saline + E. coli at a renal pelvis pressure of 75 mmHg resulted in bacteraemia and inflammatory biomarker elevations significantly greater than both E. coli irrigation with renal pelvis pressure maintained at 37 mmHg and the control. [ABSTRACT FROM AUTHOR]

Background: Dental restorative materials are recognized as artificial niches that facilitate the adherence and accumulation of oral microorganisms. To mitigate oral diseases and extend the lifespan of restorations, it is advantageous to use dental materials that exhibit low susceptibility to bacterial adhesion. Objective: To evaluate and compare bacterial adhesion on two bioactive restorative materials, a glass hybrid restorative, and an alkasite with a nanohybrid resin composite as a positive control. The secondary objectives were to compare the surface roughness (SR) of the materials and determine the correlation between the bacterial adhesion and the SR. Materials and methods: The samples consisted of 33 polished discs of each material: Group A: Tetric® N-Ceram (nanohybrid resin composite), Group B: Equia Forte™ HT Fil (glass hybrid restorative) and Group C: Cention N® (alkasite). Streptococcus mutans cultures were inoculated and after 24-hours of incubation, bacterial adhesion was measured by measuring optical density (OD) and number of colony forming units (CFUs). After 96-hours incubation, the bacterial cell count was determined using scanning electron microscopy (SEM). SR was assessed using surface profilometer. Results: Alkasite had significantly lower OD and CFUs (p < 0.001 and p = 0.015 respectively). According to the SEM analysis, the glass hybrid restorative had lower mean bacterial cell count with no significant difference between the groups. The nanohybrid composite had the smoothest surface that was significantly lower than the alkasite and glass hybrid restorative (p = 0.002). None of the groups demonstrated a correlation between bacterial adhesion and SR. Conclusion: Alkasite impedes bacterial adhesion better than the glass hybrid restorative and nanohybrid composite, while smoother surfaces are achieved with the nanohybrid composite. [ABSTRACT FROM AUTHOR]

SUMMARY: De novo shoot apical meristem (SAM) organogenesis during regeneration in tissue culture has been investigated for several decades, but the precise mechanisms governing early‐stage cell fate specification remain elusive. In contrast to SAM establishment during embryogenesis, in vitro SAM formation occurs without positional cues and is characterized by autonomous initiation of cellular patterning. Here, we report on the initial stages of SAM organogenesis and on the molecular mechanisms that orchestrate gene patterning to establish SAM homeostasis. We found that SAM organogenesis in tobacco calli starts with protuberance formation followed by the formation of an intact L1 layer covering the nascent protuberance. We also exposed a complex interdependent relationship between L1 and WUS expression and revealed that any disruption in this interplay compromises shoot formation. Silencing WUS in nascent protuberances prevented L1 formation and caused the disorganization of the outer cell layers exhibiting both anticlinal and periclinal divisions, suggesting WUS plays a critical role in the proper establishment and organization of L1 during SAM organogenesis. We further discovered that silencing TONNEAU1 prevents the exclusive occurrence of anticlinal divisions in the outermost layer of the protuberances and suppresses the acquisition of L1 cellular identity and L1 formation, ultimately impeding SAM formation and regeneration. This study provides a novel molecular framework for the characterization of a WUS/L1 interplay that mediates SAM formation during regeneration. Significance Statement: This study highlights the importance of establishing an intact L1 cell layer to promote de novo shoot apical meristem (SAM) organogenesis in vitro. It identifies a complex interdependence between WUS and ML1 expression in directing anticlinal divisions for L1 formation, which in turn initiates gene expression patterning in the SAM and its formation. Disrupting this WUS/L1 interplay impairs SAM regeneration, underscoring its central role. [ABSTRACT FROM AUTHOR]

Transwell‐based airway models have become increasingly important in studying the effects of respiratory diseases and drug treatment at the air–liquid interface of the lung epithelial barrier. However, the underlying mechanisms at the tissue and cell level often remain unclear, as transwell inserts feature limited live‐cell imaging compatibility. Here, a novel microfluidic platform is reported for the cultivation of transwell‐based lung tissues providing the possibility to alternate between air–liquid and liquid–liquid interfaces. While the air–liquid interface recapitulates physiological conditions for the lung model, the liquid–liquid interface enables live imaging of the tissue at high spatiotemporal resolution. The plastics‐based microfluidic platform enables the insertion and recuperation of the transwell inserts, which allows for tissue cultivation and analysis under standardized well plate conditions. The device is used to monitor infections of Pseudomonas aeruginosa in human stem‐cell‐derived bronchial epithelial tissue. The progression of a P. aeruginosa infection in real‐time at high resolution is continuously imaged, which provides insights into bacterial spreading and invasion on the apical tissue surface, as well as insights into tissue breaching and destruction over time. The airway tissue culture system is a powerful tool to visualize and elucidate key processes of developing respiratory diseases and to facilitate drug testing and development. [ABSTRACT FROM AUTHOR]

