Division Gene Research Articles

(p)ppGpp Buffers Cell Division When Membrane Fluidity Decreases in Escherichia coli.

Fluidity is an inherent property of biological membranes and its maintenance (homeoviscous adaptation) is important for optimal functioning of membrane-associated processes. The fluidity of bacterial cytoplasmic membrane increases with temperature or an increase in the proportion of unsaturated fatty acids and vice versa. We found that strains deficient in the synthesis of guanine nucleotide analogs (p)ppGpp and lacking FadR, a transcription factor involved in fatty acid metabolism exhibited a growth defect that was rescued by an increase in growth temperature or unsaturated fatty acid content. The strain lacking (p)ppGpp was sensitive to genetic or chemical perturbations that decrease the proportion of unsaturated fatty acids over saturated fatty acids. Microscopy showed that the growth defect was associated with cell filamentation and lysis and rescued by combined expression of cell division genes ftsQ, ftsA, and ftsZ from plasmid or the gain-of-function ftsA* allele but not over-expression of ftsN. The results implicate (p)ppGpp in positive regulation of cell division during membrane fluidity loss through enhancement of FtsZ proto-ring stability. To our knowledge, this is the first report of a (p)ppGpp-mediated regulation needed for adaptation to membrane fluidity loss in bacteria.

Clinical and immune responses to neoadjuvant fulvestrant with or without enzalutamide in ER+/Her2- breast cancer.

Most ER+ breast cancers (BC) express androgen receptors (AR). This randomized phase II trial of 4 months of neoadjuvant fulvestrant (Fulv) alone or with enzalutamide (Combo) assessed whether adding AR blockade to Fulv would limit residual tumor at the time of surgery, as measured by modified preoperative endocrine predictive index (PEPI) score. Eligible patients were women with ER+/HER2- primary BC cT2 or greater. Stratification factors were clinical node and T-stage. Fresh tumor biopsies were required at study entry, after 4 weeks on therapy (W5), and at surgery. Laboratory analyses on tumors included immunochemistry (IHC) for ER/PR/AR/GR and Ki67 protein, evaluation of gene expression, multiplex for myeloid lineage immune cells, reverse-phase protein array, and plasma metabolomic analyses. Of 69 consented patients, 59 were evaluable. Toxicity was as expected with endocrine therapy. Combo achieved PEPI = 0 more frequently (24%: 8/33) than Fulv (8%: 2/26). Ki67 was ≤10% across arms by W5 in 76% of tumors. Activation of mTOR pathway proteins was elevated in tumors with poor Ki67 response. Tumors in both arms showed decreased estrogen-regulated and cell division gene sets, while Combo arm tumors uniquely exhibited enrichment of immune activation gene sets, including interferon gamma, complement, inflammation, antigen processing, and B and T cell activation. Multiplex IHC showed significantly reduced tumor-associated macrophages and CD14+/HLADR-/CD68- MDSCs in Combo tumors at W5. In summary, Combo tumors showed a higher PEPI = 0 response, Ki67 response, and more activated tumor immune microenvironment than Fulv. The odds of response were 4.6-fold higher for patients with ILC versus IDC. (Trial registration: This trial is registered at Clinicaltrials.gov ( https://www.clinicaltrials.gov/study/NCT02955394?id=16-1042&rank=1 ). The trial registration number is NCT02955394. The full trial protocol is available under Study Details at the Clinicaltrials.gov link provided).

Genomic data provides insights into the evolutionary history and adaptive differentiation of two tetraploid strawberries.

