Artículos de revistas sobre el tema "Berry development transcriptomic route"

The progress of the grapevine genomics and the development of high-throughput technologies for gene expression analysis stimulated the investigation of the physical, biochemical and physiological changes of grape berry growth and maturation at transcriptomic level. The molecular information generated in the last decade is however still fragmented since it relies upon detailed analysis of few stages and thus lacks continuity over grape development. To identify the molecular events associated with berry development at a higher temporal resolution and define a transcriptomic map, we performed RNA-seq analysis of berry samples collected every week from fruit-set to maturity in Pinot noir and Cabernet Sauvignon for three consecutive years, resulting in 219 samples. Using the most variable portion of the transcriptome, we built a preliminary transcriptomic model of berry development based on the Cabernet Sauvignon samples. The Pinot noir samples were then aligned onto this preliminary ripening map to investigate its performance in describing the development of another grape variety. A further step for testing the model was the projection of RNA-seq samples of fruit development of five red-skin Italian cultivars. For all these surveys, the transcriptomic route allowed a precise definition of the progression of berry development during both formation and ripening phases.

<p style="text-align: justify;"><strong>Aims</strong>: The aim of this paper was to use recent transcriptomic tools available for grape in order to understand berry softening.</p><p style="text-align: justify;"><strong>Methods and results</strong>: A microarray bearing specific 50 mer oligonucleotide for 3,200 genes was used to study gene expression along 8 stages of berry development in Chardonnay and Shiraz berries. Transcripts corresponding to aquaporin genes and to genes involved in cell wall metabolism were studied in detail and ranked according to their pattern of expression.</p><p style="text-align: justify;"><strong>Conclusion</strong>: Several structural and regulatory genes whose expression pattern correlated with the late phases of ripening were identified. Significance and impact of study: This study provides a preliminary molecular basis to identify molecular markers of berry ripening.</p>

Root restriction (RR) has been reported to enhance grape berry quality in diverse aspects of grape life. In this study, RR-induced increases in the main primary metabolites in the grape berry and the expression of their related genes were studied at different developmental stages. Mainly the transcriptomic and metabolomic level were analyzed using ‘Summer Black’ grape berry as a material. The main results were as follows: A total of 11 transcripts involved in the primary metabolic pathways were significantly changed by the RR treatment. Metabolites such as sugars, organic acids, amino acids, starch, pectin, and cellulose were qualitatively and quantitatively analyzed along with their metabolic pathways. Sucrose synthase (VIT_07s0005g00750, VIT_11s0016g00470) and sucrose phosphate synthase (VIT_18s0089g00410) were inferred to play critical roles in the accumulation of starch, sucrose, glucose, and fructose, which was induced by the RR treatment. RR treatment also promoted the malic acid and tartaric acid accumulation in the young berry. In addition, the grape berries after the RR treatment tended to have lower pectin and cellulose content.

Heat stress has a strong and detrimental effect on plant growth and yield. Goji berry or wolfberry (Lycium barbarum L.) is a dual-purpose medicinal and food plant but an increase in high temperatures has caused a serious decline in wolfberry yield and quality. In this study, we first explored the heat stress responses of Goji berry, and found that heat stress adaptation mechanisms fluctuated over 48 h. Moreover, L.barbarum 1402 was more heat resistant while L.barbarum Ningqi No. 7 (N7) was sensitive to high temperatures, in which amino acids and alkaloids played key roles; expression and accumulation timing was also crucial. That is, 1402 responded to heat stress rapidly starting at 1 h under high temperature, activated related genes, and accumulated metabolites earlier in the amino acid metabolic pathway compared to N7, which responded to heat stress starting at 3 h under high temperature. Thus, 1402 resisted high temperatures much earlier and better compared to N7. Furthermore, joint transcriptome and metabolome analysis results showed that L-phenylalanine, L-tyrosine, N-benzylformamide, N-benzylmethylene isomethylamine, lysoPC 19:1, and N-acetyl-D-glucosamine-1-phosthate, as well as their related genes, were higher in content, or earlier in expression, in 1402 compared to N7 under heat treatment. This study initially elucidates that Goji berry 1402 has a better tolerance to heat stress than N7 for earlier and higher expression or accumulation of amino acids and alkaloids when related to high temperatures.

Abstract Drought events are a major challenge for many horticultural crops, including grapes, which are often cultivated in dry and warm climates. It is not understood how the cuticle contributes to the grape berry response to water deficit (WD); furthermore, the cuticular waxes and the related biosynthetic pathways are poorly characterized in this fruit. In this study, we identified candidate wax-related genes from the grapevine genome by phylogenetic and transcriptomic analyses. Developmental and stress response expression patterns of these candidates were characterized across pre-existing RNA sequencing data sets and confirmed a high responsiveness of the pathway to environmental stresses. We then characterized the developmental and WD-induced changes in berry cuticular wax composition, and quantified differences in berry transpiration. Cuticular aliphatic wax content was modulated during development and an increase was observed under WD, with wax esters being strongly up-regulated. These compositional changes were related to up-regulated candidate genes of the aliphatic wax biosynthetic pathway, including CER10, CER2, CER3, CER1, CER4, and WSD1. The effect of WD on berry transpiration was not significant. This study indicates that changes in cuticular wax amount and composition are part of the metabolic response of the grape berry to WD, but these changes do not reduce berry transpiration.

Grapevine cultivation, such as the whole horticulture, is currently challenged by several factors, among which the extreme weather events occurring under the climate change scenario are the most relevant. Within this context, the present study aims at characterizing at the berry level the physiological response of Vitis vinifera cv. Sauvignon Blanc to sequential stresses simulated under a semi-controlled environment: flooding at bud-break followed by multiple summer stress (drought plus heatwave) occurring at pre-vèraison. Transcriptomic and metabolomic assessments were performed through RNASeq and NMR, respectively. A comprehensive hormone profiling was also carried out. Results pointed out a different response to the heatwave in the two situations. Flooding caused a developmental advance, determining a different physiological background in the berry, thus affecting its response to the summer stress at both transcriptional levels, with the upregulation of genes involved in oxidative stress responses, and metabolic level, with the increase in osmoprotectants, such as proline and other amino acids. In conclusion, sequential stress, including a flooding event at bud-break followed by a summer heatwave, may impact phenological development and berry ripening, with possible consequences on berry and wine quality. A berry physiological model is presented that may support the development of sustainable vineyard management solutions to improve the water use efficiency and adaptation capacity of actual viticultural systems to future scenarios.

Aim: In the long term, genetic improvement is one of the major strategies to support sustainable wine production in a changing climate. Over the past 5 years, we have developed an interdisciplinary research program that aimed to: i) characterize the impact of temperature increase sensed by the entire plant or individual bunches on the development and functioning of the plant, ii) identify the physiological and molecular mechanisms regulating the response of vegetative and reproductive development to heat stress and iii) develop tools to map quantitative trait loci (QTLs) of plant and berry development in duly controlled, stable, and contrasting environmental conditions.Methods and results: Performing high-throughput genomic analyses combined with the use of innovative experimental designs (fruiting cuttings, microvines, single berry sampling) was critical to decipher the ecophysiological and molecular mechanisms involved in the vine response to high temperature.Conclusion: Warming promotes vegetative growth and hampers plant carbon balance, disturbing flower set and young berry development. High temperatures modify primary and secondary fruit metabolisms, desynchronizing sugar and organic acid metabolisms and delaying sugar and polyphenol accumulation during ripening. The study of day and night transcriptomic and proteomic signatures associated with heat highlighted key players of the response to temperature in the fruit. Significance and impact of the study: Capitalizing on this knowledge, a new program is being proposed for the selection of cultivars limiting the accumulation of sugars in the berry while maintaining other qualitative compounds.

Background Bud sport mutation occurs frequently in fruit plants and acts as an important approach for grapevine improvement and breeding. ‘Jinzao Wuhe’ is a bud sport of the elite cultivar ‘Himord Seedless’ with obviously enlarged organs and berries. To date, the molecular mechanisms underlying berry enlargement caused by bud sport in grapevines remain unclear. Methods Whole genome resequencing (WGRS) was performed for two pairs of bud sports and their maternal plants with similar phenotype to identify SNPs, InDels and structural variations (SVs) as well as related genes. Furthermore, transcriptomic sequencing at different developmental stages and weighted gene co-expression network analysis (WGCNA) for ‘Jinzao Wuhe’ and its maternal plant ‘Himord Seedless’ were carried out to identify the differentially expressed genes (DEGs), which were subsequently analyzed for Gene Ontology (GO) and function annotation. Results In two pairs of enlarged berry bud sports, a total of 1,334 SNPs, 272 InDels and 74 SVs, corresponding to 1,022 target genes related to symbiotic microorganisms, cell death and other processes were identified. Meanwhile, 1,149 DEGs associated with cell wall modification, stress-response and cell killing might be responsible for the phenotypic variation were also determined. As a result, 42 DEGs between ‘Himord Seedless’ and ‘Jinzao Wuhe’ harboring genetic variations were further investigated, including pectin esterase, cellulase A, cytochromes P450 (CYP), UDP-glycosyltransferase (UGT), zinc finger protein, auxin response factor (ARF), NAC transcription factor (TF), protein kinase, etc. These candidate genes offer important clues for a better understanding of developmental regulations of berry enlargement in grapevine. Conclusion Our results provide candidate genes and valuable information for dissecting the underlying mechanisms of berry development and contribute to future improvement of grapevine cultivars.

