Tissue Culture Research Articles

Increased cell killing effect in neutron capture enhanced proton beam therapy

AbstractThermal neutrons generated in the body during proton beam therapy (PBT) can be used to cause boron neutron capture reactions and have recently been proposed as neutron capture enhanced PBT (NCEPBT). However, the cell killing effect of NCEPBT remains underexplored. Here, we show an increase in the cell killing effect of NCEPBT. Using Monte Carlo simulations, we showed that neutrons generated by proton beam irradiation are uniformly spread on tissue culture plates. Human salivary gland tumor cell line (HSG), human osteosarcoma cell line (MG63), human tongue squamous cell carcinoma cell line (SAS), and human malignant melanoma cell line (G-361) were irradiated with X-rays, proton beams, and proton beams with 10B-enriched boronophenylalanine (boron concentration of 20 and 80 ppm). The relative biological effectiveness (RBE) values of proton beams alone, proton beams with 20 ppm boron, and proton beams with 80 ppm boron for HSG, MG63, SAS, and G-361 were 1.02, 1.07, and 1.23; 1.01, 1.08, and 1.44; 1.05, 1.09, and 1.46; and 1.04, 1.13, and 1.63, respectively. NCEPBT with high boron concentration showed high RBE and a high sensitizing effect. Our results confirm an increase in the cell killing effect of NCEPBT, should aid in its clinical use, and warrant its further investigation.

Abstract PR017: Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics

Abstract Introduction: Tumors systemically remodel the immune system during metastasis. Developing anti-metastatic immunotherapies that target these tumor-immune interactions to make the metastatic niche hostile colonization would represent a paradigm shift in cancer treatment. However, traditional high-throughput screening (HTS) platforms that use simple read-outs and contrived in vitro systems are ill-suited to identify such therapies. In contrast, HTS platforms that accurately model the metastatic niche and use high-dimensional read-outs such as multiplexed single-cell RNA-sequencing (scRNA-seq) can accurately profile nuanced perturbation responses in individual immune cell types and therefore have the potential to discover anti-metastatic immunotherapies. In this study, we describe the development of an ex vivo lung tissue culture HTS platform that preserves immune gene expression signatures observed in the in vivo metastatic niche and is amenable to single-cell chemical transcriptomic analysis using MULTI- seq (McGinnis et al., Nature Methods, 2019). Inspired by our recent description of myeloid cell TLR-NFκB inflammation during breast cancer lung metastasis (McGinnis et al., Cancer Cell, 2024), we then performed the first anti-metastatic immunotherapy drug screen and identified TLR-NFκB inhibitors that effectively operate on all desired myeloid cell types in the lung metastatic niche. Methods and Results: Building upon existing techniques for culturing precision-cut lung slices (Wu et al., Journal of Experimental Medicine, 2024) and patient- derived tumor fragments (Voabil et al., Nature Medicine, 2021), we established and optimized an ex vivo lung tissue culture system that enables 48-hour culturing of lung slices isolated from 4T1 tumor-bearing mice. scRNA-seq profiling revealed successful capture of all relevant immune cell types and retention of metastasis-associated gene expression programs (e.g., myeloid TLR-NFκB inflammation and neutrophil degranulation) following culture. We then scaled our platform to address how myeloid cells in the lung metastatic niche respond to perturbations targeting every component of the canonical TLR-NFκB signaling cascade. Cell-type-specific perturbation modeling and quantification of TLR-NFκB signaling inhibition identified compounds that optimally perturb all intended myeloid cell types (e.g., tissue-resident macrophages, neutrophils, and monocytes) and represent prioritized candidates for future in vivo validation studies to assess impact on metastatic disease progression. Conclusions: In this study, we describe the development and application of a novel HTS platform that couples ex vivo lung tissue cultures with single-cell chemical transcriptomics to identify anti-metastatic immunotherapy candidates. We demonstrate how our platform can identify TLR-NFκB signaling inhibitors that optimally operate in the lung metastatic niche, paving the way for future interrogation of additional metastasis-associated immune cell signaling pathways in different disease and tissue backgrounds. Citation Format: Chris McGinnis, Winnie Yao, Ansuman Satpathy. Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR017.

Establishment of callus induction and plantlet regeneration systems of Peucedanum Praeruptorum dunn based on the tissue culture method.

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. 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 0.5mg/L 6-benzyladenine (BA) and 2.0mg/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. 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.

A rare case of granulomatous mastitis by Brucella species.

