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Emilia Galdiero,1 Annarita Falanga,2 Antonietta Siciliano,1 Valeria Maselli,1 Marco Guida,1 Rosa Carotenuto,1 Margherita Tussellino,1 Lucia Lombardi,3 Giovanna Benvenuto,4 Stefania Galdiero2 1Department of Biology, 2Department of Pharmacy and CiRPEB, University of Naples Federico II, 3Department of Experimental Medicine, Second University of Naples, 4Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy Abstract: The use of quantum dots (QDs) for nanomedicine is hampered by their potential toxicologic effects and difficulties with delivery into the cell interior. We accomplished an in vivo study exploiting Daphnia magna and Xenopus laevis to evaluate both toxicity and uptake of QDs coated with the membranotropic peptide gH625 derived from the glycoprotein H of herpes simplex virus and widely used for drug delivery studies. We evaluated and compared the effects of QDs and gH625-QDs on the survival, uptake, induction of several responsive pathways and genotoxicity in D. magna, and we found that QDs coating plays a key role. Moreover, studies on X. laevis embryos allowed to better understand their cell/tissue localization and delivery efficacy. X. laevis embryos raised in Frog Embryo Teratogenesis Assay-Xenopus containing QDs or gH625-QDs showed that both nanoparticles localized in the gills, lung and intestine, but they showed different distributions, indicating that the uptake of gH625-QDs was enhanced; the functionalized QDs had a significantly lower toxic effect on embryos’ survival and phenotypes. We observed that D. magna and X. laevis are useful in vivo models for toxicity and drug delivery studies. Keywords: membranotropic peptide, delivery, blood–brain barrier, nanoparticles, genotoxicity

The present study deals with the computational simulation of a marine four-stroke dual-fuel engine, working both in diesel and natural gas modes, with a particular focus on the turbocharger system behaviour. The engine model, developed by the authors, was implemented as a computer code and used to compare the performance of the engine when using the two different fuels. The variable geometry turbine operation has been studied and simulated in detail as well as the compressor matching. The paper points out the different implications of the turbocharger functioning due to the different features of the mix air-fuel introduced in the engine. The developed computer model has been validated with good results by comparisons with reference steady state engine data, provided by the manufacturer, referred to both design and off design situations. The good results obtained from these comparisons confirm the possibility of using the calculation procedure to optimize the control logic of both the engine and the variable geometry turbine, thus extending the application possibilities of the simulation model. [ABSTRACT FROM AUTHOR]

Design and optimization of the propulsion system is a crucial task of the ship design process. The behaviour of the propulsion system, in transient conditions as well as in steady state, is greatly affected by the capability of the control system to manage the available power and to achieve the desired performance in the shortest time. The selection of a proper control scheme is a trade-off between different and conflicting needs. Two of the opposites are: increasing the ship operability by adding more functions and more controls; and reducing the control system development and installation time and cost. In this paper, the rapid prototyping and testing procedure for the development of the propulsion controller of the new Italian aircraft carrier Cavour is presented, using real-time hardware-in-the-loop (RT-HIL) simulation. The procedure is based on a wide use of simulation technology. First, a complete dynamical model of the ship propulsion plant was developed. Then, batch simulation was used to develop the best possible control scheme. Finally, RT-HIL simulation was used to debug the real controller software and to tune the controller parameters before sea trials. The application of the procedure led to a significant reduction in the development phase of the controller design. Furthermore, the adoption of the RT-HIL technology greatly reduced the time spent to tune the control system during the ship delivery phase. [ABSTRACT FROM AUTHOR]

The sequential turbocharging technique is used to improve the performance of highly rated diesel engines in particular at part loads. However, the transient behaviour of the sequential turbocharging connection/disconnection phases may be difficult to calibrate and requires an accurate study and development. This may be accomplished, in addition to the necessary experimentation, by means of dynamic simulation techniques. In this paper a model for the dynamic simulation of a sequentially turbocharged diesel engine is presented. A two-zone, non-adiabatic, actual cycle approach is used for the chemical and thermodynamic phenomena simulation in the cylinder. Fluid mass and energy accumulation in the engine volumes are evaluated by means of a filling and emptying method. The simulation of the turbocharger dynamics combines the use of the compressor and turbine maps with a model of the sequential turbocharging connection/disconnection valves and of their governor system. The procedure is applied to the simulation of the Wärtsilä18V 26X engine, a highly rated, recently developed, sequentially turbocharged marine diesel engine, whose experimental results are used for the steady state and transient validation of the simulation code with particular reference to the sequential turbocharging connection/disconnection phases. The presented results show the time histories of some important variables during typical engine load variations. [ABSTRACT FROM AUTHOR]