<p class="Abstract" style="text-align: justify;"><strong>Aim:</strong> In the long term, genetic improvement is one of the major strategies to support sustainable wine production in a changing climate. Over the past 5 years, we have developed an interdisciplinary research program that aimed to: i) characterize the impact of temperature increase sensed by the entire plant or individual bunches on the development and functioning of the plant, ii) identify the physiological and molecular mechanisms regulating the response of vegetative and reproductive development to heat stress and iii) develop tools to map quantitative trait loci (QTLs) of plant and berry development in duly controlled, stable, and contrasting environmental conditions.</p><p class="Abstract" style="text-align: justify;"><strong>Methods and results:</strong> Performing high-throughput genomic analyses combined with the use of innovative experimental designs (fruiting cuttings, microvines, single berry sampling) was critical to decipher the ecophysiological and molecular mechanisms involved in the vine response to high temperature.</p><p class="Abstract" style="text-align: justify;"><strong>Conclusion:</strong> Warming promotes vegetative growth and hampers plant carbon balance, disturbing flower set and young berry development. High temperatures modify primary and secondary fruit metabolisms, desynchronizing sugar and organic acid metabolisms and delaying sugar and polyphenol accumulation during ripening. The study of day and night transcriptomic and proteomic signatures associated with heat highlighted key players of the response to temperature in the fruit. </p><p class="Abstract" style="text-align: justify;"><strong>Significance and impact of the study:</strong> Capitalizing on this knowledge, a new program is being proposed for the selection of cultivars limiting the accumulation of sugars in the berry while maintaining other qualitative compounds.</p>

In this study, ultraviolet-C (UV-C) was utilized to improve the quality of post-harvest grape berries, and the transcriptomic and metabolomic basis of this improvement was elucidated. Berries of the red grape variety ‘Zicui’ and the white variety ‘Xiangfei’ were chosen to evaluate the effect of short- and long-term UV-C irradiation. Post-harvest UV-C application promoted malondialdehyde (MDA) and proline accumulation, and reduced the soluble solid content in berries. Both the variety and duration of irradiation could modulate the transcriptomic and metabolomic responses of berries to UV-C. Compared with the control, the differentially expressed genes (DEGs) identified under UV-C treatment were enriched in pathways related to metabolite accumulation, hormone biosynthesis and signal transduction, and reactive oxygen species (ROS) homeostasis. Flavonoid biosynthesis and biosynthesis of other secondary metabolites were the shared pathways enriched with differential metabolites. After long-term UV-C irradiation, cis-resveratrol accumulated in the berries of the two varieties, while the differential chalcone, dihydroflavone, flavonoid, flavanol, and tannin components primarily accumulated in ‘Xiangfei’, and some flavonols and anthocyanins primarily accumulated in ‘Zicui’. Based on an exhaustive survey, we made a summary for the effect of UV-C in regulating the quality development of post-harvest grape berries. The results of this study may help to elucidate the mechanism by which UV-C functions and support its efficient application.

Phytohormones strongly influence growth, development and nutritional quality of agricultural products by modulating molecular and biochemical changes. The purpose of the present study was to investigate the influence of root restriction (RR) treatment on the dynamic changes of main phytohormones during the berry development and ripening of “Summer Black” early ripening seedless grape (Vitis vinifera × V. labrusca), and to analyze the changes in the biosynthesis and signal transduction pathways of phytohormones by transcriptomics. Enzyme-linked immunosorbent assay (ELISA) and Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS) were used to quantify the phytohormone levels, and RNA-Seq was used to analyze the transcript abundance. The results showed that 23 transcripts involved in the phytohormone biosynthesis and 34 transcripts involved in the signal transduction pathways were significantly changed by RR treatment. RR also increased abscisic acid, brassinosteroid, ethylene, jasmonic acid and salicylic acid levels, while decreasing auxin, cytokinin, and gibberellin contents. The results of the present study suggest that RR treatment can accelerate the grape ripening process, and specific candidate genes were identified for further functional analysis.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

Grapevine (Vitis vinifera L.) fruit ripening is a complex biological process involving a phase transition from immature to mature. Understanding the molecular mechanism of fruit ripening is critical for grapevine fruit storage and quality improvement. However, the regulatory mechanism for the critical phase transition of fruit ripening from immature to mature in grapevine remains poorly understood. In this work, to identify the key molecular events controlling the critical phase transition of grapevine fruit ripening, we performed an integrated dynamic network analysis on time-series transcriptomic data of grapevine berry development and ripening. As a result, we identified the third time point as a critical transition point in grapevine fruit ripening, which is consistent with the onset of veraison reported in previous studies. In addition, we detected 68 genes as being key regulators involved in controlling fruit ripening. The GO (Gene Ontology) analysis showed that some of these genes participate in fruit development and seed development. This study provided dynamic network biomarkers for marking the initial transcriptional events that characterizes the transition process of fruit ripening, as well as new insights into fruit development and ripening.

Sucrose phloem unloading plays a vital role in photoassimilate distribution and storage in sink organs such as fruits and seeds. In most plants, the phloem unloading route was reported to shift between an apoplasmic and a symplasmic pattern with fruit development. However, the molecular transition mechanisms of the phloem unloading pathway still remain largely unknown. In this study, we applied RNA sequencing to profile the specific gene expression patterns for sucrose unloading in C. oleifera fruits in the apo- and symplasmic pathways that were discerned by CF fluoresce labelling. Several key structural genes were identified that participate in phloem unloading, such as PDBG11, PDBG14, SUT8, CWIN4, and CALS10. In particular, the key genes controlling the process were involved in callose metabolism, which was confirmed by callose staining. Based on the co-expression network analysis with key structural genes, a number of transcription factors belonging to the MYB, C2C2, NAC, WRKY, and AP2/ERF families were identified to be candidate regulators for the operation and transition of phloem unloading. KEGG enrichment analysis showed that some important metabolism pathways such as plant hormone metabolism, starch, and sucrose metabolism altered with the change of the sugar unloading pattern. Our study provides innovative insights into the different mechanisms responsible for apo- and symplasmic phloem unloading in oil tea fruit and represents an important step towards the omics delineation of sucrose phloem unloading transition in crops.

Abstract. The article considers gastronomic tourism and provides its definition. The peculiarities of gastrotourism in Europe are clarified, the experience of creating gastrotourism routes is analyzed, in particular the experience of Greece, Italy, Spain and Croatia in the creation of Olive Oil Roads is determined. Popular routes to Croatian and wine festivals in Slovenia are also explored. The experience of creating a gastronomic route "The Way of Wine and Taste of Ukrainian Bessarabia" is analyzed. Its prospects through the involvement of the Mykolaiv and Kherson regions are determined. Purpose. To clarify the definition of "gastronomic tourism", to study the European experience of creating gastronomic routes for further implementation in the South of Ukraine. Result. The study of world trends in gastronomic tourism allows to use them for the practice of developing the domestic tourist market, which will help grow the tourist image of the country, promote the tourist brand of the territory and increase tourist flows. Conclusions. Gastronomic tourism is a specialized type of tourism aimed at obtaining a complete picture of the country's perception by involving in the preparation of national dishes and drinks and their tasting. Tourist routes of small farms are very popular in Europe and are divided into: wine roads, taste roads, wine and taste roads - fish, cheese, honey olive, fruit and berry, etc. Gastronomic tourism will increase the tourist flow, as wine or craft farms can receive tourists all year round. According to the practice of Odessa region, despite the quarantine, the volumes of production in small farms, which became participants in the "Wine and Taste Road of Ukrainian Bessarabia", increased by 30%. The creation of a gastronomic route "The Road of Wine and Taste of the South of Ukraine", which unites Odessa, Mykolaiv and Kherson regions, is promising for the development of the Southern region. Creating a gastro route will promote the development of crafts, infrastructure, create jobs.

Orientia tsutsugamushi is an obligately intracellular bacterium with endothelial tropism and can cause mild to lethal scrub typhus in humans. No vaccine is available for this reemerging and severely neglected infection. Previous scrub typhus studies have utilized inbred mice, yet such models have intrinsic limitations. Thus, the development of suitable mouse models that better mimic human diseases is in great need for immunologic investigation and future vaccine studies. This study is aimed at establishing scrub typhus in outbred CD-1 mice and defining immune biomarkers related to disease severity. CD-1 mice received O. tsutsugamushi Karp strain via the i.v. route; major organs were harvested at 2–12 days post-infection for kinetic analyses. We found that for our given infection doses, CD-1 mice were significantly more susceptible (90–100% lethal) than were inbred C57BL/6 mice (0–10% lethal). Gross pathology of infected CD-1 mouse organs revealed features that mimicked human scrub typhus, including pulmonary edema, interstitial pneumonia, perivascular lymphocytic infiltrates, and vasculitis. Alteration in angiopoietin/receptor expression in inflamed lungs implied endothelial dysfunction. Lung immune gene profiling using NanoString analysis displayed a Th1/CD8-skewed, but Th2 repressed profile, including novel biomarkers not previously investigated in other scrub typhus models. Bio-plex analysis revealed a robust inflammatory response in CD-1 mice as evidenced by increased serum cytokine and chemokine levels, correlating with immune cell recruitment during the severe stages of the disease. This study provides an important framework indicating a value of CD-1 mice for delineating host susceptibility to O. tsutsugamushi, immune dysregulation, and disease pathogenesis. This preclinical model is particularly useful for future translational and vaccine studies for severe scrub typhus.