The emergence of methicillin‐resistant Staphylococcus aureus (MRSA) several years ago highlighted the challenge of multidrug‐resistant infections, emphasizing the critical need for innovative treatment approaches. Myrtenol, known for its antibacterial and antibiofilm properties, holds promise as a potential treatment option. This study aimed to evaluate the effectiveness of myrtenol against MRSA. The collected MRSA isolates were assessed for antimicrobial susceptibility following the Clinical and Laboratory Standards Institute (CLSI) guidelines 2023. Biofilm formation by MRSA was evaluated using the tissue culture plate (TCP) technique. The minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and minimal biofilm inhibitory concentration (MBIC) of myrtenol against MRSA were determined both individually and in combination with antibiotics. Real‐time PCR was employed to investigate the impact of myrtenol on the expression of virulence genes (sarA, agrA, and icaD) across the isolates. In this study, MRSA was identified in 90 out of 400 cases (22.5%) of hospital‐acquired pathogens. Among the collected MRSA isolates, 53 out of 90 (59%) were found to produce biofilms. The MIC of myrtenol was comparable to the MBC across all tested isolates, they were almost the same. Combinations of myrtenol with most tested antibiotics exhibited synergistic effects exceeding 60%. Among the 53 biofilm‐producing isolates, 45 isolates (85%) expressed the sarA gene, 49% expressed the agrA gene, and all biofilm‐producing MRSA isolates (100%) expressed the icaD gene. A notable reduction in the relative quantity (RQ) values of virulence gene expression was observed after treatment with the MBIC of myrtenol across all tested isolates. Myrtenol demonstrated strong antimicrobial activity against MRSA, notably reducing the expression of key virulence genes linked to biofilm formation. This suggests its potential as a therapeutic agent for treating biofilm‐associated MRSA infections. [ABSTRACT FROM AUTHOR]

Background: Recent studies have highlighted the role of miR-5195-3p in suppressing cell growth in various cancers. However, the specific functional impact of miR-5195-3p in colorectal cancer (CRC) remain to be fully clarified. The importance of miR-5195-3p in CRC was evaluated, aiming to uncover its underlying molecular mechanism and identify it as a potential therapeutic target for CRC. Results: Our research has shown that miR-5195-3p is markedly under-expressed in CRC tissues and cell cultures, with its reduced presence associated with a higher TNM stage, lymphatic invasion, and unfavorable survival outcome. Ectopic miR-5195-3p expression curtailed proliferation, migration, and invasion of SW1116 and HT29 cells. Additionally, we discovered that miR-5195-3p directly targets and negatively influences Toll-like receptor 4 (TLR4) in CRC cells. Moreover, an inverse relationship was noted between miR-5195-3p and TLR4 expression in CRC tissue samples. Notably, restoring TLR4 expression counteracted miR-5195-3p's suppressive impact on cell growth, motility, invasiveness, epithelial-mesenchymal transition (EMT), and the TLR4/MyD88 signaling pathway in SW1116 and HT29 cells. Conclusions: MiR-5195-3p suppresses the CRC cellular functions through the downregulation of TLR4/MyD88 signaling, indicating that targeting miR-5195-3p might offer a viable therapeutic strategy for CRC. [ABSTRACT FROM AUTHOR]

Background: The detection of causative pathogens plays a crucial role in the diagnosis and targeted treatment of periprosthetic joint infections (PJI). While there have been improvements in analytic methods in the past, pre-analytical procedures have not yet been sufficiently investigated. The objective of this study was to compare the culture yield of four different pre-analytical procedures. Methods: Patients with perioperative diagnosis of PJI were included in a single center cross-sectional study (2021–2022). Tissue samples (n = 20) of each patient were randomly and equally distributed to each of the four study arms. Tissue samples were either send to the laboratory without culture medium (group A) or were transported in thioglycolate medium immediately after sampling at three different temperatures (room temperature, 4 °C, 37° for 24 h; group B-D). Culture media were investigated for growth on days 1, 3, 7, 12, 14. All organisms, the number of positive samples and the time to positivity were recorded and compared between the study arms. Single positive cultures were considered as contamination. Results: In total, 71 patients were included. The proportions of culture negative samples (10–15%) and polymicrobial infections (51–54%) were comparable between the four arms. Seven patients (10%) were culture-negative in group A, but showed growth in thioglycolate media (group B-D). Furthermore, 13% of patients showed growth in all groups, but additional organisms were cultured in thioglycolate. There was growth beyond day 7 of culturing only in thioglycolate, but not in group A. A storage temperature of 4 °C showed a longer time to positivity compared to the other groups (p < 0.001). Conclusions: Pre-analytical storage of tissue samples in thioglycolate broth did not improve the culture yield and did not detect additional cases of infection compared to the standard (pre-analytical storage in sterile containers). However, including a thioglycolate medium to the sampling algorithm reduced the rate of culture-negative infections and helped to identify additional organisms. [ABSTRACT FROM AUTHOR]