Over the decades, evolutionists and ecologists have shown intense interest in the role of polyploidization in plant evolution. Without clear knowledge of the diploid ancestor(s) of polyploids, we would not be able to answer fundamental ecological questions such as the evolution of niche differences between them or its underlying genetic basis. Here, we explored the evolutionary history of two Fragaria tetraploids, Fragaria corymbosa and Fragaria moupinensis. We de novo assembled five genomes including these two tetraploids and three diploid relatives. Based on multiple lines of evidence, we found no evidence of subgenomes in either of the two tetraploids, suggesting autopolyploid origins. We determined that Fragaria chinensis was the diploid ancestor of F. corymbosa while either an extinct species affinitive to F. chinensis or an unsampled population of F. chinensis could be the progenitor of F. moupinensis. Meanwhile, we found introgression signals between F. chinensis and Fragaria pentaphylla, leading to the genomic similarity between these two diploids. Compared to F. chinensis, gene families related to high ultraviolet (UV)-B and DNA repair were expanded, while those that responded towards abiotic and biotic stresses (such as salt stress, wounding, and various pathogens) were contracted in both tetraploids. Furthermore, the two tetraploids tended to down-regulate defense response genes but up-regulate UV-B response, DNA repairing, and cell division gene expression compared to F. chinensis. These findings may reflect adaptions toward high-altitude habitats. In summary, our work provides insights into the genome evolution of wild Fragaria tetraploids and opens up an avenue for future works to answer deeper evolutionary and ecological questions regarding the strawberry genus.

The genomic database of fruits: A comprehensive fruit information database for comparative and functional genomic studies

Fruit has an important role in human nutrition and health; therefore, the systematic study of fruit genomic data is essential. The Genomic Database of Fruits (TGDF, http://tgdf.bio2db.com/), established through whole-genome analyses of 44 fruit species, is a comprehensive, user-friendly fruit database. TGDF contains a wealth of functional genes, including 11,350 flowering genes, 3161 auxin signaling genes, 2164 anthocyanin synthesis genes, 1464 abscisic acid (ABA) synthesis genes, 10,931 ​cell division and expansion genes, 1786 starch synthesis genes, 294 fruit size genes, and 6311 sugar transporter genes. Additionally, TGDF contains 1,433,368 CRISPR guide sequences from various fruit genomes, along with information on homologous genes and duplication types for the 44 fruit species. TGDF contains 6,417,060 gene annotations sourced from TrEMBL, SwissProt, Nr, and Gene Ontology databases, along with tools such as Sequence Fetch, BLAST, Synteny, and JBrowse for bioinformatics analyses. Transcriptomic data were also collected and collated from fruits, including details on instruments, tissues, or growth stages. This comprehensive, user-friendly resource is the first collection of fruit genomic data. Users can easily download genomic sequences, gene annotations, and bioinformatics analysis results from TGDF, which will be updated continually. We anticipate that TGDF will become a primary resource for fruit comparative and functional genomic studies.

Characterization of a pleiotropic regulator MtrA in Streptomyces avermitilis controlling avermectin production and morphological differentiation

BackgroundThe macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated.ResultsIn this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis.ConclusionsIn this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.

Landscape Feature Extraction and Floristic Division of Traditional Villages in the Minjiang River Basin