The aim of the present work was the development of an innovative in situ gelling system, to be applied on the mucosa of the distal colon via rectal route. The system consisted of three polymers having different functions: gellan (GG), able to jellify in presence of ions; methylcellulose (MC), a thermosensitive polymer with a gelation temperature close to 50 °C; and hydroxypropylcellulose (HPC), a mucoadhesive polymer. The three polymers were able to act synergistically, increasing the permanence of the vehicle on the mucosa and forming a protective gel layer. A DoE approach, “simplex centroid mixture design,” was used to identify the optimal quantitative composition of the vehicle. The response variables considered were: vehicle viscosity at room temperature; increase in vehicle viscosity on increasing temperature (from room to physiological value) and upon dilution with simulated colonic fluid (SCF); and viscoelastic behavior, thixotropic area, and mucoadhesion properties of the gel formed at 37 °C upon dilution in SCF. The optimized vehicle was loaded with maqui berry extract (MBE), known for its antioxidant and anti-inflammatory properties. MBE loading (0.5% w/w) into the vehicle improved rheological and mucoadhesive properties of the formulation. Both MBE and the optimized vehicle were not cytotoxic towards human fibroblasts and Caco-2 cells. Moreover, the optimized vehicle did not affect MBE antioxidant properties.

Abstract Objectives CD146 is an adhesive molecule that was originally reported on malignant melanoma cells as a protein crucial for cell adhesion. It is now known that high expression of the CD146 protein is not only characteristic of melanoma, but it occurs on a number of cancers, contributing to worse prognosis and increased aggressiveness. Independent in vitro studies in breast cancer have shown that CD146 protein alone can induce a change in epithelial to mesenchymal transcriptional profile, which is the basis of the tumor aggressiveness and metastasis. Methods In the following work, the correlation coefficients were analyzed between the genes of the mesenchymal profile and the CD146 gene in 10 independent transcriptomic data of breast cancer patients. Results The analysis confirmed the relationship between CD146 expression and mesenchymal profile genes, pointing VIMENTIN as the gene which expression is most strongly correlated with the CD146, suggesting that both genes, CD146 and VIM may be directly controlled by the same mechanism or regulate one another. Conclusions The analysis points a potential route for research on the CD146 gene expression, which may lead to understanding of its regulation in breast cancer, contributing to the development of new therapeutic strategies targeting highly metastatic breast cancer cells.

Inhalation is an important route of aldehyde exposure, and lung is one of the main targets of aldehyde toxicity. Octanal is distributed ubiquitously in the environment and is a component of indoor air pollutants. We investigated whether octanal exposure enhances the inflammatory response in the human respiratory system by increasing the expression and release of cytokines and chemokines. The effect of octanal in transcriptomic modulation was assessed in the human alveolar epithelial cell line A549 using oligonucleotide arrays. We identified a set of genes differentially expressed upon octanal exposure that may be useful for monitoring octanal pulmonary toxicity. These genes were classified according to the Gene Ontology functional category and Kyoto Encyclopedia of Genes and Genomes analysis to explore the biological processes related to octanal-induced pulmonary toxicity. The results show that octanal affects the expression of several chemokines and inflammatory cytokines and increases the levels of interleukin 6 (IL-6) and IL-8 released. In conclusion, octanal exposure modulates the expression of cytokines and chemokines important in the development of lung injury and disease. This suggests that inflammation contributes to octanal-induced lung damage and that the inflammatory genes expressed should be studied in detail, thereby laying the groundwork for future biomonitoring studies.

TPS4188 Background: The success rate of drug development in PDAC is disappointingly low.PrP is a transformative, adaptive platform clinical trial designed to continuously evaluate many novel therapeutic options while increasing the probability that patients (pts) are randomized to effective experimental therapies. It cultivates enhanced cooperation among groups representing pts advocacy, pharmaceutical companies, academia, and the FDA. This patient-centric study aims to become the largest Phase 3 registrational study in PDAC and represents a fundamental shift in drug development for PDAC in the United States (US). Methods: PrP (NCT04229004) is a platform clinical trial sponsored by the Pancreatic Cancer Action Network (PanCAN), developed based on the FDA 2020 guidance document regarding "complex innovative designs" in registration trials https://www.fda.gov/media/130897/download . It utilizes adaptive randomization and other Bayesian statistical innovations provided by Berry Consultants LLC, including the “time machine” which uses all previously randomized controls for each arm, suitably adjusted for line of therapy and the time period of the arm. Focused on 1st and 2nd line treatment of mPDAC, PrP uses an adaptive platform design with randomization to one of 2 control arms (gemcitabine + nab-paclitaxel (GA) or mFOLFIRINOX, 30% of pts) or experimental therapy (70% of pts). Candidate experimental arms are reviewed by an Arm Selection Committee based on validity of the treatment target and strength of the pre-clinical and clinical data. The primary endpoint is overall survival (OS). Pts undergo pre- and on-treatment biopsies with state-of-the-art genomic, transcriptomic, and immune analysis, along with a serial collection of blood-based research samples. Pts are managed using novel supportive care techniques; PrP contains 3 sub-protocols evaluating quality of life, sarcopenia, and actigraphy. PrP launched in 2020 and has enrolled > 130 pts; 30 US sites have been selected with 17 currently active. Current experimental arms include: (i) GA + Pamrevlumab, an anti-CTGF Ab, (ii) Racemetyrosine monotherapy, a cancer metabolism-based therapy (for follow-up of patients) and (iii) an immuno-oncology arm in activation. Other arms are in the planning stages. Compared to traditional designs, PrP offers several advantages: multiple investigational treatments evaluated in parallel over time; ̃175 pts per experimental arm required to initiate a regulatory registration; and continuous learning from every patient, resulting in significant savings of time and resources. PrP has created an entirely new learning environment for accelerating drug development in PDAC. Clinical trial information: NCT04229004.

Abstract Background: Bladder cancer is the fifth most common cancer in North America. The standard of care for high-risk non-muscle invasive bladder cancer (NMIBC) involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG). However, it is possible that the efficacy of this modality of BCG administration is suboptimal, as most patients do not respond fully to this immunotherapy. Furthermore, our current understanding of the immunotherapeutic effect of BCG is incomplete. Using a mouse model of NMIBC, we compared the tumor immune microenvironment (TiME) following intravesical versus intravenous administration of BCG, as well as changes in tumor volume and mice survival. Methods: Female C57Bl/6 mice (6-8 weeks old) were catheterized and instilled with 2.5x105 MB49 bladder cancer cells after poly-L-lysine treatment. Beginning on day 7, mice were treated with intravenous (IV) or intravesical BCG (8 mg/ml) or saline once weekly for three weeks. Ultrasound was performed weekly to monitor tumor growth. Similarly, a cohort of non-tumor bearing mice was treated with poly-L-lysine on day one followed by three weekly intravesical instillations of BCG. In both cohorts, mice were sacrificed on day 23 and bladders were harvested, enzymatically dispersed to generate single-cell suspensions for analysis by flow cytometry, or snap frozen for transcriptomic analysis using NanoString nCounter Platform. Results: Mice receiving IV BCG had better survival and their bladder tumors were significantly smaller compared with mice receiving intravesical BCG. This reduction in tumor size was associated with a significantly increased proportion of CD8+ T cells and a significantly reduced proportion of inflammatory monocytes in bladder tumors from mice treated with IV BCG compared with intravesical BCG. Whole tumor transcriptome analysis revealed alterations in various signalling pathways associated with route of BCG administration. Our results demonstrate similar trends in the distribution of immune populations in the TiME following intravesical saline and IV BCG treatment, as well as following IV saline and intravesical BCG treatment. We also found that the TiME after intravesical BCG treatment has more immune cells which are predominantly immature myeloid cells. Moreover, our results indicate that intravesical BCG treatment leads to a significant population of immature myeloid cells in the bladders of non-tumor bearing mice. Conclusion/Significance: These results provide evidence that the route of BCG administration is an important determinant of the TiME composition and may influence anti-tumor responses. Understanding the link between the TiME and BCG therapy may facilitate the development of new approaches to improve outcomes and reduce recurrence rates in patients with NMIBC. Citation Format: Aline Atallah, Arielle Grossman, Jean-Francois Pare, Robert Siemens, Tiziana Cotechini, Charles H. Graham. Effect of route of Bacillus Calmette Guerin administration on the immune microenvironment and growth of bladder tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3540.