The factors that affected callus induction, multiplication and organ differentiation of garlic were explored and the high efficient regeneration system was established. The hormone concentration and pH value of the culture medium suitable for garlic callus induction and proliferation were selected: 2 mg / L NAA,2 mg / L 6-BA,1 mg / L 2,4-D and the pH of the medium was 5. 8. The hormone concentration and pH value of the culture medium suitable for garlic callus organ differentiation were selected. The optimal medium hormone concentration and pH value for callus differentiation of Cangshan garlic were 1 mg / L NAA,3 mg / L 6-BA and the pH of the medium was 5. 8. The optimal medium concentration and pH value for callus differentiation of Soviet garlic were 2 mg / L NAA,2 mg / L 6-BA and the pH of the medium was 6. 1. The optimal medium concentration and pH value for callus rooting and differentiation of two varieties are 1 mg / L NAA and pH 5. 8. Research has also shown that high concentrations of 2,4-D were beneficial for the induction of garlic callus, but not for its differentiation. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. Key points: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread [ABSTRACT FROM AUTHOR]

Background: Cervical squamous cell carcinoma (CSCC) is a prevalent gynecological malignancy worldwide. Current treatments for CSCC can impact fertility and cause long-term complications, underscoring the need for new therapeutic strategies. Oncolytic virotherapy has emerged as a promising option for cancer treatment. Previous research has demonstrated the oncolytic activity of the coxsackievirus B3 strain 2035 A (CVB3/2035A) against various tumor types. This study aims to evaluate the clinical viability of CVB3/2035A for CSCC treatment, focusing on its oncolytic effect in patient-derived CSCC organoids. Methods: The oncolytic effects of CVB3/2035A were investigated using human CSCC cell lines in vitro and mouse xenograft models in vivo. Preliminary tests for tumor-selectivity were conducted on patient-derived CSCC tissue samples and compared to normal cervical tissues ex vivo. Three patient-derived CSCC organoid lines were developed and treated with CVB3/2035A alone and in combination with paclitaxel. Both cytotoxicity and virus replication were evaluated in vitro. Results: CVB3/2035A exhibited significant cytotoxic effects in human CSCC cell lines and xenograft mouse models. The virus selectively induced oncolysis in patient-derived CSCC tissue samples while sparing normal cervical tissues ex vivo. In patient-derived CSCC organoids, which retained the immunohistological characteristics of the original tumors, CVB3/2035A also demonstrated significant cytotoxic effects and efficient replication, as evidenced by increased viral titers and presence of viral nucleic acids and proteins. Notably, the combination of CVB3/2035A and paclitaxel resulted in enhanced cytotoxicity and viral replication. Conclusions: CVB3/2035A showed oncolytic activity in CSCC cell lines, xenografts, and patient-derived tissue cultures and organoids. Furthermore, the virus exhibited synergistic anti-tumor effects with paclitaxel against CSCC. These results suggest CVB3/2035A could serve as an alternative or adjunct to current CSCC chemotherapy regimens. [ABSTRACT FROM AUTHOR]

The study investigates the impact of polyethylene glycol (PEG), sodium nitroprusside (SNP), and different carbon sources on the in vitro callus induction and regeneration of drought-tolerant and drought-sensitive rice (Oryza sativa L.) genotypes. PEG, a known osmotic agent, mimics drought conditions, thus providing insights into the plant's response mechanisms under stress. SNP, a nitric oxide donor, is evaluated for its role in modulating stress responses and promoting growth under adverse conditions. Various carbon sources are evaluated to determine their efficacy in supporting callus induction and subsequent regeneration. Results indicate differential responses between drought-tolerant and drought-sensitive rice genotypes, highlighting the potential for optimizing tissue culture protocols to enhance the resilience and regenerative capacity of rice under drought stress. While moderate PEG (5%) stimulated callus induction and regeneration, higher concentrations resulted in adverse effects, including reduced callus mass and regeneration potential. Morphological changes were also observed in response to PEG concentrations, highlighting genotype-specific responses. Meanwhile, combined effect of carbon sources such as maltose and sorbitol along with nitrogen sources "SNP" enhanced callus induction (> 90%) and regeneration (> 90%) under 20% PEG stress in both drought-tolerant and drought-sensitive rice. [ABSTRACT FROM AUTHOR]