Traditional villages reflect the integration and collision of cultures from many aspects such as culture, function and site selection, and reflect the cooperation and struggle between man and nature. They hold outstanding historical, cultural, artistic, scientific and social values, and are important carriers of national culture. As an important birthplace of civilization in the southeastern region of China, the Minjiang River Basin has profound historical and cultural accumulation. The unique, diverse and abundant traditional village resources in the basin embody the Fujian traditional culture with distinctive local characteristics. In order to accurately identify the landscape characteristics of traditional villages in the Minjiang River Basin, the research first introduces the concept of landscape gene similarity on the basis of landscape gene theory, and quantitatively evaluates the similarity of traditional village landscapes, which reduces the influence of subjective judgment in the process of landscape gene extraction, strengthens the repeatability of the conclusions, and makes the results more accurate. Secondly, based on five batches of Chinese traditional village lists, field visits and literature review, 111 national traditional villages with complete data and well preserved in Minjiang River Basin are determined as research objects. Finally, based on the dialect and folk beliefs, the cultural characteristics of the villages are judged, and the landscape characteristics of the villages are described based on the characteristics of the residential landscape. Six types of indicators that can best reflect the characteristics of village landscape are selected to construct the index system. By calculating the similarity of each traditional village landscape and combining with spatial statistical analysis, the traditional villages in Minjiang River Basin are divided into 9 landscape regions and 4 landscape sub-regions as a whole, and construct the landscape gene map of traditional villages in Minjiang River Basin. Through the calculation of the similarity between the traditional village landscape and spatial statistical analysis, the traditional villages in the Minjiang River Basin are divided into 9 landscape areas and 4 landscape sub-areas, then mapping the landscape gene map of traditional villages in Minjiang River basin. The results show that there are differences in landscape gene composition of traditional villages in different regions in Minjiang River Basin. Based on the differences of landscape gene among traditional villages in Minjiang River Basin, combined with the administrative division and landscape gene similarity, traditional villages in Minjiang River Basin can be divided into 9 landscape areas and 4 landscape subareas which include Hakka courtyard area, Shaojiang Courtyard area, Central Fujian Courtyard area, Central Fujian enclosed building area, Eastern Fujian enclosed building area, Eastern Fujian mixed area, Southern Fujian compound area and Northern Fujian compound area, Northern Fujian gabled roof courtyard area, Northern Fujian mixed roof courtyard area, Shaojiang independent courtyard area and Shaojiang combined courtyard area. The similarity of landscape genes between two adjacent landscape areas was higher than other areas. To some extent, this is due to the role and influence of natural conditions, cultural customs and social economy in different periods. Among them, the natural conditions affecting the residence form and construction of the overall arrangement of the choice of material, culture affects the residential spatial structure characteristics such as shape, function partition, the social and economic conditions determine the scale and fineness of dwellings. Any change of conditions will produce different morphological characteristics of residential buildings, major historical events are important catalysts for this transformation process, which can accelerate or slow down the entire process. In the future, the research will take the major historical events that affect human activities in the watershed as the time node, analyze the development and evolution laws of buildings in different periods, and conduct in-depth analysis and exploration of this conclusion.

Bioprospecting of Chlamydomonas reinhardtii for boosting biofuel-related products production based on novel aggregation-induced emission active extracellular polymeric substances nanoprobes

Biofuel production from microalgae has been greatly restricted by low biomass productivity and long-term photosynthetic efficacy. Here, a novel strategy for selecting high-growing, stress-resistant algal strains with high photosynthetic capacity was proposed based on biocompatible extracellular polymeric substances (EPS) probes with aggregation-induced emission (AIE) properties. Specifically, AIE active EPS probes were synthesized for in-situ long-term monitoring of the EPS productivity at different algal growth stages. By coupling the AIE-based fluorescent techniques, algal cells were classified into four diverse populations based on their chlorophyll and EPS signals. Mechanistic studies on the sorted algal cells revealed their remarkable stress resistance and high expression of cell division, biopolymer production and photosynthesis-related genes. The sorted and subcultured algal cells consistently exhibited relatively higher growth rates and photosynthetic capacities, resulting in an increased (1.2 to 1.8-fold) algal biomass production, chlorophyll, and lipids. This study can potentially open new strategies to boost microalgal-based biofuel production.

Adaptive evolution of chloroplast division mechanisms during plant terrestrialization

Despite extensive research, the origin and evolution of the chloroplast division machinery remain unclear. Here, we employ recently sequenced genomes and transcriptomes of Archaeplastida clades to identify the core components of chloroplast division and reconstruct their evolutionary histories, respectively. Our findings show that complete division ring structures emerged in Charophytes. We find that Glaucophytes experienced strong selection pressure, generating diverse variants adapted to the changing terrestrial environments. By integrating the functions of chloroplast division genes (CDGs) annotated in a workflow developed using large-scale multi-omics data, we further show that dispersed duplications acquire more species-specific functions under stronger selection pressures. Notably, PARC6, a dispersed duplicate CDG, regulates leaf color and plant growth in Solanum lycopersicum, demonstrating neofunctionalization. Our findings provide an integrated perspective on the functional evolution of chloroplast division machinery and highlight the potential of dispersed duplicate genes as the primary source of adaptive evolution of chloroplast division.