Abstract Despite advances in the genetic characterization of chronic myelomonocytic leukemia (CMML), the molecular mechanisms that drive the disease during its distinct phases remain unclear. To uncover vulnerabilities in CMML that could be therapeutically targeted to halt its evolution, we sought to dissect at the single-cell level the cellular and transcriptomic changes that occur in the hematopoietic system at the time of CMML's initiation and its progression after hypomethylating agent (HMA) therapy. To evaluate the molecular mechanisms underlying CMML maintenance, we performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin -)CD34 + hematopoietic stem and progenitor cells (HSPCs) and bone marrow (BM) mononuclear cells (MNCs) isolated from untreated CMML patients (n=5 and 6, respectively) and age-matched healthy donors (HDs; n=2 and 3, respectively). Our integrated analysis revealed that CMML Lin -CD34 + HSPCs had a predominant granulomonocytic differentiation route with an increased frequency of early and committed myeloid-monocytic progenitors at the expense of HSCs and megakaryocyte/erythroid progenitors (Fig. 1a). Differential expression analysis among the clusters revealed that most transcriptomic differences occurred in CMML HSCs, which were characterized by the upregulation of genes involved in oxidative phosphorylation, type I interferon (IFN) and IFNγ response, myeloid development, and inflammatory signaling and had downregulated expression of genes involved in TNFα-mediated NF-κB activation (Fig. 1b). These data suggest that CMML HSCs undergo metabolic reprogramming and demand a higher level of mitochondrial activity to maintain activated monocytic differentiation in response to inflammatory signaling. Consistent with these results, scRNA-seq analysis of MNCs isolated from the same HD and CMML BM samples showed that monocytes were significantly increased at the expense of erythroid precursors and B cells in CMML (Fig. 1c). CMML monocytes had upregulated genes involved in IFNγ response, oxidative phosphorylation, MYC targets, NF-κB activation, and inflammation (e.g., S100A9, CCL3, IL1B). Interestingly, among the anti-apoptotic BCL2 family members, only the NF-κB transcriptional target BCL2A1 was significantly overexpressed. To investigate the mechanisms of resistance to HMA therapy, we performed integrated scRNA-seq analysis of sequential Lin -CD34 + cells and BM MNCs isolated from CMML patients at the time of disease initiation and progression after HMA therapy failure. CMML progression was driven by a significant expansion of lympho-myeloid progenitors (LMPPs) at the expense of earlier HSCs , which exacerbated myelomonocytic differentiation in the HSPC compartment (Fig. 1d). Expanded LMPPs were characterized by higher levels of IFNγ response, NF-κB survival signaling, and cell cycle regulators. Accordingly, scRNA-seq analysis of MNCs cells from the same patients showed significantly increased frequencies of monocytes and a reduction of naïve CD4 +/CD8 + T cells and effector memory CD8 + T cells. Differential expression analysis of the 2 sample groups in the monocyte population identified five different cellular clusters, one of which emerged only at progression (Fig. 1e). This population was characterized by high expression levels of inflammatory cytokines and the anti-apoptotic modulators MCL1 and BCL2A1. Together, these data suggest that CMML progression arises from immature myeloid progenitors at the stem cell level and that downstream monocytes undergo transcriptomic rewiring and acquire survival mechanisms that induce therapy resistance and further accelerate disease progression. In conclusion, our results elucidate the differentiation hierarchies and transcriptional programs associated with CMML's initiation and its progression after HMA therapy. Our data suggest that therapies targeting downstream effectors of NF-kB-mediated survival signaling could overcome treatment failure. Figure 1 Figure 1. Disclosures Wei: Daiichi Sanko: Research Funding. Kantarjian: AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; KAHR Medical Ltd: Honoraria; Jazz: Research Funding; Ipsen Pharmaceuticals: Honoraria; Astellas Health: Honoraria; NOVA Research: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astra Zeneca: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Abstract Background: Drug development in PDAC has been disappointing with an extremely low trial success rate despite considerable effort. PrP is a transformative, adaptive clinical trial platform that attempts to correct this by continuously and rapidly evaluating novel therapeutic options while maximizing the probability of patient (pt) randomization to an experimental treatment and nurturing enhanced cooperation among groups representing pt advocacy, pharmaceuticals, translational/clinical academia, and the FDA. This patient-centric study represents a fundamental shift in drug development for PDAC in the United States and aims to become the largest Phase 2/3 registrational study ever launched in this disease. Methods: PrP (NCT04229004) is a clinical trial platform sponsored by the Pancreatic Cancer Action Network (PanCAN) and funded solely through non-government sources. The protocol was finalized based on the FDA 2020 guidance document regarding "complex innovative designs" in registration trials https://www.fda.gov/media/130897/download. It utilizes adaptive randomization along with several trial design and Bayesian statistical innovations provided by Berry Consultants LLC. All pts undergo pre-and on-treatment biopsies with state-of-the-art genomic, transcriptomic, and immune analysis, along with collection of blood samples for research purposes throughout the study. Pts are managed using novel standardized supportive care techniques, and PrP contains 3 sub-protocols involving quality of life, sarcopenia and actigraphy. PrP was launched in 2020, and currently includes 20 US sites. Focused on both 1st and 2nd line treatment of metastatic PDAC, PrP uses an adaptive platform design with 30% of pts randomized between one of the 2 standard of care control arms (gemcitabine + nab-paclitaxel and mFOLFIRINOX) and 70% to experimental arms, currently either SM-88, a cancer metabolism-based agent (Tyme Inc); or Pamrevlumab, an antibody inhibiting the activity of the connective tissue growth factor (Fibrogen Inc.) The study is ongoing with &gt;100 pts enrolled to date. The Data and Safety Monitoring Committee regularly reviews the data and continues to recommend that the trial proceeds as planned. New study arms will be added after review by an Arm Selection Committee that assesses the validity of the treatment target and the adequacy of the preexisting pre-clinical and clinical data. An additional experimental arm is anticipated in 2021. Conclusion: Compared to traditional trial designs, PrP offers several advantages: multiple investigational treatments can be evaluated in parallel over time; only ~ 175 pts per experimental arm required to initiate a regulatory registration; and increased learning from every patient during the trial, altogether resulting in both time saving and a 30-50% cost saving. In effect, PrP has created an entirely new “learning community” and can substantially accelerate drug development for PDAC. Citation Format: Vincent J. Picozzi, Anne-Marie Duliege, Anirban Maitra, Manuel Hidalgo, Andrew Eugene Hendifar, Gregory L. Beatty, Sudheer Doss Doss, Regina Deck, Lynn M. Matrisian, Julie Fleshman, Diane M. Simeone. Precision Promise (PrP): An adaptive, multi-arm registration trial in metastatic pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-050.

Мониторинговые исследования за растительными объектами позволяют получить массив данных по особенностям роста, фенологии, развитию генеративных органов, характеру плодоношения, качеству семян, устойчивости к стресс-факторам, т.е. по показателям, которые находятся в функциональной зависимости между собой и позволяют судить о степени экологической пластичности растений. Для обоснования перспективности видов рода Juniperus, дальнейшего прогнозирования уровня адаптивности видов за пределами естественного ареала необходима достоверная и актуальная информация об успешности адаптации интродуцентов в условиях урбанизированных территорий малолесных регионов. Цель исследований – обоснование перспективности использования видов рода Juniperus на основе изучения их биологического потенциала в условиях интродукции. Объектами исследований являлись виды рода Juniperus: J. virginiana L., J. sabina L., J. communis L. и их формы, интродуцированные в дендрологических коллекциях Волгоградской области: ФНЦ агроэкологии РАН, кадастр №34:34:000000:122, 34:34:060061:10 и Нижневолжской станции по селекции древесных пород, № 34:36:0000:14:0178. Обследование проводилось маршрутным методом. Изучение сезонных ритмов развития интродуцентов проводилось методом фенологических наблюдений. Характеристика декоративности, роста и развития трех видов Juniperus L. (J. sabina L., J. virginiana L., J. communis L.) в условиях каштановых почв Волгоградской области, оценка репродуктивной способности, особенности размножения различных видов в культуре выявлялись по методикам ФНЦ агроэкологии РАН. Для определения характера плодоношения объектов исследования использовался метод определения массы 1000 семян, взвешивание проводили на весах марки «MASSA-K». Для математической обработки данных использовали стандартные алгоритмы: средняя арифметическая с абсолютной и относительной ошибками; коэффициент вариации для оценки особенностей репродуктивных процессов; достоверность различий между отдельными показателями. Приведена характеристика декоративности, роста и развития трех видов Juniperus L. (J. sabina L., J. virginiana L., J. communis L.) в условиях каштановых почв Волгоградской области. Дана оценка репродуктивной способности, выявлены особенности размножения различных видов в культуре. По комплексу изученных количественных показателей семенного материала установлено, что семена можжевельника виргинского (Juniperus virginiana L.) (10 г) меньше по массе семян обыкновенного практически в полтора раза; масса семян можжевельника казацкого (Juniperus sabina L.) (24 г) больше по массе семян можжевельника обыкновенного в полтора раза и почти в два с половиной раза больше можжевельника виргинского в сравнении с нормативными данными по можжевельнику обыкновенному (масса 1000 шт. которых составила 16 г). Изучение характера плодоношения можжевельника виргинского (J. virginiana) показало, что в 10,0 граммах шишкоягод содержится в среднем 156 ягод и 270 шт семян, выход чистых семян – 15,84 % от массы плодов. В 10,0 граммах шишкоягод можжевельника казацкого (J. sabina) содержится в среднем 86 ягод и 170 шт семян, выход чистых семян – 20,09 % от массы плодов. Установлено, что для озеленения урбанизированных территорий Волгоградской области Juniperus virginiana рекомендуется использовать в аллейных насаждениях, в чистых группах и в качестве второго яруса в насаждениях Betula, Robinia, Pseudotsuga, Larix. Juniperus sabina наиболее ценен для декорирования и укрепления склонов. В садах и парках общего пользования его применение ограничивается, а при озеленении детских учреждений исключается (хвоя и плоды ядовиты). В Волгоградской области Juniperus communis и его формы могут быть использованы на бедных песчаных почвах в групповых посадках, на опушках и в формованных живых изгородях. Пирамидальные формы пригодны на партерах, карликовые – для оформления каменистых участков. Форма с золотистой окраской в небольших группах на газоне. Monitoring studies of plant objects provide an array of data on the characteristics of growth, phenology, the development of generative organs, the specifics of fruiting, seed quality, and resistance to stress factors, i.e. indicators that are in a functional relationship with each other and allow estimating the degree of environmental plasticity of plants. To justify the prospects of species of the Juniperus genus, forecast the adaptability level of species outside the natural range, reliable and relevant information on the success of adaptation of introduced plants in urbanized areas of sparsely wooded regions is necessary. The purpose of the research is to justify the prospects of using species of the Juniperus genus based on the study of their biological potential under the conditions of introduction. The objects of research were species of the Juniperus genus: J. virginiana L., J. sabina L., J. communis L. and their forms introduced in dendrological collections of the Volgograd Region: Federal Research Center of Agroecology of the Russian Academy of Sciences, Cadastre No. 34:34:000000:122, 34:34:060061:10 and the Lower Volga tree breeding station, No. 34:36 0000:14:0178. The survey was conducted by the route method. The seasonal development rhythms of introduced plants were studied by the method of phenological observations. The characteristics of decorativeness, growth, and development of three species of Juniperus L. (J. sabina L., J. virginiana L., J. communis L.) in chestnut soils of the Volgograd Region, the assessment of reproductive ability, and the reproduction specifics of various cultivated species culture were revealed according to the methods of the Federal Scientific Center of Agroecology of the Russian Academy of Sciences. To determine the fruiting specifics of the study objects, the method of determining the mass of 1000 seeds was used; weighing was performed using a MASSA-K brand balance. For mathematical data processing, standard algorithms were used: the arithmetic mean with absolute and relative errors; the coefficient of variation to assess the characteristics of reproductive processes; the significance of differences between individual indicators. The characteristics of decorativeness, growth, and development of three species of Juniperus L. (J. sabina L., J. virginiana L., J. communis L.) in chestnut soils of the Volgograd Region are described. An assessment of reproductive ability is given, the features of reproduction of various cultivate species are revealed. According to the complex of the studied quantitative indicators of the seed material, it was found that the weight of seeds of Virginian juniper (Juniperus virginiana L.) (10 g) was almost half of the weight of common juniper seeds; the weight of the seeds of savin juniper (Juniperus sabina L.) (24 g) exceeded the weight of the seeds of common juniper by 1.5 times and that of Virginian juniper – by 2.5 times, in comparison with the standard data for common juniper (weight of 1000 pcs. – 16 g). A study of the fruiting specifics of Virginian juniper (J. virginiana) showed that 10.0 grams of conifer berries contained an average of 156 berries and 270 seeds, the yield of pure seeds was 15.84% of the fruit weight; 10.0 grams of cone berry of savin juniper (J. sabina) contained an average of 86 berries and 170 seeds, the yield of pure seeds was 20.09% of the fruit weight. It has been established that for landscaping the urban areas of the Volgograd Region, Juniperus virginiana is recommended to be used in alley stands, in clean groups, and as a second tier in the stands of Betula, Robinia, Pseudotsuga, Larix. Juniperus sabina is most valuable for decorating and strengthening slopes. In gardens and public parks, its use is limited, and when planting children's institutions, it is excluded (needles and fruits are poisonous). In the Volgograd Region, Juniperus communis and its forms can be used on poor sandy soils in group plantings, at the edges, and in molded hedges. Pyramidal forms are suitable in the level spaces, dwarf forms – for the design of rocky areas. The form with a golden color can be used in small groups on lawns.