Background: Application of plant bioregulators at specific concentration modify growth, flowering, flower yield and post harvest quality of flowers. Growth promoters like auxin, gibberellins and cytokinin modify physiological process by accelerating plant growth while growth retardant like cycocel and abscisic acid inhibit plant growth. But in open field cultivation of gerbera, little is known about use of growth regulators on flowering regulation. A good knowledge on use of growth regulators in gerbera is required before going to any recommendations. The current study aimed to standardize suitable bioregulators on flower production in gerbera in open field condition. Methods: The study was carried out in premises of Biotechnology cum Tissue Culture Centre, Odisha University of Agriculture and Technology, Bhubaneswar during 2015-16 and 2016-17. Apart from control eight treatments of growth regulators were used like GA3 @ 100 ppm and 150 ppm, cycocel @ 700 ppm and 800 ppm with or without amino acids as foliar spray. The observation were recorded from 5 randomly selected plant within each replication of treatment for different floral parameters. Result: The result revealed that days taken to flower bud initiation and days taken to flowering was minimum in GA3 @ 100 ppm + amino acid. The stalk length of flower was maximum in treatment GA3 @ 150 ppm + amino acid while largest flower diameter, maximum number of flowers and longest bloom life was observed in treatment with cycocel @ 700 ppm + amino acid. Maximum stalk thickness was found in treatment with cycocel @ 800 ppm + amino acid. The present work will be a complementary contribution to the researchers and gerbera growers in increasing productivity of this flower crops. [ABSTRACT FROM AUTHOR]

Key message: The Agrobacterium-mediated transformation system of three elite indica rice varieties was explored and two varieties yielded positive transgenic plants. Fourteen elite indica rice varieties with different genotypes were selected for callus induction, and varieties NY49, KW and CL38 were chosen for the establishment of the Agrobacterium transformation system because of their excellent tissue culture characteristics. The callus induction frequency was the highest when the concentration of 2,4-Diphenoxyacetic acid (2,4-D) was respectively at 2.5 mg/L, 3.0 mg/L and 2.8 mg/L for NY49, KW and CL38 after optimizing the tissue culture conditions. Moreover, medium types of CIM1, CIM8 and CIM7 were most suitable for the varieties NY49, KW and CL38, respectively, with high callus induction frequency. The regeneration frequency of CL38 and NY49 was as high as 73.33% and 50.00%, respectively, in MS medium containing 2 mg/L 6-benzyladenine (BAP), 2 mg/L kinetin (KT) and 0.5 mg/L naphthylacetic acid (NAA). Finally, transgenic seedlings of CL38, NY49 and NIP were obtained with infection of Agrobacterium EHA105 carrying the pYLCRISPR/Cas9PUB-H vector. The successful editing frequency detecting by sequencing in CL38 and NIP were 62.50% and 77.78%, respectively. The establishment of the transformation system provides a useful tool for gene function study and gene editing breeding in indica rice cultivars. [ABSTRACT FROM AUTHOR]

Objectives: Endothelial cells (ECs), as the main cell types in the micro- and macro-vascular structure, are crucial to many vital physiological processes occurring in human body among which are both physiologic and pathologic angiogenesis. Due to their great heterogeneity, it is recommended that the ECs of each tissue should be studied separately. Recently, targeting angiogenesis in cancer has become one of the main strategies for cancer management. Accordingly, this study aimed to introduce a simple, costeffective, and practical model for studying tumor angiogenesis, which can contribute to studies focused on antiangiogenic-based treatments for cancer. Materials and Methods: In this original study, the explants of both normal and tumor tissues of mouse lung and 4T1 mouse cancer model were cultured separately in a collagen type 1 bed. Sprouting ECs formed a network of vessel-liked structures that were photographed as a model for investigating cancerous and normal angiogenesis. These ECs were then isolated from the collagen matrix using collagenase to confirm their endothelial nature. Results: The results showed that the cells invading the collagen matrix and forming a vessel-liked network were of endothelial nature. They were also found to have EC-specific characteristics and express EC-specific markers. These embedded cells may have been treated with the desired drug or further isolated from the collagen matrix and investigated in a genome or protein level. Conclusions: In sum, this modeling had the potential to become a suitable model for studying the pathological angiogenesis such as tumors in human and animal tissues. [ABSTRACT FROM AUTHOR]