Investigation of stimulated growth effect by application of L-aspartic acid on poplar

Poplar is an important resource for the production of wood-based products. L-aspartic acid (Asp) is a proteinogenesis amino acid that plays a critical role in cell growth modulation and stress response. Also, Asp is widespread in the commonly used amino acid-based biostimulant products. However, its effects on poplars remain largely unknown. To address this issue, we applied different concentrations of Asp to poplar seedlings under N reduction conditions (simulating low N availability status), and assessed the effects of Asp on poplar growth and development. Our results showed that supplement of Asp at an appropriate concentration, that is, 0.5 mM in the present study, had the best growth-promoting effect on poplars. It significantly increased poplar biomass and height, and simultaneously elevated the photosynthetic parameters, the chlorophyll content and total free amino acid content, as well as the activity of N metabolism-related enzymes in poplars-treated with Asp in comparision with the control plants without Asp addition. Furthermore, the expression of Asp-metabolism pathway genes (AspAT1, AspAT6, AspAT10, AK1, ASS1 and ASS2), key chlorophyll synthesis gene (LHCB3) and cell division gene (CYCD3) was all dramatically upregulated in response to Asp application, suggesting that Asp-induced effect invoked photosynthesis activation and metabolism reprogramming. More interestingly, our results showed that the levels of various phytohormones, such as auxin and cytokinins were remarkably altered, and key auxin transport gene (PIN1c-2), auxin conjugation gene (GH3.6–2) were induced by Asp treatment, indicative of the possible involvement of plant hormones under the effect of Asp, especially fine-tuning auxin homeostasis by forming IAA-Asp conjugates to store auxin and catabolizing IAA-Asp to release auxin, may account for Asp-mediated growth modulation to an extent in poplars. Our study provides a novo insight into the growth promotion effect of Asp on poplars, especially, when the N source is limited, even though the underlying mechanism of Asp-induced stimulation action needs further in-depth investigation.

DNA damage response coregulator ddrR affects many cellular pathways and processes in Acinetobacter baumannii 17978.

Acinetobacter baumannii strain 17978 is an opportunistic pathogen possessing a DNA damage response (DDR) in which multiple error-prone polymerase genes are co-repressed by a UmuD homolog, UmuDAb, and the small Acinetobacter-specific protein DdrR. Additionally, these regulators coactivate nine other genes. We identified the DNA damage-inducible transcriptome for wildtype, umuDAb, and recA strains, and later established the ddrR DDR transcriptome. However, the ATCC 17978 reference genome had several assembly errors and lacked the 44 kb virulence locus, AbaAL44, that is present in the strain 17978 UN. For this project, we combined our earlier single-end read RNAseq data with the ddrR paired-end reads and aligned these data to the improved 17978 UN genome assembly that resembled our laboratory strain, 17978 JH. New DESeq2 analyses verified previous differentially expressed genes (DEGs) but also found 339 genes in 17978 JH that were not annotated or physically present in the older genome assembly. Sixty-three were differentially expressed after DNA damage, and 182 had differential basal expression when comparing umuDAb, ddrR, or recA strains to wildtype, with 94 genes' expression unchanged. This work identified and characterized the 55 gene DNA damage-repressible transcriptome, 98% of which required either umuDAb or ddrR for repression. Two-thirds of these DEGs required both regulators. We also identified 110 genes repressed only in the ddrR strain, ~50% of which were due to increased basal expression levels. Basal gene expression in the ddrR mutant was further dysregulated independent of the DDR. Over 800 genes were upregulated, and over 1200 genes were downregulated compared to wildtype expression. Half of A. baumannii's essential genes were upregulated in the ddrR strain, including cell division genes, and two-thirds of these were downregulated in the umuDAb strain. The ddrR mutant upregulated genes enriched in translation, RNA metabolism, protein metabolism, AA/FA/cell-structure synthesis, and transport, while downregulating genes enriched in quorum sensing, biofilm production, secretion systems, pilus production, cell adhesion, and aromatics and chlorine degradation. Our data underscore the need for accurate and appropriately matched genome assemblies and indicate that ddrR affects approximately 60% of the genome, rendering it a potential target for Acinetobacter baumannii infection treatment.