High-dose cyclophosphamide (HD-CTX) is largely employed in high-dose chemotherapy (HD-CHT) protocols. HD-CTX dose-limiting toxicity expresses itself as cardiac toxicity which is fatal in a minority of patients. The pathophysiology of HD-CTX-associated cardiotoxicity is still poorly understood. Autopsy studies in patients who died from acute HD-CTX-induced cardiac toxicity revealed hemorrhagic myocardial cell death and interstitial edema. Recently troponins, in particular troponin I (cTnI), have been found to represent a uniquely sensitive and specific marker of myocyte membrane integrity and therefore to increase in response to minimal myocardial cell damage in different settings, including doxorubicin-induced cardiotoxicity. We performed a multiparametric cardiologic monitoring in 16 consecutive breast cancer patients undergoing HD-CTX by means of serial ECG registrations and cardiac enzymes (CPK, CPK-MB and cTnI) determinations plus echocardiography in order to clarify acute cardiac events following HD-CTX administration. Neither overt cardiac toxicity nor cardiac enzymes elevation were recorded. Serial ECGs revealed in six cases little and reversible reduction of QRS voltage and/or ST abnormalities. Echo monitoring showed in four cases mild and transient increase of LV diastolic/systolic diameter/volume without decrease of FS% or EF% below normal values: in two of them abnormalities of diastolic function (E/A mitral doppler ratio) were also recorded. We conclude that our protocol of HD-CTX administration does not cause myocardial cell damage as analyzed by serum cTnI levels, thus suggesting that myocyte membrane injury may not be the first direct mechanism of HD-CTX cardiotoxicity. ECG (ie QRS voltages ) and Echo (ie E/A ratio) monitoring leads us to hypothesize that slight interstitial edema with reduction of LV diastolic compliance may be initial signs of cardiac dysfunction in this clinical setting. Bone Marrow Transplantation(2001) 28, 277–282. [ABSTRACT FROM AUTHOR]

An abstract of the article "Anthracyclines cardiotoxicity monitoring by conventional echo-Doppler and tissue Doppler imaging: its relationship with pro-BRAIN natriuretic peptide" by G. M. Benvenuto and colleagues is presented.

Most sea urchin species are indirect developers, going through a larval stage called pluteus. The pluteus possesses its own nervous system, consisting mainly of the apical organ neurons (controlling metamorphosis and settlement) and ciliary band neurons (controlling swimming behavior and food collection). Additional neurons are located in various areas of the gut. In recent years, the molecular complexity of this apparently "simple" nervous system has become apparent, with at least 12 neuronal populations identified through scRNA-sequencing in the species Strongylocentrotus purpuratus . Among these, there is a cluster of neurosecretory cells that produce a thyrotropin-releasing hormone-type neuropeptide (TRHergic) and that are also photosensory (expressing a Go-Opsin). However, much less is known about the organization of the nervous system in other sea urchin species. The aim of this work was to thoroughly characterize the localization of the TRHergic cells from early pluteus to juvenile stages in the Mediterranean sea urchin species Paracentrotus lividus combining immunostaining and whole mount in situ hybridization. We also compared the localization of TRHergic cells in early plutei of two other sea urchin species, Arbacia lixula and Heliocidaris tuberculata . This work provides new information on the anatomy and development of the nervous system in sea urchins. Moreover, by comparing the molecular signature of the TRHergic cells in P. lividus and S. purpuratus , we have obtained new insights how TRH-type neuropeptide signaling evolved in relatively closely related species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cocurullo, Paganos, Benvenuto and Arnone.)

