Plant developmental oddities.

MAIN CONCLUSION: Plants lacking shoot apical meristem develop with unique body shapes, suggesting rewiring of developmental genes. This loss of the meristem is likely influenced by a combination of environmental factors and evolutionary pressures. This study explores the development of plant bodies in three families (Podostemaceae, Lemnaceae, and Gesneriaceae) where the shoot apical meristem (SAM), a key structure for growth, is absent or altered. The review highlights alternative developmental strategies these plants employ. Also, we considered alternative reproduction in those species, namely through structures like turions, fronds, or modified leaves, bypassing the need for a SAM. Further, we report on studies based on the expression patterns of genes known to be involved in SAM formation and function. Interestingly, these genes are still present but expressed in atypical locations, suggesting a rewiring of developmental networks. Our view on the current literature and knowledge indicates that the loss or reduction of the SAM is driven by a combination of environmental pressures and evolutionary constraints, leading to these unique morphologies. Further research, also building on Next-Generation Sequencing, will be instrumental to explore the genetic basis for these adaptations and how environmental factors influence them.

First report of Alternaria alternata complex causing leaf spot on Vitis vinifera in Italy.

Grapevine is one of the main cultivated crops in the Northern Italy. During the summer 2023, high incidence (80% out of 350 plants) of brown, irregularly necrotic spots, measuring up to 2-3 cm, were observed on experimental potted two-years-old Vitis vinifera (cv. "Sauvignon Blanc" grafted onto K5BB (n = 180)) at Susegana (TV) (GPS coordinates: 45°51'07.7"N 12°15'19.9"E). The same symptoms were also observed on ungrafted potted two-years-old Vitis rootstocks (Kober 5BB, SO4, 110 Richter, 420A, Fercal, Rupestris Du Lot), located in the same area (n = 170).Symptomatic leaves were collected, underwent superficial sterilization in EtOH 70% and then washed twice in sterile water. Small leaf tissue fragments (± 3 mm) were cut from the margins of necrotic spots and plated on Czapek Yeast Autolysate agar (CYA) amended with Rose Bengal (Frisvad and Samson 2004; Ottow 1972). The plates were incubated at 28°C for one week in dark condition and, the fungal isolate, was sub-cultured on CYA. Fungal isolate, initially appearing greyish-white, later transitioned to black or darkish olive-green with a lighter border. Under a microscope at 40x magnification, conidia were light brown and obclavate to elliptical, divided by transverse and vertical septa. Their dimensions were 31 to 87 µm (avg. ± 56 µm) length and 11 to 32 µm (avg. ± 24.8 µm) width (n = 50). Morphological characteristics resembled those of the small-spored group of the Alternaria genus (Simmons 2007). Fungal genomic DNA was isolated from lyophilized mycelium using the phenol-chloroform method (Nerva et al. 2019). The ITS, ATPase, GAPDH, CAL and ACT sequences were amplified using primer pairs ITS1/ITS4, ATPDF1/ATPDR1, GPD-1/GPD-2, CALDF1/CALDR1 and ACTDF1/ACTDR1 respectively (Lawrence et al. 2014; White et al. 1990; Lawrence et al. 2017). Subsequently, the PCR products were Sanger-sequenced and uploaded to GenBank under the accession numbers: OR976478 (ITS), PP024642 (ATPase), PP456742 (GAPDH), PP456740 (CAL) and PP456741 (ACT). A maximum likelihood phylogenetic analysis, based on the concatenated gene sequences, was performed using W-IQ-TREE (Trifinopoulos et al. 2016). The analysis showed that the isolate was closely related to A. tenuissima and A. alternata, belonging to the A. alternata complex (Woudenberg et al. 2015). Both in vitro and in vivo pathogenicity tests have been performed. A detached leaf assay was conducted in vitro: asymptomatic grape leaves cv. "Sauvignon Blanc", previously water-washed, were placed in Petri dishes containing sterile water - 1% agar. A conidial suspension was prepared by scraping one-week old fungal cultures grown at 28°C on CYA medium with sterile water, followed by filtration. A final concentration of 1 x 105 conidia/mL was sprayed onto the leaves. Three leaves were sprayed with sterile water as a control group. All Petri dishes were enclosed in plastic bags to maintain high humidity and were incubated at 32 °C under dark conditions. After one week, leaf spots similar to the above-described symptoms appeared on the inoculated leaves, whereas no symptoms were observed in the control group. The in vivo pathogenicity test was carried out twice spraying the same inoculum concentration on leaves of two years old healthy potted grape plants (cv. "Sauvignon Blanc", n = 10), grown under greenhouse conditions (26 ± 2°C). All plants were kept in a plastic bag for seven days; three control plant were sprayed with sterile water. Irregularly brown leaf spot, similar to those observed initially, appeared on inoculated plants 15 days after inoculation. The same fungus was reisolated from lesions, confirming the Koch's postulates. To our knowledge this is the first report of A. alternata as the causal agent of leaf spot disease on Vitis vinifera in Italy. Considering the significant role of grapevine cultivation, particularly in Veneto region, it is essential to allocate greater focus on this new pathogen.

