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Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue1. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described2. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch3. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2 –/– results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

The possible regulation of the expression of uncoupling protein-2 (UCP2) mRNA by thyroid hormones in different tissues was examined in rats, Triiodothyronine (T-3) was found to produce an organ-specific enhancement of UCP2 expression in rat tissues. The effect of T-3 was markedly observed in heart, whereas a moderate effect was seen in skeletal muscle and no effect in kidney or liver, These results suggest that UCP2 is a protein that may be involved in the nuclear-mediated effect of T-3 on resting metabolic rate in the rat. (C) 1997 Federation of European Biochemical Societies.

An in vivo footprinting analysis of the ilvIH regulatory region of Escherichia coli showed that the transcription activator Lrp binds to six sites, scattered over 250 bp upstream of the transcriptional start point. When Lrp-mediated activation was impaired by the presence of exogenous leucine, only one promoter-distal site (site 2) was partially protected by Lrp binding. Equilibrium dialysis experiments showed the formation of an Lrp-leucine complex in vitro. These results suggest that leucine negatively affects ilvIH transcription because its interaction with Lrp reduces the efficiency of binding of the regulatory protein to the promoter region.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) of the blastocyst. In serum/LIF culture condition, they show variable expression of pluripotency genes that mark cell fluctuation between pluripotency and differentiation metastate. The ESCs subpopulation marked by zygotic genome activation gene (ZGA) signature, including Zscan4, retains a wider differentiation potency than epiblast-derived ESCs. We have recently shown that retinoic acid (RA) significantly enhances Zscan4 cell population. However, it remains unexplored how RA initiates the ESCs to 2-cell like reprogramming. Here we found that RA is decisive for ESCs to 2C-like cell transition, and reconstructed the gene network surrounding Zscan4. We revealed that RA regulates 2C-like population co-activating Dux and Duxbl1. We provided novel evidence that RA dependent ESCs to 2C-like cell transition is regulated by Dux, and antagonized by Duxbl1. Our suggested mechanism could shed light on the role of RA on ESC reprogramming.

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has overwhelmed the health systems worldwide. Data regarding the impact of COVID-19 on cancer patients (CPs) undergoing or candidate for immune checkpoint inhibitors (ICIs) are lacking. We depicted the practice and adaptations in the management of patients with solid tumors eligible or receiving ICIs during the COVID-19 pandemic, with a special focus on Campania region.MethodsThis survey (25 questions), promoted by the young section of SCITO (Società Campana di ImmunoTerapia Oncologica) Group, was circulated among Italian young oncologists practicing in regions variously affected by the pandemic: high (group 1), medium (group 2) and low (group 3) prevalence of SARS-CoV-2–positive patients. For Campania region, the physician responders were split into those working in cancer centers (CC), university hospitals (UH) and general hospitals (GH). Percentages of agreement, among High (H) versus Medium (M) and versus Low (L) group for Italy and among CC, UH and GH for Campania region, were compared by using Fisher’s exact tests for dichotomous answers and χ2 test for trends relative to the questions with 3 or more options.ResultsThis is the first Italian study to investigate the COVID-19 impact on cancer immunotherapy, unique in its type and very clear in the results. The COVID-19 pandemic seemed not to affect the standard practice in the prescription and delivery of ICIs in Italy. Telemedicine was widely used. There was high consensus to interrupt immunotherapy in SARS-CoV-2–positive patients and to adopt ICIs with longer schedule interval. The majority of the responders tended not to delay the start of ICIs; there were no changes in supportive treatments, but some of the physicians opted for delaying surgeries (if part of patients’ planned treatment approach). The results from responders in Campania did not differ significantly from the national ones.ConclusionOur study highlights the efforts of Italian oncologists to maintain high standards of care for CPs treated with ICIs, regardless the regional prevalence of COVID-19, suggesting the adoption of similar solutions. Research on patients treated with ICIs and experiencing COVID-19 will clarify the safety profile to continue the treatments, thus informing on the most appropriate clinical conducts.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5&ndash, 11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn&minus, /&minus, mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the "Ruggero Ceppellini Advanced School of Immunology." The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists.