Tropicoporus tropicalis (formerly Phellinus tropicalis) is a saprophytic basidiomycete that has been implicated in refractory mycoses in humans, particularly in patients with chronic granulomatous disease. Despite its clinical significance, T. tropicalis is an under‐recognised cause of eumycetoma, with no prior reports available. We present a case of white grain eumycetoma with associated osteomyelitis of the left foot, caused by T. tropicalis, confirmed through 18S‐ITS1‐5.8S‐ITS2‐28S rRNA gene amplification and sequencing. The patient was treated with itraconazole 200 mg daily, leading to gradual improvement. A review of the literature on T. tropicalis infections in humans reveals its characteristic manifestations, which include osteomyelitis, soft tissue abscesses, pulmonary nodules and keratitis. These infections are locally destructive but have the potential to disseminate. Diagnosis is often delayed and relies on molecular techniques. Amphotericin B combined with an azole appears to be the most effective treatment, often necessitating concurrent surgical drainage. In conclusion, T. tropicalis is a newly recognised pathogen associated with eumycetoma and poses an increased risk of osteomyelitis. Molecular identification, such as sequencing the internal transcribed spacer (ITS) region from cultures or tissue specimens, is crucial for accurate identification of this pathogen. [ABSTRACT FROM AUTHOR]

Curcuma longa L. is a medicinal plant that contains bioactive constituents with various pharmacological properties. However, turmeric is vulnerable to the bacterial wilt disease caused by Ralstonia solanacearum, substantially lowering yields and resulting in death. Thus, this study aims to induce mutations of turmeric cv. 'Trang 2' by applying electron beams (8 MeV) and selecting the resulting populations resistant to bacterial wilt disease. Sixty-day-old plantlets cultured in vitro were exposed to 0, 50, 100 and 150 Gy electron beams with a 240 Gy/min dose rate. The experiment was performed at the Thailand Institute of Nuclear Technology. To explore the potential of electron beam sensitivity in the survival and growth rate, unirradiated plantlets were used to normalize the radiation treatments. Plantlet survival was used to calculate the lethal dose (LD), and the number of new shoots was used to estimate the growth reduction (GR) dose through regression analysis. The survival and growth rates of the plantlets decreased as the radiation dose increased. At doses of 100 and 150 Gy, the turmeric plantlets could not produce new shoots. The median lethal dose (LD50) was 58.6 Gy. A 50 % growth reduction dose (GR50) was observed at 45.3 Gy. Symptom severity ranged from 11.7 to 91.7 %, demonstrating significantly lower levels in the EBRTP-2 and EBRTP-4 M1V5 electron-beam-irradiated populations. Furthermore, the disease incidence was 33.3 % in the EBRTP-6 population. Finally, turmeric populations resistant to bacterial wilt (EBRTP-2, EBRTP-4 and EBRTP-6) were isolated after electron-beam-induced mutation at 50 Gy. [ABSTRACT FROM AUTHOR]

In the present study, a customized device (Epi-ExPer) was designed and fabricated to facilitate an epithelial organ culture, allowing for controlled exposure to exogenous chemical stimuli and accommodating the evaluation of permeation of the tissue after treatment. The Epi-ExPer system was fabricated using a stereolithography (SLA)-based additive manufacturing (AM) method. Human and porcine oral epithelial mucosa tissues were inserted into the device and exposed to resinous monomers commonly released by dental restorative materials. The effect of these xenobiotics on the morphology, viability, permeability, and expression of relevant markers of the oral epithelium was evaluated. Tissue culture could be performed with the desired orientation of air-liquid interface (ALI) conditions, and exposure to xenobiotics was undertaken in a spatially guarded and reproducible manner. Among the selected monomers, HEMA and TEGDMA reduced tissue viability at high concentrations, while tissue permeability was increased by the latter. Xenobiotics affected the histological image by introducing the vacuolar degeneration of epithelial cells and increasing the expression of panCytokeratin (pCK). Epi-ExPer device offers a simple, precise, and reproducible study system to evaluate interactions of oral mucosa with external stimuli, providing a biocompatibility and permeability assessment tool aiming to an enhanced in vitro/ex vivo-to-in vivo extrapolation (IVIVE) that complies with European Union (EU) and Food and Durg Administration (FDI) policies. [ABSTRACT FROM AUTHOR]