Diatoms can survive long periods in dark, anoxic sediments by forming resting spores or resting cells. These have been considered dormant until recently when resting cells of Skeletonema marinoi were shown to assimilate nitrate and ammonium from the ambient environment in dark, anoxic conditions. Here, we show that resting cells of S. marinoi can also perform dissimilatory nitrate reduction to ammonium (DNRA), in dark, anoxic conditions. Transmission electron microscope analyses showed that chloroplasts were compacted, and few large mitochondria had visible cristae within resting cells. Using secondary ion mass spectrometry and isotope ratio mass spectrometry combined with stable isotopic tracers, we measured assimilatory and dissimilatory processes carried out by resting cells of S. marinoi under dark, anoxic conditions. Nitrate was both respired by DNRA and assimilated into biomass by resting cells. Cells assimilated nitrogen from urea and carbon from acetate, both of which are sources of dissolved organic matter produced in sediments. Carbon and nitrogen assimilation rates corresponded to turnover rates of cellular carbon and nitrogen content ranging between 469 and 10,000 years. Hence, diatom resting cells can sustain their cells in dark, anoxic sediments by slowly assimilating and respiring substrates from the ambient environment., (© 2024 The Authors. Environmental Microbiology published by John Wiley & Sons Ltd.)

The "ribbon," a structural arrangement in which Golgi stacks connect to each other, is considered to be restricted to vertebrate cells. Although ribbon disruption is linked to various human pathologies, its functional role in cellular processes remains unclear. In this study, we investigate the evolutionary origin of the Golgi ribbon. We observe a ribbon-like architecture in the cells of several metazoan taxa suggesting its early emergence in animal evolution predating the appearance of vertebrates. Supported by AlphaFold2 modeling, we propose that the evolution of Golgi reassembly and stacking protein (GRASP) binding by golgin tethers may have driven the joining of Golgi stacks resulting in the ribbon-like configuration. Additionally, we find that Golgi ribbon assembly is a shared developmental feature of deuterostomes, implying a role in embryogenesis. Overall, our study points to the functional significance of the Golgi ribbon beyond vertebrates and underscores the need for further investigations to unravel its elusive biological roles., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the fecal bacterial microbiota at the time of diagnosis (T0) and after 1 month of therapy (T1) in cats diagnosed with lymphoplasmacytic enteritis (LPE) or cats with low-grade intestinal T-cell lymphoma (LGITL) and to compare these findings with those of healthy cats., Animals: 5 healthy cats, 13 cats with LPE, and 7 cats with LGITL were prospectively enrolled between June 2020 and June 2021., Methods: Fecal samples were collected at T0 and T1, and DNA was extracted for 16S ribosomal amplicon sequencing. Alpha diversity and beta diversity were computed. The taxonomic assignment was performed using sequences from the Silva v138 formatted reference database. Differential abundant taxa were selected in each taxonomic level, with the P value adjusted < .05, as the cut-off., Results: No significant differences in alpha and beta diversity were found either at T0 or T1 between healthy and diseased cats or between cats with LPE and LGITL. Beta-diversity analysis showed an increase in the Fusobacteriaceae family in cats with LGITL at T0, compared to cats with LPE. Regardless of histological diagnosis, several microbiota differences were found at T0 based on serum cobalamin levels., Clinical Relevance: Fecal samples were successfully used to characterize the bacteriome of the intestinal tract in cats by 16S rRNA gene sequencing. However, results highlighted that the metagenomic evaluation was not useful to discriminate between LPE and LGITL nor to predict the therapeutic response in this study population.

The early identification of a subclinical rejection (SCR) can improve the long-term outcome of the transplanted kidney through intensified immunosuppression. However, the only approved diagnostic method is the protocol biopsy, which remains an invasive method and not without minor and/or major complications. The protocol biopsy is defined as the sampling of allograft tissue at pre-established times even in the absence of an impaired renal function; however, it does not avoid histological damage. Therefore, the discovery of new possible biomarkers useful in the prevention of SCR has gained great interest. Among all the possible candidates, there are microRNAs (miRNAs), which are short, noncoding RNA sequences, that are involved in mediating numerous post-transcriptional pathways. They can be found not only in tissues, but also in different biological fluids, both as free particles and contained in extracellular vesicles (EVs) released by different cell types. In this study, we firstly performed a retrospective miRNA screening analysis on biopsies and serum EV samples of 20 pediatric transplanted patients, followed by a second screening on another 10 pediatric transplanted patients' urine samples at one year post-transplant. In both cohorts, we divided the patients into two groups: patients with histological SCR and patients without histological SCR at one year post-transplantation. The isolated miRNAs were analyzed in an NGS platform to identify different expressions in the two allograft states. Although no statistical data were found in sera, in the tissue and urinary EVs, we highlighted signatures of miRNAs associated with the histological SCR state.