Abstract Background High temperature during grape berry ripening impairs the quality of fruits and wines. Veraison time, which marks ripening onset, is a key factor for determining climatic conditions during berry ripening. Understanding its genetic control is crucial to successfully breed varieties more adapted to a changing climate. Quantitative trait loci (QTL) studies attempting to elucidate the genetic determinism of developmental stages in grapevine have identified wide genomic regions. Broad scale transcriptomic studies, by identifying sets of genes modulated during berry development and ripening, also highlighted a huge number of putative candidates. Results With the final aim of providing an overview about available information on the genetic control of grapevine veraison time, and prioritizing candidates, we applied a meta-QTL analysis for grapevine phenology-related traits and checked for co-localization of transcriptomic candidates. A consensus genetic map including 3130 markers anchored to the grapevine genome assembly was compiled starting from 39 genetic maps. Two thousand ninety-three QTLs from 47 QTL studies were projected onto the consensus map, providing a comprehensive overview about distribution of available QTLs and revealing extensive co-localization especially across phenology related traits. From 141 phenology related QTLs we generated 4 veraison meta-QTLs located on linkage group (LG) 1 and 2, and 13 additional meta-QTLs connected to the veraison time genetic control, among which the most relevant were located on LG 14, 16 and 18. Functional candidates in these intervals were inspected. Lastly, taking advantage of available transcriptomic datasets, expression data along berry development were integrated, in order to pinpoint among positional candidates, those differentially expressed across the veraison transition. Conclusion Integration of meta-QTLs analysis on available phenology related QTLs and data from transcriptomic dataset allowed to strongly reduce the number of candidate genes for the genetic control of the veraison transition, prioritizing a list of 272 genes, among which 78 involved in regulation of gene expression, signal transduction or development.

Abstract Background Grapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars ‘Pinot Noir’ (PN) and ‘Pinot Noir Precoce’ (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years. Results The difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designated VviRTIC1 and VviRTIC2 (VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP. Conclusions Many of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.

Abstract In viticulture, grafting has been practiced widely and influences grape development as well as berry and wine quality. However, limited knowledge is present about the effects of rootstocks on grape phenolic compounds, which locate primarily in the berry skin and contribute to certain sensory attributes of wine. In this study, scion-rootstock interactions were investigated at green-berry stage and veraison stage when grapevines were hetero-grafted with three commonly used rootstock genotypes (5BB, 101-14MG, and SO4). The physiological investigations showed that hetero-grafts especially for CS/5BB presented higher concentrations of total proanthocyanidins (PAs) and various PA components in the berry skins when compared to the auto-grafted grapevines. Further metabolomics analysis identified 105 differentially accumulated flavonoid compounds, and the majority of them including anthocyanins, PAs and flavonols were significantly increased in the berry skins of hetero-grafted grapevines compared to the auto-grafted control. In addition, transcriptomic analysis using the same samples totally identified several thousand differentially expressed genes between hetero-grafted and auto-grafted vines. The three rootstocks not only increased the transcription levels of stilbene, anthocyanin, PA, and flavonol synthetic genes, but also affected the transcript abundance of a large number of transcription factors. Taken together, our results jointly supported that hetero-grafting could promote phenolic compound accumulation in the grape berry skin during development, which provide new insights for improving the application value of grafting by enhancing the accumulation of the nutritious phenolic components in the grape.

Abstract Background Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds. Results To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige. Conclusions We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.

AbstractGrapevine (Vitis vinifera) is widely applicated in food industry, which shows high economical and nutritional values. However, growth of grapevine was usually affected by various environmental stresses, such as salt, drought and disease. Ubiquitin fusion degradation protein 1 (UFD1) is an essential ubiquitin-recognition protein facilitates regulation of stress response through ERAD pathway. Even though, a comprehensive investigation of UFD1 genes in the plant species is still lacking. Here we identified three VvUFD1 proteins from genome of grapevine, which were assigned into different subgroups. All VvUFD1 genes contain highly conserved motifs in structure. Several cis-elements that related to fruit development and stress response were found in the promoter regions of VvUFD1 genes, including bHLH, NCA, MYB, HD-ZIP, GATA and AP2. Expression analysis found VvUFD1 genes showed different expression patterns in different tissues. Most importantly, VvUFD1 genes were found to be involved in salt stress response during growth of grapevine. Transcriptomic analyses were investigated for further understanding the genes’ function. Expression of VvUFD1 were increased at late stage of berry ripening. In addition, expression of VvUFD1 were also regulated by elevated light treatment and pathogen Neofusicoccum parvum infection. Co-expression network analysis revealed several major transcription factors that co-expressed with VvUFD1 genes. These results provide a basis for investigating the function of UFD1 genes in plant species and expand understanding of the regulation of berry development and salt stress response in grapevine.

AbstractBackgroundGrapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections.ResultsWe identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data.ConclusionsOur observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, theVvAGL11mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

AbstractOzonated water has become an innovative, environmentally friendly tool for controlling the development of fungal diseases in the vineyard or during grape postharvest conservation. However, little information is currently available on the effects of ozonated water sprayings on the grapevine physiology and metabolism. Using the microvine model, we studied the transcriptomic response of leaf and fruit organs to this treatment. The response to ozone was observed to be organ and developmental stage-dependent, with a decrease of the number of DEGs (differentially expressed genes) in the fruit from the onset of ripening to later stages. The most highly up-regulated gene families were heat-shock proteins and chaperones. Other up-regulated genes were involved in oxidative stress homeostasis such as those of the ascorbate–glutathione cycle and glutathione S-transferases. In contrast, genes related to cell wall development and secondary metabolites (carotenoids, terpenoids, phenylpropanoids / flavonoids) were generally down-regulated after ozone treatment, mainly in the early stage of fruit ripening. This down-regulation may indicate a possible carbon competition favouring the re-establishment and maintenance of the redox homeostasis rather than the synthesis of secondary metabolites at the beginning of ripening, the most ozone responsive developmental stage.