A conserved graft formation process in Norway spruce and Arabidopsis identifies the PAT gene family as central regulators of wound healing

The widespread use of plant grafting enables eudicots and gymnosperms to join with closely related species and grow as one. Gymnosperms have dominated forests for over 200 million years, and despite their economic and ecological relevance, we know little about how they graft. Here we developed a micrografting method in conifers using young tissues that allowed efficient grafting with closely related species and between distantly related genera. Conifer graft junctions rapidly connected vasculature and differentially expressed thousands of genes including auxin and cell-wall-related genes. By comparing these genes to those induced during Arabidopsis thaliana graft formation, we found a common activation of cambium, cell division, phloem and xylem-related genes. A gene regulatory network analysis in Norway spruce (Picea abies) predicted that PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) acted as a core regulator of graft healing. This gene was strongly up-regulated during both spruce and Arabidopsis grafting, and Arabidopsis mutants lacking PAT genes failed to attach tissues or successfully graft. Complementing Arabidopsis PAT mutants with the spruce PAT1 homolog rescued tissue attachment and enhanced callus formation. Together, our data show an ability for young tissues to graft with distantly related species and identifies the PAT gene family as conserved regulators of graft healing and tissue regeneration.

Transcriptome and Secretome Analyses of Endophyte Methylobacterium mesophilicum and Pathogen Xylella fastidiosa Interacting Show Nutrient Competition.

Xylella fastidiosa is the causal agent of several plant diseases affecting fruit and nut crops. Methylobacterium mesophilicum strain SR1.6/6 was isolated from Citrus sinensis and shown to promote plant growth by producing phytohormones, providing nutrients, inhibiting X. fastidiosa, and preventing Citrus Variegated Chlorosis. However, the molecular mechanisms involved in the interaction among these microbes are still unclear. The present work aimed to analyze physiological and molecular aspects of M. mesophilicum SR1.6/6 and X. fastidiosa 9a5c in co-culture. The transcriptome and secretome analyses indicated that X. fastidiosa down-regulates cell division and transport genes and up-regulates stress via induction of chaperones and pathogenicity-related genes including, the lipase-esterase LesA, a protease, as well as an oligopeptidase in response to M. mesophilicum competition. On the other hand, M. mesophilicum also down-regulated transport genes, except for iron uptake, which was up-regulated. Secretome analysis identified four proteins in M. mesophilicum exclusively produced in co-culture with X. fastidiosa, among these, three are related to phosphorous uptake. These results suggest that M. mesophilicum inhibits X. fastidiosa growth mainly due to nutrient competition for iron and phosphorous, thus promoting X. fastidiosa starvation, besides producing enzymes that degrade X. fastidiosa cell wall, mainly hydrolases. The understanding of these interactions provides a direction for control and management of the phytopathogen X. fastidiosa, and consequently, helps to improve citrus growth and productivity.

The two-component system CpxAR is required for the high potassium stress survival of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen, which can cause porcine contagious pleuropneumonia and lead to great economic losses to worldwide swine industry. High potassium is an adverse environment for bacteria, which is not conducive to providing turgor pressure for cell growth and division. Two-component system CpxAR is an important regulatory system of bacteria in response to environmental changes, which is involved in a variety of biological activities, such as antibiotic resistance, periplasmic protein folding, peptidoglycan metabolism and so on. However, little is known about the role of CpxAR in high potassium stress in A. pleuropneumoniae. Here, we showed that CpxAR is critical for cell division of A. pleuropneumoniae under high potassium (K+) stress. qRT-PCR analysis found that CpxAR positively regulated the cell division genes ftsEX. In addition, we also demonstrated that CpxR-P could directly bind the promoter region of the cell division gene ftsE by EMSA. In conclusion, our results described a mechanism where CpxAR adjusts A. pleuropneumoniae survival under high-K+ stress by upregulating the expression of the cell division proteins FtsE and FtsX. These findings are the first to directly demonstrate CpxAR-mediated high-K+ tolerance, and to investigate the detailed molecular mechanism.

The minimal intrinsic stochasticity of constitutively expressed eukaryotic genes is sub-Poissonian.