An Integrative Approach to Selected Species of Tanacetum L. (Asteraceae): Insights into Morphology and Phytochemistry.

In this work, we studied Tanacetum vulgare, Tanacetum parthenium, and Tanacetum corymbosum (Asteraceae) cultivated at the Ghirardi Botanic Garden (Toscolano Maderno, Brescia, Northern Italy) of the University of Milan. An integrative research approach was adopted: microscopic and histochemical, with special focus on the secretory structures responsible for the productivity of secondary metabolites; phytochemical, with the analysis of the essential oil (EO) profiles from the air-dried, flowered aerial parts collected in June 2021; bio-ecological, with emphasis, based on literature data, on the ecology and biological activity of the main EO components. In all three species, two basic trichome morphotypes (flagellar non-glandular and biseriate glandular) occurred with different distribution patterns. The glandular ones produced terpenes, along with flavonoids. A high level of chemical variability in the EO compositions emerged, specifically for qualitative data. T. vulgare profile was more complex and heterogeneous than those obtained from T. parthenium and T. corymbosum, with camphor as the predominant compound, followed by farnesol and α-santalone, respectively. Finally, the obtained scientific findings were made available to the visitors of the botanic garden through new dissemination labeling that highlights the "invisible", microscopic features of the plants, from an Open Science perspective ("Botanic Garden, factories of molecules…work in progress"-Lombardy Region Project Lr. 25/2016, year 2021).

Rice florigens control a common set of genes at the shoot apical meristem including the F-BOX BROADER TILLER ANGLE 1 that regulates tiller angle and spikelet development.

Rice flowering is triggered by transcriptional reprogramming at the shoot apical meristem (SAM) mediated by florigenic proteins produced in leaves in response to changes in photoperiod. Florigens are more rapidly expressed under short days (SDs) compared to long days (LDs) and include the HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) phosphatidylethanolamine binding proteins. Hd3a and RFT1 are largely redundant at converting the SAM into an inflorescence, but whether they activate the same target genes and convey all photoperiodic information that modifies gene expression at the SAM is currently unclear. We uncoupled the contribution of Hd3a and RFT1 to transcriptome reprogramming at the SAM by RNA sequencing of dexamethasone-inducible over-expressors of single florigens and wild-type plants exposed to photoperiodic induction. Fifteen highly differentially expressed genes common to Hd3a, RFT1, and SDs were retrieved, 10 of which still uncharacterized. Detailed functional studies on some candidates revealed a role for LOC_Os04g13150 in determining tiller angle and spikelet development and the gene was renamed BROADER TILLER ANGLE 1 (BRT1). We identified a core set of genes controlled by florigen-mediated photoperiodic induction and defined the function of a novel florigen target controlling tiller angle and spikelet development.

Teucrium fruticans L., a Multi-Scale Study: From Trichomes to Essential Oil.