Distributed generation from wind and solar acts on regional electric demand as a reduced consumption, giving rise to a “load shadowing effect”. The net load becomes much more difficult to predict due to its dependence on the meteorological conditions. As a consequence, the growing penetration of variable generation increases the imbalance between demand and scheduled supply (net load forecast) and the reserve margins (net load uncertainty). The aim of this work is to quantify the benefit of the use of advanced probabilistic approaches rather than a traditional time-series method to assess the day-ahead reserves. For this purpose, several methods for load and net load uncertainty assessment have been developed and applied to a real case study considering also future solar penetration scenarios. The results show that, when forecasting only the load both traditional and probabilistic methods exhibit similar accuracy. Instead, in the case of net load prediction, i.e. when solar power is present, the probabilistic forecast can effectively limit the reserve margin needed to arrange the imbalance between residual demand and supply. The developed probabilistic approach provides a notable reduction of the Following Reserve which increases with the solar penetration: from 32.5% to 68.3% at 7% and 45% of penetration.

The development of successful climate services faces a number of challenges, including the identification of the target audience and their needs and requirements, and the effective communication of complex climate information, through engagement with a range of stakeholders. This paper describes how these challenges were tackled during the European Climatic Energy Mixes (ECEM) project, part of the Copernicus Climate Change Service (C3S), in order to deliver a pre-operational, proof-of-concept climate service for the European renewable energy sector. The process of iterative user engagement adopted in ECEM is described, from the initial presentation of the team’s first vision for such a service to support external stakeholders, through to evaluation of the final interactive tool for visualisation, data download and supporting documentation (the C3S ECEM Demonstrator). The outcomes of this evaluation are outlined, together with a retrospective reflection on the engagement and development process. The extent to which co-production and co-design were achieved in practice is assessed. The paper also highlights the distance travelled from the start to end of ECEM in terms of building capacity, developing a community of practice, and raising the Technology Readiness Level. The relevance of ECEM for the European climate services market is briefly considered, including the development of downstream commercial services which build upon the public C3S services. Keywords: Climate services, Sectoral information system, Renewable energy, User needs and requirements, Stakeholder engagement, Co-production

Solar photovoltaic energy is widespread worldwide and particularly in Europe, which became in 2016 the first region in the world to pass the 100 GW of installed capacity. As with all the renewable energy sources, for an effective management of solar power, it is essential to have reliable and accurate information about weather/climate conditions that affect the production of electricity. Operations in the solar energy industry are normally based on daily (or intra-daily) forecasts. Nevertheless, information about the incoming months can be relevant to support and inform operational and maintenance activities. This paper discusses a methodology to assess whether a seasonal climate forecast can provide a useful prediction for a specific sector, in this paper the European solar power industry. After evaluating the quality of the forecasts in providing probabilistic information for solar radiation, we describe how to assess their potential usefulness for a generic user by proposing an approach that takes into account not only their accuracy but also other potentially relevant factors. This approach is called index of opportunity and is then illustrated by presenting an example for the European solar power sector. The index of opportunity provides indications about where and when seasonal climate forecasts can benefit the decision-making in the photovoltaic sector. Even more importantly, it suggests an approach on how to evaluate their usefulness for the user's decision-making. This approach has the advantage of not limiting the definition of the usefulness only to the quality of the forecasts but rather considering, in an explicit way, all the factors that must be combined with the forecast's quality to define what is useful or not for the user.

Forkhead box E1 (FOXE1) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1-induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1. Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1-expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1. These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.

The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/&minus, ) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/&minus, genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.