Various rapid propagation strategies have been discovered, which has facilitated large-scale plant reproduction and cultivar development. These methods, in many plant species, are used to rapidly generate large quantities (900 mini-tubers/m2) of high-quality propagule (free from contamination) at a relatively low cost in a small space. They are also used for plant preservation. This review article aims to provide potential applications for regeneration and clonal propagation. Plant propagation using advanced agrotechnology, such as aeroponics, is becoming increasingly popular among academics and industrialists. The advancement of asexual aeroponic propagation has been achieved through advancements in monitoring and control systems using IoT and smart sensor technology. New sensor technology systems have gained substantial interest in agriculture in recent years. It is used in agriculture to precisely arrange various operations and objectives while harnessing limited resources with minimal human intervention. Modern intelligent technologies and control systems simplify sensor data collection, making it more efficient than manual data collection, which can be slow and prone to errors. Specific ambient variables like temperature, humidity, light intensity, stock solution concentrations (nutrient water), EC (electrical conductivity), pH values, CO2 content, and atomization parameters (frequency and interval) are collected more effectively through these systems. The use of intelligent technologies provides complete control over the system. When combined with IoT, it aids in boosting crop quality and yield while also lowering production costs and providing data directly to tablets and smartphones in aeroponic propagation systems. It can potentially increase the system's productivity and usefulness compared to the older manual monitoring and operating methods. [ABSTRACT FROM AUTHOR]

In Australia, native possums are a major wildlife reservoir for Mycobacterium ulcerans, the causative agent of the neglected tropical skin disease Buruli ulcer (BU). Large-scale possum excreta surveys that use PCR to detect M. ulcerans in 100-1,000 s of excreta specimens are an important tool that can inform geospatial modeling and predict locations of future human BU risk. However, the significant expense of commercial kits used to extract DNA from specimens is a major barrier to routine implementation. Here, we developed a low-cost method for DNA extraction from possum excreta, possum tissue, and pure mycobacterial cultures, using a guanidinium isothiocyanate lysis solution and paramagnetic beads. In a 96-well plate format for high-throughput processing, the paramagnetic bead DNA extraction method was threefold less sensitive but only 1/6 the cost of a commonly used commercial kit. Applied to tissue swabs, the method was fourfold more sensitive and 1/5 the cost of a commercial kit. When used for preparing DNA from pure mycobacterial cultures, the method yielded purified genomic DNA with quality metrics comparable to more lengthy techniques. Our paramagnetic bead method is an economical means to undertake large-scale M. ulcerans environmental surveillance that will directly inform efforts to halt the spread of BU in Victoria, Australia, with potential for applicability in other endemic countries. IMPORTANCE Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria. [ABSTRACT FROM AUTHOR]

Regulation (EU) 2017/745 on medical devices (MDR) has significantly modified the rules to be adopted for MD qualifications and classification. New requirements require robust evidence on mechanisms of action (MoAs) that cannot be produced by existing common EU or ISO standards. Therefore, on a "caseby- case basis," a new evidence-based non-clinical approach to MD qualification must be defined. In this study, an in vitro experimental approach is described to assess the physicochemical and mechanical MoA of two hyaluronic acid (HA)- based medical devices: Mucogyne® Gel and Mucogyne® Ovule for vaginal use. They both act as moisturizers and lubricants as well as a healing adjuvant by promoting the continued moisture of the vulvovaginal area. The MoA of these two products has been demonstrated by using a 3D reconstructed human vaginal epithelium (HVE) model in a homeostatic physiological state and in stressed conditions. Film forming and persistency properties were assessed on intact HVE tissues by caffeine permeation assay and Lucifer Yellow (LY) localization on HVE vertical sections. Healing properties were assessed on injured HVE tissues by trans-electrical epithelial resistance (TEER) measurements associated with histomorphological analysis (H&E), and moisturizing efficacy was evaluated on HVE tissues cultured in dry conditions by histomorphological analysis (H&E) and aquaporin 3 (AQP3) expression and localization by immunohistochemistry (IHC). Using the same "dry" HVE model, the non-pharmacological action of the two products was addressed by CD44 (hyaluronic acid receptor) expression and localization. The results suggest that in vitro evaluations can provide robust results on a human-relevant experimental model for the intended use of the products and supports clinical data with mechanistic information which may not be achieved with in vivo studies but are particularly important for product qualification. The results also underline the specific relative efficacy of the mechanisms investigated for Mucogyne® Gel and Mucogyne® Ovule in line with their different formulation types (respectively, hydrophilic and lipophilic) that influence the action of the active ingredient HA. The present in vitro non-clinical evaluation of HVE combined with clinical investigation data obtained in women explain why Mucogyne MDs provide significant benefits in various physiological or pathological situations, including vaginal dryness and healing. [ABSTRACT FROM AUTHOR]