Abstract Pearl of Csaba (PC) is a valuable backbone parent for early-ripening grapevine (Vitis vinifera) breeding, from which many excellent early-ripening varieties have been bred. However, the genetic basis of the stable inheritance of its early-ripening trait remains largely unknown. Here, the pedigree, consisting of 40 varieties derived from PC, was re-sequenced for an average depth of ∼30×. Combined with the resequencing data of 24 other late-ripening varieties, 5,795,881 high-quality single nucleotide polymorphisms (SNPs) were identified following a strict filtering pipeline. The population genetic analysis showed that these varieties could be distinguished clearly, and the pedigree was characterized by lower nucleotide diversity and stronger linkage disequilibrium than the non-pedigree varieties. The conserved haplotypes (CHs) transmitted in the pedigree were obtained via identity-by-descent analysis. Subsequently, the key genomic segments were identified based on the combination analysis of haplotypes, selective signatures, known ripening-related quantitative trait loci (QTLs) and transcriptomic data. The results demonstrated that varieties with a superior haplotype, H1, significantly (one-way ANNOVA, p &lt; 0.001) exhibited early grapevine berry development. Further analyses indicated that H1 encompassed VIT_16s0039g00720 encoding a folate/biopterin transporter protein (VvFBT) with a missense mutation. VvFBT was specifically and highly expressed during grapevine berry development, particularly at veraison. Exogenous folate treatment advanced the veraison of ‘Kyoho’. This work uncovered core haplotypes and genomic segments related to the early-ripening trait of PC and provided an important reference for the molecular breeding of early-ripening grapevine varieties.

Separation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the “Double negative” (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterize cells based on their transcriptome. To maximize immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamic transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. A T-independent branch that involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other, T-dependent, branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and closely linked to the classical/precursor memory branch suggesting an IgE specific T-dependent cell population.

Leaves are the primary photosynthetic structures, while photosynthesis is the direct motivation of crop yield formation. As a legume plant, peanut (Arachis hypogaea) is one of the most economically essential crops as well as an important source of edible oil and protein. The leaves of A. hypogaea are in the tetrafoliate form, which is different from the trifoliate leaf pattern of Medicago truncatula, a model legume species. In A. hypogaea, an even-pinnate leaf with a pair of proximal and distal leaflets was developed; however, only a single terminal leaflet and a pair of lateral leaflets were formed in the odd-pinnate leaf in M. truncatula. In this study, the development of compound leaf in A. hypogaea was investigated. Transcriptomic profiles revealed that the common and unique differentially expressed genes were identified in a proximal leaflet and a distal leaflet, which provided a research route to understand the leaf development in A. hypogaea. Then, a naturally occurring mutant line with leaf developmental defects in A. hypogaea was obtained, which displayed a pentafoliate form with an extra terminal leaflet. The characterization of the mutant indicated that cytokinin and class I KNOTTED-LIKE HOMEOBOX were involved in the control of compound leaf pattern in A. hypogaea. These results expand our knowledge and provide insights into the molecular mechanism underlying the formation of different compound leaf patterns among species.

Transcriptomics and metabolomics are methodologies being increasingly chosen to perform molecular studies in grapevine (Vitis vinifera L.), focusing either on plant and fruit development or on interaction with abiotic or biotic factors. Currently, the integration of these approaches has become of utmost relevance when studying key plant physiological and metabolic processes. The results from these analyses can undoubtedly be incorporated in breeding programs whereby genes associated with better fruit quality (e.g., those enhancing the accumulation of health-promoting compounds) or with stress resistance (e.g., those regulating beneficial responses to environmental transition) can be used as selection markers in crop improvement programs. Despite the vast amount of data being generated, integrative transcriptome/metabolome meta-analyses (i.e., the joint analysis of several studies) have not yet been fully accomplished in this species, mainly due to particular specificities of metabolomic studies, such as differences in data acquisition (i.e., different compounds being investigated), unappropriated and unstandardized metadata, or simply no deposition of data in public repositories. These meta-analyses require a high computational capacity for data mining a priori, but they also need appropriate tools to explore and visualize the integrated results. This perspective article explores the universe of omics studies conducted in V. vinifera, focusing on fruit-transcriptome and metabolome analyses as leading approaches to understand berry physiology, secondary metabolism, and quality. Moreover, we show how omics data can be integrated in a simple format and offered to the research community as a web resource, giving the chance to inspect potential gene-to-gene and gene-to-metabolite relationships that can later be tested in hypothesis-driven research. In the frame of the activities promoted by the COST Action CA17111 INTEGRAPE, we present the first grapevine transcriptomic and metabolomic integrated database (TransMetaDb) developed within the Vitis Visualization (VitViz) platform (https://tomsbiolab.com/vitviz). This tool also enables the user to conduct and explore meta-analyses utilizing different experiments, therefore hopefully motivating the community to generate Findable, Accessible, Interoperable and Reusable (F.A.I.R.) data to be included in the future.

Abstract Epithelial–mesenchymal transition (EMT) is a cellular process involved in development and disease progression. Intermediate EMT states were observed in tumors and fibrotic tissues, but previous in vitro studies focused on time-dependent responses with single doses of signals; it was unclear whether single-cell transcriptomes support stable intermediates observed in diseases. Here, we performed single-cell RNA-sequencing with human mammary epithelial cells treated with multiple doses of TGF-β. We found that dose-dependent EMT harbors multiple intermediate states at nearly steady state. Comparisons of dose- and time-dependent EMT transcriptomes revealed that the dose-dependent data enable higher sensitivity to detect genes associated with EMT. We identified cell clusters unique to time-dependent EMT, reflecting cells en route to stable states. Combining dose- and time-dependent cell clusters gave rise to accurate prognosis for cancer patients. Our transcriptomic data and analyses uncover a stable EMT continuum at the single-cell resolution, and complementary information of two types of single-cell experiments.

Abstract Background A major route for cell-to-cell signalling in plants is mediated by cell wall-embedded pores termed plasmodesmata forming the symplasm. Plasmodesmata regulate the plant development and responses to the environment; however, our understanding of what factors or regulatory cues affect their structure and permeability is still limited. In this paper, a meta-analysis was carried out for the identification of conditions affecting plasmodesmata transport and for the in silico prediction of plasmodesmata proteins in species for which the plasmodesmata proteome has not been experimentally determined. Results Using the information obtained from experimental proteomes, an analysis pipeline (named plasmodesmata in silico proteome 1 or PIP1) was developed to rapidly generate candidate plasmodesmata proteomes for 22 plant species. Using the in silico proteomes to interrogate published transcriptomes, gene interaction networks were identified pointing to conditions likely affecting plasmodesmata transport capacity. High salinity, drought and osmotic stress regulate the expression of clusters enriched in genes encoding plasmodesmata proteins, including those involved in the metabolism of the cell wall polysaccharide callose. Experimental determinations showed restriction in the intercellular transport of the symplasmic reporter GFP and enhanced callose deposition in Arabidopsis roots exposed to 75-mM NaCl and 3% PEG (polyethylene glycol). Using PIP1 and transcriptome meta-analyses, candidate plasmodesmata proteins for the legume Medicago truncatula were generated, leading to the identification of Medtr1g073320, a novel receptor-like protein that localises at plasmodesmata. Expression of Medtr1g073320 affects callose deposition and the root response to infection with the soil-borne bacteria rhizobia in the presence of nitrate. Conclusions Our study shows that combining proteomic meta-analysis and transcriptomic data can be a valuable tool for the identification of new proteins and regulatory mechanisms affecting plasmodesmata function. We have created the freely accessible pipeline PIP1 as a resource for the screening of experimental proteomes and for the in silico prediction of PD proteins in diverse plant species.

IntroductionMyocardial infarction (MI) is a deadly medical condition leading to irreversible damage to the inflicted cardiac tissue. Elevated inflammatory response marks the severity of MI and is associated with the development of heart failure (HF), a long-term adverse outcome of MI. However, the efficacy of anti-inflammatory therapies for MI remains controversial. Deciphering the dynamic transcriptional signatures in peripheral blood mononuclear cells (PBMCs) is a viable and translatable route to better understand post-MI inflammation, which may help guide post-MI anti-inflammatory treatments.MethodsIn this work, integrated whole-genome gene expression analysis was performed to explore dynamic immune landscapes associated with MI.ResultsGSEA and GSVA showed that pathways involved in the inflammatory response and metabolic reprogramming were significantly enriched in PBMCs from MI patients. Based on leukocyte profiles generated by xCell algorithm, the relative abundance of monocytes and neutrophils was significantly increased in PBMCs from MI patients and had positive correlations with typical inflammation-associated transcripts. Mfuzz clustering revealed temporal gene expression profiles of PBMCs during the 6-month post-MI follow-up. Analysis of DEGs and gene sets indicated that PBMCs from HF group were characterized by elevated and lasting expression of genes implicated in inflammation and coagulation. Consensus clustering generated 4 metabolic subtypes of PBMCs with molecular heterogeneity in HF patients.DiscussionIn summary, integrated whole-genome gene expression analysis here outlines a transcriptomic framework that may improve the understanding of dynamic signatures present in PBMCs, as well as the heterogeneity of PBMCs in MI patients with or without long-term clinical outcome of HF. Moreover, the work here uncovers the diversity and heterogeneity of PBMCs from HF patients, providing novel bioinformatic evidence supporting the mechanistic implications of metabolic reprogramming and mitochondrial dysfunction in the post-MI inflammation and HF. Therefore, our work here supports the notion that individualized anti-inflammatory therapies are needed to improve the clinical management of post-MI patients.