Gene expression inherently gives rise to stochastic variation ("noise") in the production of gene products. Minimizing noise is crucial for ensuring reliable cellular functions. However, noise cannot be suppressed below a certain intrinsic limit. For constitutively expressed genes, this limit is typically assumed to be Poissonian noise, wherein the variance in mRNA numbers is equal to their mean. Here, we demonstrate that several cell division genes in fission yeast exhibit mRNA variances significantly below this limit. The reduced variance can be explained by a gene expression model incorporating multiple transcription and mRNA degradation steps. Notably, in this sub-Poissonian regime, distinct from Poissonian or super-Poissonian regimes, cytoplasmic noise is effectively suppressed through a higher mRNA export rate. Our findings redefine the lower limit of eukaryotic gene expression noise and uncover molecular requirements for achieving ultralow noise, which is expected to be important for vital cellular functions.

P-Aminobenzoic acid inhibits the growth of soybean pathogen Xanthomonas axonopodis pv. glycines by altering outer membrane integrity.

p-Aminobenzoic acid (pABA) is an environmentally friendly bioactive metabolite synthesized by Lysobacter antibioticus. This compound showed an unusual antifungal mode of action based on cytokinesis inhibition. However, the potential antibacterial properties of pABA remain unexplored. In this study, pABA showed antibacterial activity against Gram-negative bacteria. This metabolite inhibited growth (EC50 = 4.02 mM), and reduced swimming motility, extracellular protease activity, and biofilm formation in the soybean pathogen Xanthomonas axonopodis pv. glycines (Xag). Although pABA was previously reported to inhibit fungal cell division, no apparent effect was observed on Xag cell division genes. Instead, pABA reduced the expression of various membrane integrity-related genes, such as cirA, czcA, czcB, emrE, and tolC. Consistently, scanning electron microscopy observations revealed that pABA caused major alternations in Xag morphology and blocked the formation of bacterial consortiums. In addition, pABA reduced the content and profile of outer membrane proteins and lipopolysaccharides in Xag, which may explain the observed effects. Preventive and curative applications of 10 mM pABA reduced Xag symptoms in soybean plants by 52.1% and 75.2%, respectively. The antibacterial properties of pABA were studied for the first time, revealing new insights into its potential application for the management of bacterial pathogens. Although pABA was previously reported to show an antifungal mode of action based on cytokinesis inhibition, this compound inhibited Xag growth by altering the outer membrane's integrity. © 2023 Society of Chemical Industry.

Is chloroplast size optimal for photosynthetic efficiency?

Improving photosynthetic efficiency has recently emerged as a promising way to increase crop production in a sustainable manner. While chloroplast size may affect photosynthetic efficiency in several ways, we aimed to explore whether chloroplast size manipulation can be a viable approach to improving photosynthetic performance. Several tobacco (Nicotiana tabacum) lines with contrasting chloroplast sizes were generated via manipulation of chloroplast division genes to assess photosynthetic performance under steady-state and fluctuating light. A selection of lines was included in a field trial to explore productivity. Lines with enlarged chloroplasts underperformed in most of the measured traits. Lines with smaller and more numerous chloroplasts showed a similar efficiency compared with wild-type (WT) tobacco. Chloroplast size only weakly affected light absorptance and light profiles within the leaf. Increasing chloroplast size decreased mesophyll conductance (gm ) but decreased chloroplast size did not increase gm . Increasing chloroplast size reduced chloroplast movements and enhanced non-photochemical quenching. The chloroplast smaller than WT appeared to be no better than WT for photosynthetic efficiency and productivity under field conditions. The results indicate that chloroplast size manipulations are therefore unlikely to lead to higher photosynthetic efficiency or growth.

The paralogues MAGOH and MAGOHB are oncogenic factors in high-grade gliomas and safeguard the splicing of cell division and cell cycle genes

ABSTRACT The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.