This work represents the first multi-scale study on Teucrium fruticans L. cultivated at the Ghirardi Botanic Garden (Lombardy, Northern Italy), combining a micromorphological and a phytochemical survey on the plant's aerial parts. Micromorphological investigations, performed by Light Microscopy, Fluorescence Microscopy and Scanning Electron Microscopy, highlighted the presence of five trichomes morphotypes, distinguished by a different distribution pattern: peltates, short-stalked and ball-like medium-stalked capitates, ubiquitous on the whole plant, medium-stalked and long-stalked capitates, exclusive to the floral whorls. Both peltates and medium-stalked capitates were recognized as the main terpene production sites. Phytochemical characterization focused on the essential oils (EOs), obtained by Clevenger-type hydrodistillation in February and April 2022 and characterized by Gas Chromatography-Mass Spectrometry (GC/MS), which resulted mainly formed by sesquiterpene hydrocarbons. The February EO profile was characterized by ß-caryophyllene (28.30 %) and germacrene D (19.16 %) as main compounds, while in April ß-myrcene was detected at high percentage (13.77 %), in addition to the previous two components (15.72 % and 11.55 %, respectively). Literature data, dealing with the biological activities of the main oil constituents, highlighted an anti-microbial, anti-inflammatory, and anti-tumor potential, due to the high content in sesquiterpenes and, particularly, of ß-caryophyllene and germacrene D.

Achillea moschata Wulfen: From Ethnobotany to Phytochemistry, Morphology, and Biological Activity.

A multidisciplinary investigation on Achillea moschata Wulfen (Asteraceae) is outlined herein. This work, part of the European Interreg Italy-Switzerland B-ICE project, originated from an ethnobotanical survey performed in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy) in 2019-2021 which highlighted this species' relevance of use in folk medicine to treat gastrointestinal diseases. In addition, this contribution included analyses of the: (a) phytochemical profile of the aqueous and methanolic extracts of the dried flower heads using LC-MS/MS; (b) morpho-anatomy and histochemistry of the vegetative and reproductive organs through Light, Fluorescence, and Scanning Electron Microscopy; (c) biological activity of the aqueous extract concerning the antioxidant and anti-inflammatory potential through cell-based in vitro models. A total of 31 compounds (5 phenolic acids, 13 flavonols, and 13 flavones) were detected, 28 of which included in both extracts. Covering and secreting trichomes were observed: the biseriate 10-celled glandular trichomes prevailing on the inflorescences represented the main sites of synthesis of the polyphenols and flavonoids detected in the extracts, along with volatile terpenoids. Finally, significant antioxidant and anti-inflammatory activities of the aqueous extract were documented, even at very low concentrations; for the first time, the in vitro tests allowed us to formulate hypotheses about the mechanism of action. This work brings an element of novelty due to the faithful reproduction of the traditional aqueous preparation and the combination of phytochemical and micromorphological research approaches.

Curation of a reference database of COI sequences for insect identification through DNA metabarcoding: COins.

DNA metabarcoding is a widespread approach for the molecular identification of organisms. While the associated wet-lab and data processing procedures are well established and highly efficient, the reference databases for taxonomic assignment can be implemented to improve the accuracy of identifications. Insects are among the organisms for which DNA-based identification is most commonly used; yet, a DNA-metabarcoding reference database specifically curated for their species identification using software requiring local databases is lacking. Here, we present COins, a database of 5' region cytochrome c oxidase subunit I sequences (COI-5P) of insects that includes over 532 000 representative sequences of >106 000 species specifically formatted for the QIIME2 software platform. Through a combination of automated and manually curated steps, we developed this database starting from all COI sequences available in the Barcode of Life Data System for insects, focusing on sequences that comply with several standards, including a species-level identification. COins was validated on previously published DNA-metabarcoding sequences data (bulk samples from Malaise traps) and its efficiency compared with other publicly available reference databases (not specific for insects). COins can allow an increase of up to 30% of species-level identifications and thus can represent a valuable resource for the taxonomic assignment of insects' DNA-metabarcoding data, especially when species-level identification is needed https://doi.org/10.6084/m9.figshare.19130465.v1.