Abstract Administration of recombinant glial cell line-derived neurotrophic factor into the putamen has been tested in preclinical and clinical studies to evaluate its neuroprotective effects on the progressive dopaminergic neuronal degeneration that characterizes Parkinson’s disease. However, intracerebral glial cell line-derived neurotrophic factor infusion is a challenging therapeutic strategy, with numerous potential technical and medical limitations. Most of these limitations could be avoided if the production of endogenous glial cell line-derived neurotrophic factor could be increased. Glial cell line-derived neurotrophic factor is naturally produced in the striatum from where it exerts a trophic action on the nigrostriatal dopaminergic pathway. Most of striatal glial cell line-derived neurotrophic factor is synthesized by a subset of GABAergic interneurons characterized by the expression of parvalbumin. We sought to identify molecular targets specific to those neurons and which are putatively associated with glial cell line-derived neurotrophic factor synthesis. To this end, the transcriptomic differences between glial cell line-derived neurotrophic factor-positive parvalbumin neurons in the striatum and parvalbumin neurons located in the nearby cortex, which do not express glial cell line-derived neurotrophic factor, were analysed. Using mouse reporter models, we have defined the genomic signature of striatal parvalbumin interneurons obtained by fluorescence-activated cell sorting followed by microarray comparison. Short-listed genes were validated by additional histological and molecular analyses. These genes code for membrane receptors (Kit, Gpr83, Tacr1, Tacr3, Mc3r), cytosolic proteins (Pde3a, Crabp1, Rarres2, Moxd1) and a transcription factor (Lhx8). We also found the proto-oncogene cKit to be highly specific of parvalbumin interneurons in the non-human primate striatum, thus highlighting a conserved expression between species and suggesting that specific genes identified in mouse parvalbumin neurons could be putative targets in the human brain. Pharmacological stimulation of four G-protein-coupled receptors enriched in the striatal parvalbumin interneurons inhibited Gdnf expression presumably by decreasing cyclic adenosine monophosphate formation. Additional experiments with pharmacological modulators of adenylyl cyclase and protein kinase A indicated that this pathway is a relevant intracellular route to induce Gdnf gene activation. This preclinical study is an important step in the ongoing development of a specific pro-endo-glial cell line-derived neurotrophic factor pharmacological strategy to treat Parkinson’s disease.