Exposure to metal nanoparticles is potentially harmful, particularly when occurring during embryogenesis. In this study, we tested the effects of commercial AuNPs and AgNPs, widely used in many fields for their features, on the early development of Xenopus laevis , an anuran amphibian key model species in toxicity testing. Through the Frog Embryo Teratogenesis Assay- Xenopus test (FETAX), we ascertained that both nanoparticles did not influence the survival rate but induced morphological anomalies like modifications of head and branchial arch cartilages, depigmentation of the dorsal area, damage to the intestinal brush border, and heart rate alteration. The expression of genes involved in the early pathways of embryo development was also modified. This study suggests that both types of nanoparticles are toxic though nonlethal, thus indicating that their use requires attention and further study to better clarify their activity in animals and, more importantly, in humans.

Purpose: Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs)., Methods: 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively., Results: Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S - adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways., Conclusion: The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM., (© 2023. The Author(s).)

Down syndrome is a common genetic disorder caused by partial or complete triplication of chromosome 21. This syndrome shows an overall and progressive impairment of olfactory function, detected early in adulthood. The olfactory neuronal cells are located in the nasal olfactory mucosa and represent the first sensory neurons of the olfactory pathway. Herein, we applied the olfactory swabbing procedure to allow a gentle collection of olfactory epithelial cells in seven individuals with Down syndrome and in ten euploid controls. The aim of this research was to investigate the peripheral gene expression pattern in olfactory epithelial cells through RNAseq analysis. Validated tests (Sniffin' Sticks Extended test) were used to assess olfactory function. Olfactory scores were correlated with RNAseq results and cognitive scores (Vineland II and Leiter scales). All Down syndrome individuals showed both olfactory deficit and intellectual disability. Down syndrome individuals and euploid controls exhibited clear expression differences in genes located in and outside the chromosome 21. In addition, a significant correlation was found between olfactory test scores and gene expression, while a non-significant correlation emerged between olfactory and cognitive scores. This first preliminary step gives new insights into the Down syndrome olfactory system research, starting from the olfactory neuroepithelium, the first cellular step on the olfactory way., (© 2022. The Author(s).)

Transcription factors are crucial drivers of cellular differentiation during animal development and often share ancient evolutionary origins. The T-box transcription factor Brachyury plays a pivotal role as an early mesoderm determinant and neural repressor in vertebrates; yet, the ancestral function and key evolutionary transitions of the role of this transcription factor remain obscure. Here, we present a genome-wide target-gene screen using chromatin immunoprecipitation sequencing in the sea anemone Nematostella vectensis, an early branching non-bilaterian, and the sea urchin Strongylocentrotus purpuratus, a representative of the sister lineage of chordates. Our analysis reveals an ancestral gene regulatory feedback loop connecting Brachyury, FoxA and canonical Wnt signalling involved in axial patterning that predates the cnidarian-bilaterian split about 700 million years ago. Surprisingly, we also found that part of the gene regulatory network controlling the fate of neuromesodermal progenitors in vertebrates was already present in the common ancestor of cnidarians and bilaterians. However, while several endodermal and neuronal Brachyury target genes are ancestrally shared, hardly any of the key mesodermal downstream targets in vertebrates are found in the sea anemone or the sea urchin. Our study suggests that a limited number of target genes involved in mesoderm formation were newly acquired in the vertebrate lineage, leading to a dramatic shift in the function of this ancestral developmental regulator., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The identity and function of a given cell type relies on the differential expression of gene batteries that promote diverse phenotypes and functional specificities. Therefore, the identification of the molecular and morphological fingerprints of cell types across taxa is essential for untangling their evolution. Here we use a multidisciplinary approach to identify the molecular and morphological features of an exocrine, pancreas-like cell type harbored within the sea urchin larval gut. Using single cell transcriptomics, we identify various cell populations with a pancreatic-like molecular fingerprint that are enriched within the S. purpuratus larva digestive tract. Among these, in the region where they reside, the midgut/stomach domain, we find that populations of exocrine pancreas-like cells have a unique regulatory wiring distinct from the rest the of the cell types of the same region. Furthermore, Serial Block-face scanning Electron Microscopy (SBEM) of the exocrine cells shows that this reported molecular diversity is associated to distinct morphological features that reflect the physiological and functional properties of this cell type. Therefore, we propose that these sea urchin exocrine cells are homologous to the well-known mammalian pancreatic acinar cells and thus we trace the origin of this particular cell type to the time of deuterostome diversification. Overall, our approach allows a thorough characterization of a complex cell type and shows how both the transcriptomic and morphological information contribute to disentangling the evolution of cell types and organs such as the pancreatic cells and pancreas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Paganos, Ronchi, Carl, Mizzon, Martinez, Benvenuto and Arnone.)