Miniaturized 3D bone marrow tissue model to assess response to Thrombopoietin-receptor agonists in patients.

Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias.

Platelets are tiny cell fragments essential for blood to clot. They are created and released into the bloodstream by megakaryocytes, giant cells that live in the bone marrow. In certain genetic diseases, such as Inherited Thrombocytopenia, the bone marrow fails to produce enough platelets: this leaves patients extremely susceptible to bruising, bleeding, and poor clotting after an injury or surgery. Certain patients with Inherited Thrombocytopenia respond well to treatments designed to boost platelet production, but others do not. Why these differences exist could be investigated by designing new test systems that recreate the form and function of bone marrow in the laboratory. However, it is challenging to build the complex and poorly understood bone marrow environment outside of the body. Here, Di Buduo et al. have developed an artificial three-dimensional miniature organ bioreactor system that recreates the key features of bone marrow. In this system, megakaryocytes were grown from patient blood samples, and hooked up to a tissue scaffold made of silk. The cells were able to grow as if they were in their normal environment, and they could shed platelets into an artificial bloodstream. After treating megakaryocytes with drugs to stimulate platelet production, Di Buduo et al. found that the number of platelets recovered from the bioreactor could accurately predict which patients would respond to these drugs in the clinic. This new test system enables researchers to predict how a patient will respond to treatment, and to tailor therapy options to each individual. This technology could also be used to test new drugs for Inherited Thrombocytopenias and other blood-related diseases; if scaled-up, it could also, one day, generate large quantities of lab-grown blood cells for transfusion.

Association Between Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Genetic Variants and Clinical Progression of Huntington's Disease.

BACKGROUND: Although Huntington's disease (HD) is caused by a single dominant gene, it is clear that there are genetic modifiers that may influence the age of onset and disease progression. OBJECTIVES: We sought to investigate whether new inflammation-related genetic variants may contribute to the onset and progression of HD. METHODS: We first used postmortem brain material from patients at different stages of HD to look at the protein expression of toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells 2 (TREM2). We then genotyped the TREM2 R47H gene variant and 3 TLR4 single nucleotide polymorphisms in a large cohort of HD patients from the European Huntington's Disease Network REGISTRY. RESULTS: We found an increase in the number of cells expressing TREM2 and TLR4 in postmortem brain samples from patients dying with HD. We also found that the TREM2 R47H gene variant was associated with changes in cognitive decline in the large cohort of HD patients, whereas 2 of 3 TLR4 single nucleotide polymorphisms assessed were associated with changes in motor progression in this same group. CONCLUSIONS: These findings identify TREM2 and TLR4 as potential genetic modifiers for HD and suggest that inflammation influences disease progression in this condition. © 2019 International Parkinson and Movement Disorder Society.

Sex change in kiwifruit (Actinidia chinensis Planch.): a developmental framework for the bisexual to unisexual floral transition.

KEY MESSAGE: The developmental morphology of male and female kiwifruit flowers is tracked to delimit a framework of events to aid the study of divergence in floral gene expression. The transition from hermaphrodite to unisexual development of kiwifruit (Actinidia chinensis Planch) flowers has been reported previously, but differences in gene expression controlling sexual development for this species have not been associated with the major developmental changes occurring within pistils. We investigated the key stages in male and female flower development to define the point at which meristematic activities diverge in the two sexes. A combination of scanning electron microscopy and light microscopy was used to investigate pistil development from the earliest stages. We identified seven distinct stages characterized by differences in ovary size and shape, macrosporogenesis, ovule primordium development, anther locule lengthening, microspore wall thickening, and pollen degeneration. Sex differences were evident from the initial stage of development, with a laterally compacted gynoecium in male flowers. However, the key developmental stage, at which tissue differentiation clearly deviated between the two sexes, was stage 3, when flowers were 3.5 to 4.5 mm in length at approximately 10 d from initiation of stamen development. At this stage, male flowers lacked evident carpel meristem development as denoted by a lack of ovule primordium formation. Pollen degeneration in female flowers, probably driven by programmed cell death, occurred at the late stage 6, while the final stage 7 was represented by pollen release. As the seven developmental stages are associated with specific morphological differences, including flower size, the scheme suggested here can provide the required framework for the future study of gene expression during the regulation of flower development in this crop species.