Abstract B065: Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer

Abstract The phosphoinositide 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is dysregulated in the majority of advanced prostate cancer patients, often linked to phosphate and tensin homolog (PTEN) inactivation. An increasing number of studies demonstrates the reciprocal crosstalk between the PI3K/AKT/mTOR and the androgen receptor (AR) pathways with the inhibition of one of them leading to the compensatory activation of the other one. We compared the effects of different PI3K/AKT/mTOR pathway inhibitors as single agents or in combination with the AR inhibitor darolutamide.In vitro impact of the compounds was evaluated in prostate cancer cell lines by proliferation and apoptosis assays, and by RNA-seq analysis. In vivo effects were determined in a patient-derived (PDX) prostate cancer model in an efficacy study followed by immunohistochemistry analysis. Analysis of VCaP cells treated with the androgen R1881 showed that beside androgen response, the PI3K/AKT/mTOR pathway was an essential hallmark characteristic of androgen action. Additional darolutamide treatment opposed androgen effects on both pathways. We therefore evaluated the impact of selective inhibitors of PI3K or AKT and saw strong in vitro antiproliferative effects in the prostate cancer cell lines tested. We then focused on the pan-PI3K inhibitor copanlisib and observed pronounced synergistic effects with darolutamide in the VCaP model. This was accompanied by induction of PARP cleavage and activation of caspase 3/7. A detailed transcriptomic analysis revealed that the combination of both inhibitors resulted in more pronounced transcript changes, compared to individual treatments. Principal component analysis furthermore showed the combination to be closer to the DMSO control along the axis that represents androgen regulation. We observed a pronounced downregulation of cell proliferation and cell division genes following combination treatment compared to individual treatments, a prominent example being the proliferation marker KI67. In vivo studies performed with the PDX LuCaP 35 model revealed a superior inhibitory effect of the combination treatment with darolutamide and copanlisib when compared to single agents. Immunohistochemistry analysis revealed a significant upregulation of a pro-apoptotic pathway in tumors treated with darolutamide and copanlisib which was not observed in single treatment arms, when compared to vehicle control. In summary, we found that different PI3K and AKT inhibitors impaired the in vitro proliferation of prostate cancer cell lines. Combining the AR inhibitor darolutamide with the pan-PI3K inhibitor copanlisib led to increased apoptosis and down-regulation of cell division and proliferation genes. Importantly, the improved tumor inhibition by the combination was also observed in vivo. These results warrant further analysis of the impact of combined AR and PI3K pathway inhibition in prostate cancer, especially when aberrations in the PI3K/AKT/mTOR pathway or PTEN loss are observed. Citation Format: Simon Heller, Tatsuo Sugawara, Ekaterina Nevedomskaya, Simon J. Baumgart, Holly Nguyen, Eva Corey, Annika Böhme, Oliver von Ahsen, Oliver Politz, Bernard Haendler. Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B065.

Influences of flavonoids from Sedum aizoon L. on biofilm formation of Pseudomonas fragi.

Pseudomonas fragi (P. fragi) is one of the main categories of bacteria responsible for the spoilage of chilled meat. In the processing and preservation of chilled meat, it is easy to form biofilms on the meat, leading to the development of slime on the meat, which becomes a major quality defect. Flavonoids, as one of the critical components of secondary plant metabolites, are receiving increasing attention for their antibacterial activity. Flavonoids in Sedum aizoon L. (FSAL), relying on its prominent antibacterial activity, are of research importance in food preservation and other applications. This article aims to investigate the effect of FSAL on the biofilm formation of P. fragi, to better apply FSAL to the processing and preservation of meat products. The disruption of cellular structure and aggregation properties by FSAL was demonstrated by the observation of the cellular state within the biofilm. The amount of biofilm formation was determined by crystal violet staining, and the content of polysaccharides and proteins in the extracellular wrapped material was determined. It was shown that the experimental concentrations of FSAL (1.0 MIC) was able to inhibit biofilm formation and reduce the main components in the extracellular secretion. The swimming motility assay and the downregulation of flagellin-related genes confirmed that FSAL reduced cell motility and adhesion. The downregulation of cell division genes and the lowering of bacterial metabolic activity suggested that FSAL could hinder bacterial growth and reproduction within P. fragi biofilms. KEY POINTS: • FSAL inhibited the activity of Pseudomonas fragi in the dominant meat strain • The absence of EPS components affected the formation of P. fragi biofilms • P. fragi has reduced adhesion capacity due to impaired flagellin function.

Anti-Bacterial and Anti-Biofilm Activities of Anandamide against the Cariogenic Streptococcus mutans

Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.