Background: Antibiotic-responsive enteropathy (ARE) is diagnosed by excluding other causes of diarrhea and when there is a short-term response to administration of antibiotics., Objectives: To characterize the gut microbiota and clinical trend of dogs with suspected ARE and to evaluate the variation in microbiota before (T0), after 30 days (T30) of tylosin treatment, and 30 days after discontinuation of treatment (T60). A further objective was to evaluate whether changes in gut microbiota are related to relapses of diarrhea when the therapy is tapered., Animals: Study sample (group A) was composed of 15 dogs with chronic diarrhea, group B was composed of 15 healthy dogs. Group A was given tylosin for 30 days., Methods: A multicentric prospective study. Clinical Indexes, fecal score, and samples for microbiota analysis were collected at T0, T30, and T60 in group A and T0 and T30 in group B. The gut microbiota was analyzed via 16S ribosomal RNA gene. Qiime2 version 2020.2 was used to perform bioinformatic analyses, and Alpha- and Beta-diversity were computed., Results: Diarrhea recurred after T30 in 9 of 14 dogs, which were classified as affected by ARE. At T0, a difference was noted in the beta-diversity between groups (Bray Curtis metric P = .006). A T0-T30 difference in alpha-diversity was noted in group A (Shannon index P = .001, Faith PD P = .007)., Conclusions and Clinical Importance: Although tylosin influences the microbiota of dogs with ARE, we failed to find any specific characteristic in the microbiota of dogs with ARE., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

The exposure of organisms to the nanoparticulate is potentially hazardous, particularly when it occurs during embryogenesis. The effects of commercial SiO 2 NPs in early development were studied, using Xenopus laevis as a model to investigate their possible future employment by means of the Frog Embryo Teratogenesis Assay-Xenopus test (FETAX). The SiO 2 NPs did not change the survival but produced several abnormalities in developing embryos, in particular, the dorsal pigmentation, the cartilages of the head and branchial arches were modified; the encephalon, spinal cord and nerves are anomalous and the intestinal brush border show signs of suffering; these embryos are also bradycardic. In addition, the expression of genes involved in the early pathways of embryo development was modified. Treated embryos showed an increase of reactive oxygen species. This study suggests that SiO 2 NPs are toxic but non-lethal and showed potential teratogenic effects in Xenopus. The latter may be due to their cellular accumulation and/or to the effect caused by the interaction of SiO 2 NPs with cytoplasmic and/or nuclear components. ROS production could contribute to the observed effects. In conclusion, the data indicates that the use of SiO 2 NPs requires close attention and further studies to better clarify their activity in animals, including humans., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tattooing is a technique that introduces colored substances under the skin in order to color it permanently. Decomposition products of tattoo pigments produce numerous damages for the skin and other organs. We studied the effects of a commercial red ink tattoo, PR170, on Xenopus laevis embryos and Daphnia magna nauplii using concentrations of 10, 20, and 40 mg/L. For Xenopus , we applied the FETAX protocol analyzing survival, malformations, growth, heart rate, and the expression of genes involved in the development. In D. magna , we evaluated the toxicity with an immobilization test. Moreover, we investigated the production of ROS, antioxidant enzymes, and the expression of the ATP-binding cassette in both models. Our results indicate that PR170 pigment has nanoparticle dimensions, modifies the survival and the ATP-binding cassette activity, and induces oxidative stress that probably produces the observed effects in both models. Deformed embryos were observed in Xenopus , probably due to the modification of expression of genes involved in development. The expression of pro-inflammatory cytokines was also modified in this amphibian. We think that these effects are due to the accumulation of PR170 and, in particular, to the presence of the azoic group in the chemical structure of this pigment. Further studies needed to better understand the effects of commercial tattoo inks.