Trehalose to cryopreserve human pluripotent stem cells.

The successful exploitation of human pluripotent stem cells (hPSCs) for research, translational or commercial reasons requires the implementation of a simple and efficient cryopreservation method. Cryopreservation is usually performed with dimethylsulphoxide (DMSO), in addition to animal proteins. However, even at sub-toxic levels, DMSO diminishes the pluripotency capacity of hPSCs and affects their epigenetic system by acting on the three DNA methyltransferases (Dnmts) and histone modification enzymes. Our study aimed to test trehalose-based cryosolutions containing ethylene glycol (EG) or glycerol (GLY) on hESCs RC17, hiPSCs CTR2#6 and long-term neuroepithelial-like stem cells (lt-NES) AF22. Here, we demostrate the effectiveness of these cryosolutions in hPSCs by showing an acceptable rate of cell viability and high stability compared to standard 10% DMSO freezing medium (CS10). All cell lines retained their morphology, self renewal potential and pluripotency, and none of the cryosolutions affected their differentiation potential. Genotoxicity varied among different stem cells types, while trehalose-based cryopreservation did not sensibly alter the homeostasis of endoplasmic reticulum (ER). This study provides evidence that pluripotent and neural stem cells stored in trehalose alone or with other cryoprotectants (CPAs) maintain their functional properties, indicating their potential use in cell therapies if produced in good manufacturing practice (GMP) facility.

Generation of Human-Induced Pluripotent Stem Cells from Wolfram Syndrome Type 2 Patients Bearing the c.103 + 1G>A CISD2 Mutation for Disease Modeling.

Wolfram syndrome (WFS) is a rare autosomal premature aging syndrome that shows signs of diabetes mellitus, optic atrophy, and deafness in addition to central nervous system and endocrine complications. The frequent form of WFS type 1 (WFS1) harbors causative mutations in the WFS1 gene, whereas the rare form or WFS type 2 (WFS2) involves CISD2. Mutations in these two genes are recognized by a subset of variable clinical symptoms and a set of overlapping features. In this study, we report on the generation of stable human-induced pluripotent stem cells (hiPSCs) derived from primary fibroblasts of a previously reported Italian family with CISD2 mutation (c.103 + 1G>A), occurring in the consensus intron 1 splicing site in two sisters, deleting the first exon of the transcript. The generated hiPSCs provide a cell model system to study the mutation's role in the multisystemic clinical disorders previously described and test eventual drug effects on the specific and associated clinical phenotype.

Epigenetic and transcriptional modulation of WDR5, a chromatin remodeling protein, in Huntington's disease human induced pluripotent stem cell (hiPSC) model.

DNA methylation (DNAm) changes are of increasing relevance to neurodegenerative disorders, including Huntington's disease (HD). We performed genome-wide screening of possible DNAm changes occurring during striatal differentiation in human induced pluripotent stem cells derived from a HD patient (HD-hiPSCs) as cellular model. We identified 240 differentially methylated regions (DMRs) at promoters in fully differentiated HD-hiPSCs. Subsequently, we focused on the methylation differences in a subcluster of genes related to Jumonji Domain Containing 3 (JMJD3), a demethylase that epigenetically regulates neuronal differentiation and activates neuronal progenitor associated genes, which are indispensable for neuronal fate acquisition. Noticeably among these genes, WD repeat-containing protein 5 (WDR5) promoter was found hypermethylated in HD-hiPSCs, resulting in a significant down-modulation in its expression and of the encoded protein. A similar WDR5 expression decrease was seen in a small series of HD-hiPSC lines characterized by different CAG length. The decrease in WDR5 expression was particularly evident in HD-hiPSCs compared to hESCs and control-hiPSCs from healthy subjects. WDR5 is a core component of the MLL/SET1 chromatin remodeling complexes essential for H3K4me3, previously reported to play an important role in stem cells self-renewal and differentiation. These results suggest the existence of epigenetic mechanisms in HD and the identification of genes, which are able to modulate HD phenotype, is important both for biomarker discovery and therapeutic interventions.