Granulomatous mastitis (GM) is a rare, chronic, benign inflammatory disease of the breast. Here, we present a rare case of GM caused by brucellosis and present the first review to compile the cases in the literature. The diagnosis was confirmed by the patient's serological and histopathological results. The patient was successfully treated with doxycycline+rifampicin combination therapy for six weeks. In conclusion, infectious agents, especially brucellosis, should be considered in the differential diagnosis of GM in endemic regions. Diagnostic methods, such as tissue culture and serological tests, should be used to detect possible infectious agents if necessary.

Enabling biocontained plant virus transmission studies through establishment of an axenic whitefly (Bemisia tabaci) colony on plant tissue culture.

Whiteflies (Bemisia tabaci) and the diseases they transmit are a major detriment to crop yields and a significant contributor to world hunger. The highly evolved interactions of host plant, phloem-feeding insect vector with endosymbionts and persistently transmitted virus represent a tremendous challenge for interdisciplinary study. Presented here is the establishment of a colony of axenic whiteflies on tissue-cultured plants. Efficient colony establishment was achieved by a surface sterilization of eggs laid on axenic phototrophically tissue-cultured plants. The transfer of emerging whiteflies through coupled tissue culture vessels to new axenic plants facilitates robust subculturing and produces hundreds of whitefly adults per month. Whitefly proliferation on more than two dozen plant species is shown as well as in vitro testing of whitefly preference for different plants. This novel multi-organism system provides the high-level of biocontainment required by Federal permitting to conduct virus transmission experiments. Axenic whitefly adults were able to acquire and transmit a begomovirus into tissue-cultured plants, indicating that culturable gut microorganisms are not required for virus transmission. The approach described enables a wide range of hypotheses regarding whitefly phytopathology without the expense, facilities, and contamination ambiguity associated with current approaches.

Leveraging Walnut Somatic Embryos as a Biomanufacturing Platform for Recombinant Proteins and Metabolites

Biomanufacturing enables novel sources of compounds with constant demand, such as food coloring and preservatives, as well as new compounds with peak demand, such as diagnostics and vaccines. The COVID-19 pandemic has highlighted the need for alternative sources of research materials, thrusting research on diversification of biomanufacturing platforms. Here, we show initial results exploring the walnut somatic embryogenic system expressing the recombinant receptor binding domain (RBD) and ectodomain of the spike protein (Spike) from the SARS-CoV-2 virus. Stably transformed walnut embryo lines were selected and propagated in vitro. Both recombinant proteins were detected at 3–14 µg/g dry weight of tissue culture material. Although higher yields of recombinant protein have been obtained using more conventional biomanufacturing platforms, we also report on the production of the red pigment betanin in somatic embryos, reaching yields of 650 mg/g, even higher than red beet Beta vulgaris. This first iteration shows the potential of biomanufacturing using somatic walnut embryos that can now be further optimized for different applications sourcing specialized proteins and metabolites.

Pomegranate plant tissue culture technique - a review

‌Background: The pomegranate, or Punica granatum L., is a member of the "Punicaceae" family, which also includes Punica protopunica and Punica granatum. Originating in Afghanistan and Iran, it has spread over Asia, Africa, and Europe's Mediterranean region. The traditional method of pomegranate propagation is laborious and time-consuming. It does not guarantee healthy, disease-free plants. Its shortcomings include poor success rates, extremely sluggish replication, and a one-year establishing period for new plants. Tissue culture is a technique that can be used to grow plants in all seasons without restrictions. Several pomegranate-growing nations have tried in vitro propagation to generate virus-free, similar-type plants, and it has been widely used for propagating many fruit trees. Research Purpose: The goal of this essay was to examine how to do plant tissue culture method and get familiar with the techniques. with an emphasis on the pomegranate micropropagation. The only aspect of plant tissue culture that may be able to get around these issues is in vitro technology. Therefore, there is plenty of room for employing micropropagation to multiply desired genotypes on a wide scale. Research Method: This review article was conducted based on scientific websites in Persian and English languages, including SID, Magiran, Google Scholar, Web of Science, and Library Studies, Results: Pomegranate plants multiply quickly through micropropagation, producing plants (clones) that are genetically similar. In a short amount of time, a single explant can yield thousands of plants. These plants match exactly. The study provides a standardized protocol for in vitro regeneration of nodal explants, which could be useful in large-scale production of uniform and disease-free plantlets. The process of plant tissue culture reduces the need for importing expensive plant seeds.