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction ( lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0-55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii , an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

Metastasis is responsible for the majority of cancer-related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin-fixed paraffin-embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Cancer is considered a high-risk condition for severe illness resulting from COVID-19. The interaction between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and human metabolism is key to elucidating the risk posed by COVID-19 for cancer patients and identifying effective treatments, yet it is largely uncharacterised on a mechanistic level. We present a genome-scale map of short-term metabolic alterations triggered by SARS-CoV-2 infection of cancer cells. Through transcriptomic- and proteomic-informed genome-scale metabolic modelling, we characterise the role of RNA and fatty acid biosynthesis in conjunction with a rewiring in energy production pathways and enhanced cytokine secretion. These findings link together complementary aspects of viral invasion of cancer cells, while providing mechanistic insights that can inform the development of treatment strategies., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The inhibitory effect of extracellular DNA (exDNA) on the growth of conspecific individuals was demonstrated in different kingdoms. In plants, the inhibition has been observed on root growth and seed germination, demonstrating its role in plant-soil negative feedback. Several hypotheses have been proposed to explain the early response to exDNA and the inhibitory effect of conspecific exDNA. We here contribute with a whole-plant transcriptome profiling in the model species Arabidopsis thaliana exposed to extracellular self- (conspecific) and nonself- (heterologous) DNA. The results highlight that cells distinguish self- from nonself-DNA. Moreover, confocal microscopy analyses reveal that nonself-DNA enters root tissues and cells, while self-DNA remains outside. Specifically, exposure to self-DNA limits cell permeability, affecting chloroplast functioning and reactive oxygen species (ROS) production, eventually causing cell cycle arrest, consistently with macroscopic observations of root apex necrosis, increased root hair density and leaf chlorosis. In contrast, nonself-DNA enters the cells triggering the activation of a hypersensitive response and evolving into systemic acquired resistance. Complex and different cascades of events emerge from exposure to extracellular self- or nonself-DNA and are discussed in the context of Damage- and Pathogen-Associated Molecular Patterns (DAMP and PAMP, respectively) responses.

Background: Understanding the molecular and cellular processes that underpin animal development are crucial for understanding the diversity of body plans found on the planet today. Because of their abundance in the fossil record, and tractability as a model system in the lab, skeletons provide an ideal experimental model to understand the origins of animal diversity. We herein use molecular and cellular markers to understand the growth and development of the juvenile sea urchin (echinoid) skeleton., Results: We developed a detailed staging scheme based off of the first ~ 4 weeks of post-metamorphic life of the regular echinoid Paracentrotus lividus. We paired this scheme with immunohistochemical staining for neuronal, muscular, and skeletal tissues, and fluorescent assays of skeletal growth and cell proliferation to understand the molecular and cellular mechanisms underlying skeletal growth and development of the sea urchin body plan., Conclusions: Our experiments highlight the role of skeletogenic proteins in accretionary skeletal growth and cell proliferation in the addition of new metameric tissues. Furthermore, this work provides a framework for understanding the developmental evolution of sea urchin body plans on macroevolutionary timescales.

Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH 4 F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.

High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches., (© 2020 UICC.)

Stage I epithelial ovarian cancer (EOC) represents about 10% of all EOCs. It is characterized by a complex histopathological and molecular heterogeneity, and it is composed of five main histological subtypes (mucinous, endometrioid, clear cell and high, and low grade serous), which have peculiar genetic, molecular, and clinical characteristics. As it occurs less frequently than advanced-stage EOC, its molecular features have not been thoroughly investigated. In this study, using in silico approaches and gene expression data, on a multicentric cohort composed of 208 snap-frozen tumor biopsies, we explored the subtype-specific molecular alterations that regulate tumor aggressiveness in stage I EOC. We found that single genes rather than pathways are responsible for histotype specificities and that a cAMP-PKA-CREB1 signaling axis seems to play a central role in histotype differentiation. Moreover, our results indicate that immune response seems to be, at least in part, involved in histotype differences, as a higher immune-reactive behavior of serous and mucinous samples was observed with respect to other histotypes.