Purpose: Infectious spondylitis is caused by hematogenous seeding or adjacent soft tissue infection. No study has provided evidence that incubating biopsy specimens in blood culture bottles could enhance detection rates, nor has any study compared this method with conventional culture techniques. We aimed to assess the diagnostic yield of open microsurgical biopsies for infectious spondylitis and the efficacy of various culture media in the presence and absence of pre-biopsy antibiotic therapy. Methods: This retrospective study, which was conducted at a university-affiliated teaching hospital in Korea, enrolled 165 adult patients with suspected infectious spondylitis between February 2014 and September 2020. The diagnostic yield of open biopsy was compared among three culture media, namely, blood culture bottles, swab culture using transport media, and tissue culture using plain tubes, while considering preoperative antibiotic exposure. Results: Causative bacteria were identified in 84.2% of all cases. Blood culture bottles had the highest positivity rate (83.5%), followed by swab cultures (64.4%) and tissue cultures (44.9%). The differences in positivity rates were significant (P < 0.001). Preoperative antibiotic therapy reduced detection rates across all media, particularly in tissue cultures. Conclusions: We established the high diagnostic yield of open microsurgical biopsy using blood culture bottles, suggesting that pre-biopsy antibiotic therapy significantly affects bacterial detection, thereby underscoring the importance of culture medium selection in the diagnosis of infectious spondylitis. [ABSTRACT FROM AUTHOR]

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of the dietary addition of lavender essential oil (LEO) on mitigating the adverse effects of high stocking densities (HSD) in the Nile tilapia, Oreochromis niloticus. We used 216 fingerlings and randomly distributed them according to a 2 × 3 factorial design, comprising two stocking densities: a low stocking density (LSD, 2.92 kg/m3) and a high stocking density (5.85 kg/m3) and three levels of LEO, 0, 0.5%, and 1%, for 70 days. We evaluated their growth, digestive enzyme activity, blood biochemical parameters, immune/antioxidant indices, and intestinal morphology. All fish were intraperitoneally inoculated with Aeromonas veronii for a bacterial challenge test at the end of the feeding trial and monitored for 14 days. After 14 days, head kidney samples were collected from all fish to assess the level of expression of immune-related and apoptosis-related genes. Additionally, tissue samples were collected from the liver, kidney, and intestine to investigate the immunohistochemistry and histopathological alterations. The results showed that the final body weight and weight gain were lower and the feed conversion ratio was higher in fish cultured under HSD compared to those cultured under LSD. The fish cultured under LSD and HSD had higher final body weight when administered dietary LEO compared to the fish that were fed the non-supplemented diet. Adding 1% LEO increased the intestinal villus height and muscular coat thickness (MT). The MT was lower in fish cultured under HSD compared to those cultured under LSD. The amylase and lipase activities were lower in the fish cultured under HSD than in those cultured under LSD. However, LEO addition increased the lipase activity in fish cultured under LSD and HSD. Fish cultured under HSD showed lower immune/antioxidant indices (lysozymes, nitric oxide, complement 3, catalase, superoxide dismutase, and glutathione-S transferase) than those cultured under LSD. However, LEO addition significantly increased the immune response and antioxidant activity in fish cultured under LSD and HSD (P < 0.01). The level of expression of the IL-1β, IL-6, IL-8, NF-kB, and TLR-4 B genes was upregulated in the LSD + LEO1, LSD + LEO0.5, and HSD + LEO1 groups. The expression of TGF-β and IL-10 was upregulated in fish that received 1% and 0.5% LEO, irrespective of whether they were cultured under LSD or HSD. The level of expression of apoptosis-related genes (caspase 3 and BAX) was upregulated in the HSD + LEO0 group and modified by the addition of LEO in their diet. The immunoexpression of NF-kB and IL-1β after bacterial challenge increased in the tissues of fish cultured under LSD and HSD after LEO was administered. These results indicated that the dietary addition of 1% LEO mitigated the harmful effects of HSD on the growth, immunity, antioxidant activity, and disease resistance of the Nile tilapia. [ABSTRACT FROM AUTHOR]