An Arteriovenous Bioreactor Perfusion System for Physiological In Vitro Culture of Complex Vascularized Tissue Constructs

Background: The generation and perfusion of complex vascularized tissues in vitro requires sophisticated perfusion techniques. For multiscale arteriovenous networks, not only the arterial, but also the venous, biomechanical and biochemical conditions that physiologically exist in the human body must be accurately emulated. For this, we here present a modular arteriovenous perfusion system for the in vitro culture of a multi-scale bioartificial vascular network. Methods: The custom-built perfusion system consisted of two circuits: in the arterial circuit, physiological arterial biomechanical and biochemical conditions were simulated using a modular set-up with a pulsatile peristaltic pump, compliance chambers, and resistors. In the venous circuit, venous conditions were emulated accordingly. In the center of the system, a bioartificial multi-scale vascularized fibrin-based tissue was perfused by both circuits simultaneously under biomimetic arteriovenous conditions. Culture conditions were monitored continuously using a multi-sensor monitoring system. Results: The physiological arterial and venous pressure- and flow-curves, as well as the microvascular arteriovenous oxygen partial pressure gradient, were accurately emulated in the perfusion system. The multi-sensor monitoring system facilitated live monitoring of the respective parameters and data-logging. In a proof-of-concept experiment, vascularized three-dimensional fibrin tissues showed sustained cell viability and homogenous microvessel formation after culture in the perfusion system. Conclusions: The arteriovenous perfusion system facilitated the in vitro culture of a multiscale vascularized tissue under physiological pressure-, flow-, and oxygen-gradient conditions. With that, it presents a promising technique for the in vitro generation and culture of complex large-scale vascularized tissues.

Survey on Influence of Cultural Practices on Banana Pseudostem Weevil, Odoiporus longicollis Olivier (Coleoptera: Curculionidae) Infestation in Major Banana Growing Districts of Northern Karnataka, India

The production and productivity of banana is governed by many biotic and abiotic factors. Among biotic factors, incidence of insect-pests is considered as one of the major impedimentin successful cultivation of banana. With respect to the Indian condition around eight pests commonly infest the banana crop. However, the banana pseudostem weevil Odoiporus longicollis is cosmopolite insect pest that threatens the banana cultivation. The most common management practices followed for managing this pest includes cultural practices. Diverse cultural practices have been communicated to and/or applied by resource-poor households over the past two decades to improve the overall farm health and crop tolerance to biotic/abiotic factors. Therefore, cultural practices are the only measures available to small-scale farmers for the control of banana pseudostem weevil. Hence the survey was conducted during 2020-21 to study the influence of cultural practices on O. longicollis infestation in major banana growing districts of north Karnataka. From the survey results on the cultural practices reveals that the pest severity was more severe on ratoon crop fields and the frequency recorded was 37.82%. However, the frequency recorded was 1.68% in first crop, wherein no pest infestation was recorded in main crop. Whereas, good number of farmers could not make a difference in infestation of pest between main and ratoon crop (60.50%) and in such fields infestation level was 4.11%. The banana farmers who were interacted mainly used suckers (36.13%) and tissue culture plants (36.13%) for planting followed by rhizome (24.37%), whereas few farmers used suckers and rhizomes (3.36%) for planting in a single piece of land. Accordingly, the per cent infestation of BPW was less when both tissue cultured plants (4.84%) and suckers (5.53%) used for planting. Whereas, infestation was more severe when both suckers and rhizomes were used (27.50%) on same piece of land and which was followed by rhizomes alone (16.00%).

JAAD Game Changers: "Diagnosis of deep cutaneous fungal infections: Correlation between skin tissue culture and histopathology".

JAAD Game Changers: "Diagnosis of deep cutaneous fungal infections: Correlation between skin tissue culture and histopathology".

Multi-omics analysis reveals the positive impact of differential chloroplast activity during in vitro regeneration of barley.

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. In this study, two contrasting cultivars differencing in their in vitro regeneration efficiency were identified. Tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency through multiomics analysis. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MS/MS revealed 3062 protein groups and 16,989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. TEM analysis also revealed differential plastid development. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. A comprehensive examination of gene expression, protein profiling and metabolite patterns unveiled a significant increase in the genes encompassing various functions, such as ion homeostasis, chlorophyll metabolic process, ROS regulation, and the secondary metabolic pathway.