A Review of Research-Grade Human Induced Pluripotent Stem Cells Qualification and Biobanking Processes.

Human induced pluripotent stem cell (hiPSC) biobanks are invaluable resources for basic and clinical research, since they provide a sustainable supply of accessible cell lines that meet high quality and safety standards. hiPSCs are particularly useful for understanding disease mechanisms, creating cell models for drug development, and generating novel clinical therapies. For clinical applications and drug discovery, it is fundamental that the acquired pluripotent cell lines never touch animal-derived products nor xenogeneic reagents (Good Manufacturing Practice-grade); whereas for research grade, it is sufficient to operate under Good Laboratory Practice conditions. However, regardless of the end use, it is important that every step in the whole process, starting from the original cells throughout expansion and manipulation, must be performed and recorded rigorously. Here, we describe our biobanking management system that is applied specifically to human pluripotent stem cells.

Occurrence of Toxigenic Fungi and Aflatoxin Potential of Aspergillus spp. Strains Associated with Subsistence Farmed Crops in Haiti.

Subsistence farming and poor storage facilities favor toxigenic fungal contamination and mycotoxin accumulation in staple foods from tropical countries such as Haiti. The present preliminary study was designed to evaluate the occurrence of toxigenic fungi in Haitian foodstuffs to define the mycotoxin risk associated with Haitian crops. The objectives of this research were to determine the distribution of toxigenic fungi in the Haitian crops maize, moringa, and peanut seeds and to screen Aspergillus section Flavi (ASF) isolates for production of aflatoxins B1 and G1 in vitro. Maize, moringa, and peanut samples were contaminated by potential toxigenic fungal taxa, mainly ASF and Fusarium spp. The isolation frequency of Aspergillus spp. and Fusarium spp. was influenced by locality and thus by farming systems, storage systems, and weather conditions. Particularly for ASF in peanut and maize samples, isolation frequencies were directly related to the growing season length. The present study represents the first report of contamination by toxigenic fungi and aflatoxin in moringa seeds, posing concerns about the safety of these seeds, which people in Haiti commonly consume. Most (80%) of the Haitian ASF strains were capable of producing aflatoxins, indicating that Haitian conditions clearly favor the colonization of toxigenic ASF strains over atoxigenic strains. ASF strains producing both aflatoxins B1 and G1 were found. Understanding the distribution of toxigenic ASF in Haitian crops and foodstuffs is important for determining accurate toxicological risks because the toxic profile of ASF is species specific. The occurrence of toxigenic fungi and the profiles of the ASF found in various crops highlight the need to prevent formation of aflatoxins in Haitian crops. This study provides relevant preliminary baseline data for guiding the development of legislation regulating the quality and safety of crops in this low-income country.

Moringa oleifera Seeds and Oil: Characteristics and Uses for Human Health.

Moringa oleifera seeds are a promising resource for food and non-food applications, due to their content of monounsaturated fatty acids with a high monounsaturated/saturated fatty acids (MUFA/SFA) ratio, sterols and tocopherols, as well as proteins rich in sulfated amino acids. The rapid growth of Moringa trees in subtropical and tropical areas, even under conditions of prolonged drought, makes this plant a reliable resource to enhance the nutritional status of local populations and, if rationalized cultivation practices are exploited, their economy, given that a biodiesel fuel could be produced from a source not in competition with human food crops. Despite the relatively diffuse use of Moringa seeds and their oil in traditional medicine, no pharmacological activity study has been conducted on humans. Some encouraging evidence, however, justifies new efforts to obtain clear and definitive information on the benefits to human health arising from seed consumption. A critical review of literature data concerning the composition of Moringa oil has set in motion a plan for future investigations. Such investigations, using the seeds and oil, will focus on cultivation conditions to improve plant production, and will study the health effects on human consumers of Moringa seeds and their oil.