Handroanthus chrysanthus Jacq. S. O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC are two forest species that grow in the coastal region of Ecuador and are threatened with extinction. A protocol for the mass multiplication of these species was developed using in vitro culture techniques. The cultures were initiated from seeds, and the effect of two culture media: Woody Plant Medium (WPM) and Murashige-Skoog (MS), two concentrations of NaClO (0.5% and 1%), and two disinfection times (3 and 5 min) was studied. During multiplication, the effect of three concentrations of two cytokinins: 6-benzylaminopurine (6-BAP) 4.4, 5.5, or 6.6 μM; kinetin 4.6, 5.75, or 6.9 μM) on the number of shoots, their length, and diameter was analyzed. This phase of the experiment was carried out in two successive multiplications. For rooting, two concentrations of indole-3-butyric acid (IBA) (2.45 and 4.9 μM) were tested, and the number of roots formed and their length were determined. It was demonstrated that the WPM medium is the most suitable for the in vitro culture of both species and that disinfection time and NaClO concentration affect each species differently. For the multiplication of H. chrysanthus, the most suitable cytokinin was 6-BAP 6.6 μM; T. rosea performed better in the absence of cytokinins. IBA 2.45 μM produced the best results for the rooting of H. chrysanthus, while for T. rosea, IBA 4.9 μM was the most suitable. The acclimatized plants showed a high survival rate, demonstrating the feasibility of using this methodology for the accelerated propagation of these endangered species. Key message: In this research, the culture medium requirements and conditions for the micropropagation of H. chrysanthus and T. rosea were fine-tuned. This technique can be implemented to obtain plants for use in reforestation. [ABSTRACT FROM AUTHOR]

Background: Photorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide; however, actual clinical infections have so far only been identified in North America, Australia, and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells. Methods: In this study, we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. Pathogenicity was analysed using infection assays with both cultured cell lines (THP-1, CHO, and HEK cells) and primary immune cells, and peripheral blood mononuclear cells (PBMCs) isolated from human blood. Results: Here, we present the findings from the Australian (Kingscliff) and North American (ATCC43949) clinical isolates, and non-clinical soilborne nematode isolates from Thailand (PB68) and Northern Europe (HIT and JUN) of P. asymbiotica. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells. Conclusion: Here for the first time, we show how P. asymbiotica selectively infects certain immune cells while avoiding others and that infectivity varies depending on growth temperature. We also show that the tropism varies depending on the geographic location a strain is isolated from, with only the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture. [ABSTRACT FROM AUTHOR]

The orchid Dendrobium moniliforme faces endangerment due to habitat loss and illegal harvesting, necessitating the development of an optimized artificial propagation system to aid conservation and reintroduction efforts. This study evaluated the effects of three plant growth media, namely Murashige and Skoog (MS), Hyponex, and Orchid Maintenance Medium (OMM) (P668), and various organic additives (apple homogenate, banana homogenate, and coconut water) on the in vitro seedling growth of D. moniliforme. The results reveal that, in early postgermination stages, seedlings achieve maximum growth in the Hyponex medium, with a fresh weight (92 mg) and root length (2.7 cm) approximately 20-fold greater than those in the MS medium and OMM. After 6 months, for seedlings grown in MS medium and OMM with banana (50 g·L−1), the mean fresh weights were 29 and 107 mg, respectively; however, the highest biomass was observed in seedlings grown in the Hyponex medium with coconut water (50 mL·L−1), exhibiting a mean fresh weight of 201 mg. This study highlights Hyponex medium with coconut water as the most effective combination for promoting D. moniliforme growth and identifies suitable organic supplements for the in vitro cultivation of seedlings from asymbiotic seed culture. This propagation system offers valuable technical support for the mass production and conservation of this epiphytic orchid. [ABSTRACT FROM AUTHOR]

Vitis vinifera is an important fruit crop which is mainly consumed fresh or used for the production of wine. Genetic improvement programs through New Genomic Techniques (NGTs) aim to develop grapevine varieties resistant to biotic and abiotic stresses or enhancing nutraceutical properties. In order to apply NGTs, maintaining embryogenic calluses from flower tissues is critical. Optimizing culture conditions—pH, gelling agents, temperature, light, growth regulators, and gas composition—is essential for inducing efficient embryogenic responses tailored to each genotype/explant. Ethylene, a pivotal gaseous plant hormone, significantly influences tissue culture by affecting organogenesis and embryogenesis processes in several plants. Modulating ethylene levels shows promise for improving tissue culture vitality. This study evaluates in Vitis vinifera the effects of silver thiosulfate (STS) and salicylic acid (SA) on embryogenic callus growth, specifically investigating their roles in maintaining and inducing embryogenic competence. STS, particularly at 40 µM and 60 µM concentrations, effectively preserved embryogenic competence in Italia and Red Globe calluses, while high SA concentrations showed varied and occasionally adverse effects. At the same time, STS markedly suppressed the non-embryogenic callus growth in recalcitrant variety Italia, potentially increasing the ratio between embryogenic to non-embryogenic calluses development. [ABSTRACT FROM AUTHOR]