OBTAINING ASEPTIC CULTURE IN VITRO OF RHODIOLA SEMENOWII L. FROM VARIOUS TYPES OF EXPLANTS

In vitro cultivation of callus cells allows for the stable production of secondary metabolites, being an alternative to the use of medicinal plant biomass from natural populations. Standardization of cultivation protocols and technological processes for the synthesis of secondary metabolites in vitro conditions determines significant interest in the use of tissue culture methods (PTC – Plant Tissue Culture). The aim of the study was to obtain an aseptic culture of Rhodiola semenowii in vitro from various types of explants, as well as the effect of the hormonal composition of the nutrient medium on the induction of callusogenesis in the culture of isolated organs of R. semenowii. As a result of the research, the optimal mode of obtaining R. semenovii aseptic cultures from various explants was determined. It has been shown that the use of 70% EtOH in combination with 0.01% HgCl2 or 20% NaClO makes it possible to obtain aseptic cultures from any type of R. semenovii explants. It has been established that the type of explant is R. semenovii influenced both the frequency of callus formation and the beginning of its formation. The least optimal types of R. semenovii explants are stem and rhizome segments, the frequency of callusogenesis varied from 5.8% to 75.6%. The induction of callusogenesis also depended on the hormonal composition of the nutrient medium: the highest percentage of callus formation in all types of explants was on media containing 0.5mg L−1 thidiazuron (TDZ) + 1.0 mg L−1 1-naphthaleneacetic acid (NAA) and 0.5mg L−12,4D + 2.0 mg L−1 6-Benzylaminopurine (BAP).

Identification of ERAD-dependent degrons for the endoplasmic reticulum lumen

Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the endoplasmic reticulum associated degradation (ERAD) system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly conserved ERAD system.

Multi-Indicator Comprehensive Quality Evaluation of Turpinia arguta (Lindl.) Seem Herbs at Different Harvesting Periods

This study aimed to investigate differences in the leaf biomasses, chemical compositions, and pharmacological activities of Turpinia arguta histocultured seedlings and live seedlings at different harvesting periods (S1, S2, S3, and S4) in order to determine the optimal harvesting period. Eleven indexes, including biomass, key active components, secondary metabolites, nutrient content, and antioxidant activity, were evaluated by high-performance liquid chromatography and colorimetric methods during different harvesting periods. The weights of the 11 indexes were calculated by principal component analysis, and then a comprehensive quality evaluation was performed. The results showed significant differences in leaf biomasses, key active components, secondary metabolites, nutrient contents, and antioxidant activities between the different harvesting periods. The highest quality score was obtained for the S4 period, indicating that the quality of T. arguta was the best at this time and that the S4 period was the most suitable harvesting period. At this period (S4), the medicinal component content, antioxidant activity, and comprehensive quality score of the histocultured seedlings of T. arguta were higher than those of the live seedlings, indicating the importance of tissue culture technology in enhancing the quality of T. arguta. This study provides more novel and abundant information and reference for determining the appropriate harvesting period of T. arguta, with the aim of providing newer scientific guidance for the management of herbs.

Identification of ERAD-dependent degrons for the endoplasmic reticulum lumen.

Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the endoplasmic reticulum associated degradation (ERAD) system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly conserved ERAD system.

CSIG-25. MAPK/ERK SIGNALING IN GLIOMAS MODULATES INTERFERON RESPONSES, PROMOTES TUMOR-MICROGLIA CROSSTALK, AND CONFERS SUSCEPTIBILITY TO ANTI-PD-1 AND ANTI-CTLA-4 IMMUNOTHERAPY

Abstract While immune checkpoint blockade (ICB) is not effective in unselected glioblastoma (GBM) patients, we previously established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GBM. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain to be determined. We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T-cell response and performed survival studies to validate the most relevant hits. Additionally, paired scRNA-seq with p-ERK staining, spatial transcriptomics, and ex-vivo slice culture of BRAFV600E mutant GBM tumors treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Our CRISPR/Cas9 screening results identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to anti-tumoral immunity across all kinases. Survival studies showed that mice bearing high p-ERK gliomas exhibited prolonged survival with anti-PD-1 treatment, which was not seen when ERK phosphorylation diminished. MAPK activation in murine gliomas led to durable anti-tumoral immunity upon implant re-challenge, with memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells was associated with interferon responses and antigen presentation. Moreover, in BRAFV600E human GBM cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GBM tissue analyzed by spatial transcriptomics, we observed modulation of interferon responses by the MAPK/ERK pathway. Notably, in slice cultures from BRAFV600E mutant GBM, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia. In conclusion, the MAPK/ERK pathway is a critical driver of GBM susceptibility to anti-tumoral immunity, modulating the interferon responses and antigen-presenting machinery in glioma cells, as well as tumor cell interaction with the microenvironment.