Cell Line Macroarray: An Alternative High-Throughput Platform to Analyze hiPSC Lines.

In the past decade, tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly, enormous amount of data can be obtained from the cell line macroarray (CLMA) technology, which developed from the TMA using formalin-fixed, paraffin-embedded cell pellets. Here, we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones, which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here, we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer, TissueQuest, as a reliable tool to quickly select the best clones, based upon the level of expression of multiple pluripotent biomarkers.

Hd3a, RFT1 and Ehd1 integrate photoperiodic and drought stress signals to delay the floral transition in rice.

Plants show a high degree of developmental plasticity in response to external cues, including day length and environmental stress. Water scarcity in particular can interfere with photoperiodic flowering, resulting in the acceleration of the switch to reproductive growth in several species, a process called drought escape. However, other strategies are possible and drought stress can also delay flowering, albeit the underlying mechanisms have never been addressed at the molecular level. We investigated these interactions in rice, a short day species in which drought stress delays flowering. A protocol that allows the synchronization of drought with the floral transition was set up to profile the transcriptome of leaves subjected to stress under distinct photoperiods. We identified clusters of genes that responded to drought differently depending on day length. Exposure to drought stress under floral-inductive photoperiods strongly reduced transcription of EARLY HEADING DATE 1 (Ehd1), HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1), primary integrators of day length signals, providing a molecular connection between stress and the photoperiodic pathway. However, phenotypic and transcriptional analyses suggested that OsGIGANTEA (OsGI) does not integrate drought and photoperiodic signals as in Arabidopsis, highlighting molecular differences between long and short day model species.

Investigating DNA methylation dynamics and safety of human embryonic stem cell differentiation toward striatal neurons.

The potential use of human embryonic stem cells (hESCs) in cell-based therapies points out the critical importance of epigenomic evaluation for cell-based therapies. Specifically, DNA methylation appears to be a crucial player in establishing cell fate commitment and lineage choices. In this study, we report the global changes observed on the CpG islands distributed in promoters, gene bodies, and intergenic regions and the major biochemical pathways and genes involved in methylation changes as H9-hESCs turn into a neuronal culture containing medium-sized spiny striatal neurons (MSNs). Using an ontogeny-recapitulating protocol of striatal neuron differentiation, we analyzed DNA methylation profiles during the conversion from pluripotency to neuropotency up to the acquisition of a mature neuronal phenotype. H9-hESCs changed the methylation pattern both through de novo methylation and hypomethylation of specific gene promoters. Bioinformatic analysis allowed us to identify a panel of striatal-associated genes, which were regulated by DNA methylation and differentially expressed during striatal commitment. Importantly, DNA methylation analysis revealed that H9-hESCs did not acquire methylation-based oncogenic properties after differentiation. Indeed, hypermethylation of cancer-associated genes that characterize transformed cells, such as Polycomb repressive complex-associated genes, was not detected in the neuronal cultures. However, the oncosuppressor gene, BCL2L11, became hypermethylated in H9-hESC-derived mature neurons. Whole-genome DNA methylation profiling could become a technological platform to predict the differentiative potential of hESC-derived cultures and establish further biosafety assessment quality control tools of the cell-based products.

Evaluation of cross-sample contamination in tissue microarrays by polymerase chain reaction.

In the past decade, the popularity and power of Tissue Microarray (TMA) technology has increased since it provides a method to detect diagnostic and prognostic markers in an array of clinical tissue specimens collected for translational research. TMAs allow for rapid and cost-effective analysis of hundreds of molecular markers at the nucleic acid and protein levels. This technology is particularly useful in the realization of the Human Protein Atlas Project, since it aims to create a reference database of non-redundant human proteins. In this context, it is important to assure the lack of cross-sample contamination due to the repeated use of the same needle in consecutive coring. Here we show that carry-over contamination from one tissue core to another does not occur, reinforcing the accuracy of the TMA technology in the simultaneous testing of multiple bio-samples.