IntroductionThe walking tour is an enduring feature of cities. Fuelled by a desire to learn more about the hidden and unknown spaces of the city, the walking tour has moved beyond its historical role as tourist attraction to play a key role in the transformation of urban space through gentrification. Conversely, the walking tour has a counter-history as part of a critical urban praxis. This article reflects on historical examples, as well as our own experience of conducting Field Trip, a critical geographical walking tour through an industrial precinct in Marrickville, a suburb of Sydney that is set to undergo rapid change as a result of high-rise residential apartment construction (Gibson et al.). This precinct, known as Carrington Road, is located on the unceded land of the Cadigal and Wangal people of the Eora nation who call the area Bulanaming.Drawing on a long history of philosophical walking, many contemporary writers (Solnit; Gros; Bendiner-Viani) have described walking as a practice that can open different ways of thinking, observing and being in the world. Some have focused on the value of walking to the study of place (Hall; Philips; Heddon), and have underscored its relationship to established research methods, such as sensory ethnography (Springgay and Truman). The work of Michel de Certeau pays particular attention to the relationship between walking and the city. In particular, the concepts of tactics and strategy have been applied in a variety of ways across cultural studies, cultural geography, and urban studies (Morris). In line with de Certeau’s thinking, we view walking as an example of a tactic – a routine and often unconscious practice that can become a form of creative resistance.In this sense, walking can be a way to engage in and design the city by opposing its structures, or strategies. For example, walking in a city such as Sydney that is designed for cars requires choosing alternative paths, redirecting flows of people and traffic, and creating custom shortcuts. Choosing pedestrianism in Sydney can certainly feel like a form of resistance, and we make the argument that Field Trip – and walking tours more generally – can be a way of doing this collectively, firstly by moving in opposite directions, and secondly, at incongruent speeds to those for whom the scale and style of strategic urban development is inevitable. How such tactical walking relates to the design of cities, however, is less clear. Walking is a generally described in the literature as an individual act, while the design of cities is, at its best participatory, and always involving multiple stakeholders. This reveals a tension between the practice of walking as a détournement or appropriation of urban space, and its relationship to existing built form. Field Trip, as an example of collective walking, is one such appropriation of urban space – one designed to lead to more democratic decision making around the planning and design of cities. Given the anti-democratic, “post-political” nature of contemporary “consultation” processes, this is a seemingly huge task (Legacy et al.; Ruming). We make the argument that Field Trip – and walking tours more generally – can be a form of collective resistance to top-down urban planning.By using an open-source wiki in combination with the Internet Archive, Field Trip also seeks to collectively document and make public the local knowledge generated by walking at the frontier of gentrification. We discuss these digital choices as oppositional practice, and consider the idea of tactical media (Lovink and Garcia; Raley) in order to connect knowledge sharing with the practice of walking.This article is structured in four parts. Firstly, we provide a historical introduction to the relationship between walking tours and gentrification of global cities. Secondly, we examine the significance of walking tours in Sydney and then specifically within Marrickville. Thirdly, we discuss the Field Trip project as a citizen-led walking tour and, finally, elaborate on its role as tactical media project and offer some conclusions.The Walking Tour and Gentrification From the outset, people have been walking the city in their own ways and creating their own systems of navigation, often in spite of the plans of officialdom. The rapid expansion of cities following the Industrial Revolution led to the emergence of “imaginative geographies”, where mediated representations of different urban conditions became a stand-in for lived experience (Steinbrink 219). The urban walking tour as mediated political tactic was utilised as far back as Victorian England, for reasons including the celebration of public works like the sewer system (Garrett), and the “othering” of the working class through upper- and middle-class “slum tourism” in London’s East End (Steinbrink 220). The influence of the Situationist theory of dérive has been immense upon those interested in walking the city, and we borrow from the dérive a desire to report on the under-reported spaces of the city, and to articulate alternative voices within the city in this project. It should be noted, however, that as Field Trip was developed for general public participation, and was organised with institutional support, some aspects of the dérive – particularly its disregard for formal structure – were unable to be incorporated into the project. Our responsibility to the participants of Field Trip, moreover, required the imposition of structure and timetable upon the walk. However, our individual and collective preparation for Field Trip, as well as our collective understanding of the area to be examined, has been heavily informed by psychogeographic methods that focus on quotidian and informal urban practices (Crosby and Searle; Iveson et al).In post-war American cities, walking tours were utilised in the service of gentrification. Many tours were organised by real estate agents with the express purpose of selling devalorised inner-city real estate to urban “pioneers” for renovation, including in Boston’s South End (Tissot) and Brooklyn’s Park Slope, among others (Lees et al 25). These tours focused on a symbolic revalorisation of “slum neighbourhoods” through a focus on “high culture”, with architectural and design heritage featuring prominently. At the same time, urban socio-economic and cultural issues – poverty, homelessness, income disparity, displacement – were downplayed or overlooked. These tours contributed to a climate in which property speculation and displacement through gentrification practices were normalised. To this day, “ghetto tours” operate in minority neighbourhoods in Brooklyn, serving as a beachhead for gentrification.Elsewhere in the world, walking tours are often voyeuristic, featuring “locals” guiding well-meaning tourists through the neighbourhoods of some of the world’s most impoverished communities. Examples include the long runningKlong Toei Private Tour, through “Bangkok’s oldest and largest slum”, or the now-ceased Jakarta Hidden Tours, which took tourists to the riverbanks of Jakarta to see the city’s poorest before they were displaced by gentrification.More recently, all over the world activists have engaged in walking tours to provide their own perspective on urban change, attempting to direct the gentrifier’s gaze inward. Whilst the most confrontational of these might be the Yuppie Gazing Tour of Vancouver’s historically marginalised Downtown Eastside, other tours have highlighted the deleterious effects of gentrification in Williamsburg, San Francisco, Oakland, and Surabaya, among others. In smaller towns, walking tours have been utilised to highlight the erasure of marginalised scenes and subcultures, including underground creative spaces, migrant enclaves, alternative and queer spaces. Walking Sydney, Walking Marrickville In many cities, there are now both walking tours that intend to scaffold urban renewal, and those that resist gentrification with alternative narratives. There are also some that unwittingly do both simultaneously. Marrickville is a historically working-class and migrant suburb with sizeable populations of Greek and Vietnamese migrants (Graham and Connell), as well as a strong history of manufacturing (Castles et al.), which has been undergoing gentrification for some time, with the arts playing an often contradictory role in its transformation (Gibson and Homan). More recently, as the suburb experiences rampant, financialised property development driven by global flows of capital, property developers have organised their own self-guided walking tours, deployed to facilitate the familiarisation of potential purchasers of dwellings with local amenities and ‘character’ in precincts where redevelopment is set to occur. Mirvac, Marrickville’s most active developer, has designed its own self-guided walking tour Hit the Marrickville Pavement to “explore what’s on offer” and “chat to locals”: just 7km from the CBD, Marrickville is fast becoming one of Sydney’s most iconic suburbs – a melting pot of cuisines, creative arts and characters founded on a rich multicultural heritage.The perfect introduction, this self-guided walking tour explores Marrickville’s historical architecture at a leisurely pace, finishing up at the pub.So, strap on your walking shoes; you're in for a treat.Other walking tours in the area seek to highlight political, ecological, and architectural dimension of Marrickville. For example, Marrickville Maps: Tropical Imaginaries of Abundance provides a series of plant-led walks in the suburb; The Warren Walk is a tour organised by local Australian Labor Party MP Anthony Albanese highlighting “the influence of early settlers such as the Schwebel family on the area’s history” whilst presenting a “political snapshot” of ALP history in the area. The Australian Ugliness, in contrast, was a walking tour organised by Thomas Lee in 2016 that offered an insight into the relationships between the visual amenity of the streetscape, aesthetic judgments of an ambiguous nature, and the discursive and archival potentialities afforded by camera-equipped smartphones and photo-sharing services like Instagram. Figure 1: Thomas Lee points out canals under the street of Marrickville during The Australian Ugliness, 2016.Sydney is a city adept at erasing its past through poorly designed mega-projects like freeways and office towers, and memorialisation of lost landscapes has tended towards the literary (Berry; Mudie). Resistance to redevelopment, however, has often taken the form of spectacular public intervention, in which public knowledge sharing was a key goal. The Green Bans of the 1970s were partially spurred by redevelopment plans for places like the Rocks and Woolloomooloo (Cook; Iveson), while the remaking of Sydney around the 2000 Olympics led to anti-gentrification actions such as SquatSpace and the Tour of Beauty, an “aesthetic activist” tour of sites in the suburbs of Redfern and Waterloo threatened with “revitalisation.” Figure 2: "Tour of Beauty", Redfern-Waterloo 2016. What marks the Tour of Beauty as significant in this context is the participatory nature of knowledge production: participants in the tours were addressed by representatives of the local community – the Aboriginal Housing Company, the local Indigenous Women’s Centre, REDWatch activist group, architects, designers and more. Each speaker presented their perspective on the rapidly gentrifying suburb, demonstrating how urban space is made an remade through processes of contestation. This differentiation is particularly relevant when considering the basis for Sydney-centric walking tours. Mirvac’s self-guided tour focuses on the easy-to-see historical “high culture” of Marrickville, and encourages participants to “chat to locals” at the pub. It is a highly filtered approach that does not consider broader relations of class, race and gender that constitute Marrickville. A more intense exploration of the social fabric of the city – providing a glimpse of the hidden or unknown spaces – uncovers the layers of social, cultural, and economic history that produce urban space, and fosters a deeper engagement with questions of urban socio-spatial justice.Solnit argues that walking can allow us to encounter “new thoughts and possibilities.” To walk, she writes, is to take a “subversive detour… the scenic route through a half-abandoned landscape of ideas and experiences” (13). In this way, tactical activist walking tours aim to make visible what cannot be seen, in a way that considers the polysemic nature of place, and in doing so, they make visible the hidden relations of power that produce the contemporary city. In contrast, developer-led walking tours are singularly focussed, seeking to attract inflows of capital to neighbourhoods undergoing “renewal.” These tours encourage participants to adopt the position of urban voyeur, whilst activist-led walking tours encourage collaboration and participation in urban struggles to protect and preserve the contested spaces of the city. It is in this context that we sought to devise our own walking tour – Field Trip – to encourage active participation in issues of urban renewal.In organising this walking tour, however, we acknowledge our own entanglements within processes of gentrification. As designers, musicians, writers, academics, researchers, venue managers, artists, and activists, in organising Field Trip, we could easily be identified as “creatives”, implicated in Marrickville’s ongoing transformation. All of us have ongoing and deep-rooted connections to various Sydney subcultures – the same subcultures so routinely splashed across developer advertising material. This project was borne out of Frontyard – a community not-just-art space, and has been supported by the local Inner West Council. As such, Field Trip cannot be divorced from the highly contentious processes of redevelopment and gentrification that are always simmering in the background of discussions about Marrickville. We hope, however, that in this project we have started to highlight alternative voices in those redevelopment processes – and that this may contribute towards a “method of equality” for an ongoing democratisation of those processes (Davidson and Iveson).Field Trip: Urban Geographical Enquiry as Activism Given this context, Field Trip was designed as a public knowledge project that would connect local residents, workers, researchers, and decision-makers to share their experiences living and working in various parts of Sydney that are undergoing rapid change. The site of our project – Carrington Road, Marrickville in Sydney’s inner-west – has been earmarked for major redevelopment in coming years and is quickly becoming a flashpoint for the debates that permeate throughout the whole of Sydney: housing affordability, employment accessibility, gentrification and displacement. To date, public engagement and consultation regarding proposed development at Carrington Road has been limited. A major landholder in the area has engaged a consultancy firm to establish a community reference group (CRG) the help guide the project. The CRG arose after public outcry at an original $1.3 billion proposal to build 2,616 units in twenty towers of up to 105m in height (up to thirty-five storeys) in a predominantly low-rise residential suburb. Save Marrickville, a community group created in response to the proposal, has representatives on this reference group, and has endeavoured to make this process public. Ruming (181) has described these forms of consultation as “post-political,” stating thatin a universe of consensual decision-making among diverse interests, spaces for democratic contest and antagonistic politics are downplayed and technocratic policy development is deployed to support market and development outcomes.Given the notable deficit of spaces for democratic contest, Field Trip was devised as a way to reframe the debate outside of State- and developer-led consultation regimes that guide participants towards accepting the supposed inevitability of redevelopment. We invited a number of people affected by the proposed plans to speak during the walking tour at a location of their choosing, to discuss the work they do, the effect that redevelopment would have on their work, and their hopes and plans for the future. The walking tour was advertised publicly and the talks were recorded, edited and released as freely available podcasts. The proposed redevelopment of Carrington Road provided us with a unique opportunity to develop and operate our own walking tour. The linear street created an obvious “circuit” to the tour – up one side of the road, and down the other. We selected speakers based on pre-existing relationships, some formed during prior rounds of research (Gibson et al.). Speakers included a local Aboriginal elder, a representative from the Marrickville Historical Society, two workers (who also gave tours of their workplaces), the Lead Heritage Adviser at Sydney Water, who gave us a tour of the Carrington Road pumping station, and a representative from the Save Marrickville residents’ group. Whilst this provided a number of perspectives on the day, regrettably some groups were unrepresented, most notably the perspective of migrant groups who have a long-standing association with industrial precincts in Marrickville. It is hoped that further community input and collaboration in future iterations of Field Trip will address these issues of representation in community-led walking tours.A number of new understandings became apparent during the walking tour. For instance, the heritage-listed Carrington Road sewage pumping station, which is of “historic and aesthetic significance”, is unable to cope with the proposed level of residential development. According to Philip Bennett, Lead Heritage Adviser at Sydney Water, the best way to maintain this piece of heritage infrastructure is to keep it running. While this issue had been discussed in private meetings between Sydney Water and the developer, there is no formal mechanism to make this expert knowledge public or accessible. Similarly, through the Acknowledgement of Country for Field Trip, undertaken by Donna Ingram, Cultural Representative and a member of the Metropolitan Local Aboriginal Land Council, it became clear that the local Indigenous community had not been consulted in the development proposals for Carrington Road. This information, while not necessary secret, had also not been made public. Finally, the inclusion of knowledgeable local workers whose businesses are located on Carrington Road provided an insight into the “everyday.” They talked of community and collaboration, of site-specificity, the importance of clustering within their niche industries, and their fears for of displacement should redevelopment proceed.Via a community-led, participatory walking tour like Field Trip, threads of knowledge and new information are uncovered. These help create new spatial stories and readings of the landscape, broadening the scope of possibility for democratic participation in cities. Figure 3: Donna Ingram at Field Trip 2018.Tactical Walking, Tactical Media Stories connected to walking provide an opportunity for people to read the landscape differently (Mitchell). One of the goals of Field Trip was to begin a public knowledge exchange about Carrington Road so that spatial stories could be shared, and new readings of urban development could spread beyond the confines of the self-contained tour. Once shared, this knowledge becomes a story, and once remixed into existing stories and integrated into the way we understand the neighbourhood, a collective spatial practice is generated. “Every story is a travel story – a spatial practice”, says de Certeau in “Spatial Stories”. “In reality, they organise walks” (72). As well as taking a tactical approach to walking, we took a tactical approach to the mediation of the knowledge, by recording and broadcasting the voices on the walk and feeding information to a publicly accessible wiki. The term “tactical media” is an extension of de Certeau’s concept of tactics. David Garcia and Geert Lovink applied de Certeau’s concept of tactics to the field of media activism in their manifesto of tactical media, identifying a class of producers who amplify temporary reversals in the flow of power by exploiting the spaces, channels and platforms necessary for their practices. Tactical media has been used since the late nineties to help explain a range of open-source practices that appropriate technological tools for political purposes. While pointing out the many material distinctions between different types of tactical media projects within the arts, Rita Raley describes them as “forms of critical intervention, dissent and resistance” (6). The term has also been adopted by media activists engaged in a range of practices all over the world, including the Tactical Technology Collective. For Field Trip, tactical media is a way of creating representations that help navigate neighbourhoods as well as alternative political processes that shape them. In this sense, tactical representations do not “offer the omniscient point of view we associate with Cartesian cartographic practice” (Raley 2). Rather these representations are politically subjective systems of navigation that make visible hidden information and connect people to the decisions affecting their lives. Conclusion We have shown that the walking tour can be a tourist attraction, a catalyst to the transformation of urban space through gentrification, and an activist intervention into processes of urban renewal that exclude people and alternative ways of being in the city. This article presents practice-led research through the design of Field Trip. By walking collectively, we have focused on tactical ways of opening up participation in the future of neighbourhoods, and more broadly in designing the city. 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