Matrine regulates autophagy in ileal epithelial cells in a porcine circovirus type 2-infected murine model

IntroductionPorcine circovirus type 2 (PCV2) is an important pathogen that causes diarrhea in nursery and fattening pigs, resulting in huge economic losses for commercial pig farms. Protective efficacy of vaccines is compromised by mutations in pathogens. There is an urgent need to articulate the mechanism by which PCV2 destroys the host’s intestinal mucosal barrier and to find effective therapeutic drugs. Increasing attention has been paid to the natural antiviral compounds extracted from traditional Chinese medicines. In the present study, we investigated the role of Matrine in mitigating PCV2-induced intestinal damage and enhancing autophagy as a potential therapeutic strategy in mice.MethodsA total of 40 female, specific-pathogen-free-grade Kunming mice were randomly divided into four groups with 10 mice in each group: control, PCV2 infection, Matrine treatment (40 mg/kg Matrine), and Ribavirin treatment (40 mg/kg Ribavirin). Except for the control group, all mice were injected intraperitoneally with 0.5 mL 105.4 50% tissue culture infectious dose (TCID50)/mL PCV2.ResultsWhile attenuating PCV2-induced downregulation of ZO-1 and occludin and restoring intestinal barrier function in a PCV2 Kunming mouse model, treatment with Matrine (40 mg/kg) attenuated ultrastructural damage and improved intestinal morphology. Mechanistically, Matrine reversed PCV2-induced autophagosome accumulation by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation and upregulating Beclin1 protein expression, thus resisting viral hijacking of enterocyte autophagy.DiscussionOur findings demonstrate that Matrine may be a novel, potential antiviral agent against PCV2 by activating intestine cellular autophagy, which provides a new strategy for host-directed drug discovery.

Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission. In vitro models using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturation in vivo. However, culture conditions in vitro should remain as close to those in vivo as possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.IMPORTANCEEstablishing models to investigate host-pathogen interactions requires mimicking physiological conditions as closely as possible. One consideration is the oxygen concentration used for in vitro tissue culture experiments. Standard incubators do not regulate oxygen levels, exposing cells to oxygen concentrations of approximately 18%. However, cells within the body are exposed to much lower oxygen concentrations, with physiological oxygen concentrations in the brain being 0.55%-8% oxygen. Here, we describe a model for herpes simplex virus 1 (HSV-1) latent infection using neurons cultured in 5% oxygen. We show that culturing neurons in more physiological oxygen concentrations improves neuronal health to permit long-term studies of virus-cell interactions and the impact on reactivation.

A quest for the potato of the future: Characterization of wild tuber-bearing Solanum species for de novo domestication.

The potato (Solanum tuberosum) is a staple food worldwide, but modern potato cultivation relies heavily on the use of pesticides to control pests and diseases. However, many wild Solanum species are highly resistant to biotic and abiotic stresses relevant for potato production. Several of those species have been used in potato breeding to confer resistances which has only been moderately successful. Instead, we propose an alternative approach to utilize the potential of wild Solanum germplasm. Recently, de novo domestication has been suggested to create more resilient crops: Instead of introducing resistance genes into existing crops, domestication traits could be introduced into resistant wild crop relatives to create new crops. Therefore, we selected ten promising species from the 107 known wild tuber-bearing Solanum species for their resistance to biotic and abiotic stresses based on existing scientific literature for experimental characterization looking at tuberization under short and long-day conditions, tuber glycoalkaloid content, starch digestibility and performance in tissue culture. Based on this, the highly pest and disease resistant S. bulbocastanaum was chosen. Our results showed that it produced relatively large tubers, also under long-day conditions and performed exceptionally well in tissue culture.

Epigenetic Regulation of Anthocyanin Biosynthesis in Betula pendula ‘Purple Rain’

Betula pendula ‘Purple Rain’ is characterized by its purple leaves and has ornamental applications. A green mutant line NL, which was mutated by line NZ of B. pendula ‘Purple Rain’ during tissue culture, shows green leaves instead of the typical purple color of B. pendula ‘Purple Rain’. This study quantified the leaf color traits of NL and a normal B. pendula ‘Purple Rain’ line NZ, and uncovered differentially expressed genes involved in flavonoid biosynthesis pathway genes in NL through RNA-Seq analysis. Compared to NZ, reduced levels of six anthocyanins contained in NL were revealed via flavonoids-targeted metabolomics. Sequence mutations in transcription factors that could explain NL’s phenotype failed to be screened via whole-genome resequencing, suggesting an epigenetic basis for this variant. Therefore, a key gene, BpMYB113, was identified in NL via the combined analysis of small RNA sequencing, whole-genome methylation sequencing, and transcriptomics. In NL, this gene features a hyper CHH context methylation site and a lower transcription level compared to NZ, disrupting the expression of downstream genes in the phenylalanine metabolism pathway, and thereby reducing flavonoid biosynthesis. Our study elucidates an epigenetic mechanism underlying color variation in variegated trees, providing pivotal insights for the breeding and propagation of colored-leaf tree species.