In this study, we use Escherichia coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on monomethylether poly(ethylene glycol), methyl methacrylate, and 2-dimethyl(aminoethyl) methacrylate, whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups that can generate a charged surface by protonation when in contact with water. For this purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that the polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from large unilamellar vesicles at different phospholipid compositions indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that the polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of the outer membrane structure ( fabR, fadR, fabA, waaA, waaC, kdsA, pldA, and pagP), as well as regulators of cellular response to oxidative stress ( soxS), are more expressed when bacteria are exposed to the polymer surface. All together these results identified the outer membrane as the main cellular target of the antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In this perspective, data reported here could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.

Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp-dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr, which was recruited as an Esrp target in stem chordates and subsequently co-opted into the development of many novel traits in vertebrates.

Bacteria are used in ecotoxicology for their important role in marine ecosystems and their quick, reproducible responses. Here we applied a recently proposed method to assess the ecotoxicity of nanomaterials on the ubiquitous marine bacterium Vibrio anguillarum , as representative of brackish and marine ecosystems. The test allows the determination of 6-h EC 50 in a wide range of salinity, by assessing the reduction of bacteria actively replicating and forming colonies. The toxicity of copper oxide nanoparticles (CuO NPs) at different salinities (5-20-35 ‰) was evaluated. CuSO 4 5H 2 O and CuO bulk were used as reference toxicants (solubility and size control, respectively). Aggregation and stability of CuO NP in final testing dispersions were characterized; Cu 2+ dissolution and the physical interactions between Vibrio and CuO NPs were also investigated. All the chemical forms of copper showed a clear dose-response relationship, although their toxicity was different. The order of decreasing toxicity was: CuSO 4 5H 2 O > CuO NP > CuO bulk. As expected, the size of CuO NP aggregates increased with salinity and, concurrently, their toxicity decreased. Results confirmed the intrinsic toxicity of CuO NPs, showing modest Cu 2+ dissolution and no evidence of CuO NP internalization or induction of bacterial morphological alterations. This study showed the V. anguillarum bioassay as an effective tool for the risk assessment of nanomaterials in marine and brackish environments.

Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100nM and 4μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

H-Prune hydrolyzes short-chain polyphosphates (PPase activity) together with an hitherto cAMP-phosphodiesterase (PDE), the latest influencing different human cancers by its overexpression. H-Prune promotes cell migration in cooperation with glycogen synthase kinase-3 (Gsk-3β). Gsk-3β is a negative regulator of canonical WNT/β-catenin signaling. Here, we investigate the role of Gsk-3β/h-Prune complex in the regulation of WNT/β-catenin signaling, demonstrating the h-Prune capability to activate WNT signaling also in a paracrine manner, through Wnt3a secretion. In vivo study demonstrates that h-Prune silencing inhibits lung metastasis formation, increasing mouse survival. We assessed h-Prune levels in peripheral blood of lung cancer patients using ELISA assay, showing that h-Prune is an early diagnostic marker for lung cancer. Our study dissects out the mechanism of action of h-Prune in tumorigenic cells and also sheds light on the identification of a new therapeutic target in non-small-cell lung cancer.

The toxic benthic dinoflagellate Ostreopsis cf. ovata has considerably expanded its distribution range in the last decade, posing risks to human health. Several aspects of this species are still poorly known. We studied ultrastructural features of cultivated and natural populations of Ostreopsis cf. ovata from the Gulf of Naples (Mediterranean Sea) using confocal laser scanning, and scanning and transmission electron microscopy. New information on the morphology and location of several sulcal plates was gained and a new plate designation is suggested that better fits the one applied to other Gonyaulacales. The microtubular component of the cytoskeleton, revealed using an anti-β-tubulin antibody, consisted of a cortical layer of microtubules arranged asymmetrically in the episome and in the hyposome, complemented by a complex inner microtubular system running from the sulcal area towards the internal part of the cell. The conspicuous canal was delimited by two thick, burin-shaped lobes ending in a tubular ventral opening. The canal was surrounded by mucocysts discharging their content into it. A similar structure has been reported in other benthic and planktonic dinoflagellates and may be interpreted as an example of convergent evolution in species producing large amounts of mucus., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE-ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light-driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4-based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4-dependent targeted degradation of the UV-lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4-dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.

In recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations in many different organisms, including plants. Because of their versatility and ease of use, these methods have gained favor in low- and medium-throughput investigations as well. However, due to the recombination step, the resulting fusion proteins contain long and often highly charged polylinker sequences that can interfere with their physiological function. Furthermore, in some cases the gene of interest must be cloned twice (once with and once without a stop codon) for N- and C-terminal tagging. Here, we present a hybrid combinatorial cloning strategy that overcomes many of these limitations. In the first step, the gene of interest is cloned into an entry vector containing standardized cloning sites with the desired N- or C-terminal tag and an optimized polylinker sequence. A Gateway recombination reaction is used to transfer the protein-tag fusion from the entry clone to a Gateway destination vector with the desired promoter and selectable marker for the organism of interest. As experimental requirements evolve, constructs for expressing the protein of interest with the desired tag, promoter, and selectable marker or other features can rapidly and easily be created.

Background: Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale., Results: Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations., Conclusion: This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.

Homologous recombination (HR) serves a dual role in providing genetic flexibility and in maintaining genome integrity. Little is known about the regulation of HR and other repair pathways in the context of chromatin. We report on a mutant affected in the expression of the Arabidopsis INO80 ortholog of the SWI/SNF ATPase family, which shows a reduction of the HR frequency to 15% of that in wild-type plants. In contrast, sensitivity to genotoxic agents and efficiency of T-DNA integration remain unaffected, suggesting that INO80 is a positive regulator of HR, while not affecting other repair pathways. So far, INO80 function has only been reported in a lower eukaryote. Profiling studies on three ino80 allelic mutants show that INO80 regulates nearly 100 Arabidopsis genes. However, the transcriptional regulation of repair-related genes is unaffected in the mutant. This suggests a dual role for INO80 in transcription and DNA repair by HR.

A large number of recent studies have demonstrated that many important aspects of plant development are regulated by heritable changes in gene expression that do not involve changes in DNA sequence. Rather, these regulatory mechanisms involve modifications of chromatin structure that affect the accessibility of target genes to regulatory factors that can control their expression. The central component of chromatin is the nucleosome, containing the highly conserved histone proteins that are known to be subject to a wide range of post-translational modifications, which act as recognition codes for the binding of chromatin-associated factors. In addition to these histone modifications, DNA methylation can also have a dramatic influence on gene expression. To accommodate the burgeoning interest of the plant science community in the epigenetic control of plant development, a series of methods used routinely in our laboratories have been compiled that can facilitate the characterization of putative chromatin-binding factors at the biochemical, molecular and cellular levels.

Background: Monitoring patients on oral anticoagulation is essential to prevent haemorrhage and recurrent thrombosis, but it is still difficult. We studied a group of 348 patients with mechanical heart valve prostheses to verify whether the use of a new portable prothrombin time device might improve the management of oral anticoagulant therapy., Methods and Material: We used a new portable prothrombin time device to check the anticoagulation therapy in a group of 348 patients with mechanical heart valve prostheses to validate its results by comparing to routine I.N.R. determinations (28 pts) and verify its user-friendliness by a face-to-face interview. Furthermore, the incidence of haemorrhagic and thromboembolic events has been studied by a two-year follow-up., Results: Pearson correlation indicated an R2 = 0.9 between I.N.R. values determined by routine or by a new portable prothrombin time device. All patients agreed to replace the routine I.N.R. determinations with those by the new procedure, particularly female, young and those with usually difficult blood collections. During the two-year follow-up period, 21 haemorrhagic (2.6/100/patients/year) and 5 thromboembolic events (0.6/100/patients/year) occurred; but neither replaced valves' thrombi nor fatal events were found out., Conclusions: I.N.R.s determined using a new portable prothrombin time device are convincing as routine determinations. Such new procedure improves the quality of life, and the therapeutical range of anticoagulation is maintained safer and longer than by routine determinations. These results suggest that the control of anticoagulation in patients to a set I.N.R. therapeutic range is improved by using portable prothrombin time device in expert anticoagulation clinics, such improvement may result in reducing mortality as well as morbidity in patients treated with oral anticoagulants.

Light provides a major source of information from the environment during plant growth and development. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., ). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis.

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.