The aim of the present study was to evaluate the primary stability of a two-miniscrew system inserted into a synthetic bone and to compare the system with the traditional one. Forty-five bi-layered polyurethane blocks were used to simulate maxillary cancellous and cortical bone densities. Samples were randomly assigned to three groups&mdash, one-miniscrew system (Group A, N = 23), two-miniscrew system (Group B, N = 22) and archwire-only (Group C, N = 10). A total of 67 new miniscrews were subdivided into Group A (23 singles) and Group B (22 couples). 30 mm of 19&Prime, &times, 25&Prime, archwires were tied to the miniscrew. The load was applied perpendicularly to the archwire. Maximum Load Value (MLV), Yield Load (YL) and Loosening Load (LL) were recorded for each group. The YL of Group B and C had a mean value respectively of 4.189 &plusmn, 0.390 N and 3.652 &plusmn, 0.064 N. The MLV of Group A, B and C had a mean value respectively of 1.871 &plusmn, 0.318N, of 4.843 &plusmn, 0.515 N and 4.150 &plusmn, 0.086 N. The LL of Group A and B had a mean value respectively of 1.871 &plusmn, 0.318 N and of 2.294 &plusmn, 0.333 N. A two- temporary anchorage device (TAD) system is on average stiffer than a one-TAD system under orthodontic loading.

DNAJC17 is a heat shock protein (HSP40) family member, identified in mouse as susceptibility gene for congenital hypothyroidism. DNAJC17 knockout mouse embryos die prior to implantation. In humans, germline homozygous mutations in DNAJC17 have been found in syndromic retinal dystrophy patients, while heterozygous mutations represent candidate pathogenic events for myeloproliferative disorders. Despite widespread expression and involvement in human diseases, DNAJC17 function is still poorly understood. Herein, we have investigated its function through high-throughput transcriptomic and proteomic approaches. DNAJC17-depleted cells transcriptome highlighted genes involved in general functional categories, mainly related to gene expression. Conversely, DNAJC17 interactome can be classified in very specific functional networks, with the most enriched one including proteins involved in splicing. Furthermore, several splicing-related interactors, were independently validated by co-immunoprecipitation and in vivo co-localization. Accordingly, co-localization of DNAJC17 with SC35, a marker of nuclear speckles, further supported its interaction with spliceosomal components. Lastly, DNAJC17 up-regulation enhanced splicing efficiency of minigene reporter in live cells, while its knockdown induced perturbations of splicing efficiency at whole genome level, as demonstrated by specific analysis of RNAseq data. In conclusion, our study strongly suggests a role of DNAJC17 in splicing-related processes and provides support to its recognized essential function in early development.

The increased penetration of photovoltaic power introduces new challenges for the stability of the electrical grid, both at the local and national level. Many different effects are caused by high solar power injection into the electric grid. Among them, the increased risk of imbalance between the actual and scheduled power transmission is of particular relevance. The consequence is the need to exchange larger amounts of dispatchable power on the balancing energy market. The aim of this work is to analyze and quantify the effects of PV penetration in a target region and to evaluate the energy and economic benefits of using day-ahead PV forecast for power transmission scheduling. For this purpose, we developed several data-driven methods for transmission scheduling that include day-ahead PV power forecasts. We compared the resulting operational imbalances from these new models against two reference models currently used by the local grid operators. In the case of no PV generation in the target area, the more accurate reference model leads to an imbalance of 3.6% of the peak power transmission while more accurate data-driven method reduces the imbalance to 3.2%. When the distributed PV capacity is not zero, the imbalance of the reference model grows from 5.15% (at the current penetration of 7%) to 9.8% (at the maximum planned regional penetration of 45%). When we apply the new scheduling model, imbalances are reduced to respectively 3.5% and 5.8% at 7% and 45% of penetration. Since in Italy the costs of imbalances resulting from distributed PV are borne by ratepayers, these costs are estimated to be respectively 2.3% and 15% of the average electricity bill at 7% and 45% penetration if the reference scheduling is used. When applying the new model these costs are respectively reduced to 1.2% and 8.5%. © 2018 Elsevier Ltd

This study investigated differences in periodontal health variables between buccally impacted maxillary canines (BIMC) and palatally impacted maxillary canines (PIMC) after surgical-orthodontic treatment with open technique. Nineteen patients were enrolled: 10 with unilateral BIMC (5 men, 5 women; mean age 18.50 +/- 1.96 years) and 9 with unilateral PIMC (4 men, 5 women; mean age 19.44 +/- 2.40 years). Probing depth and keratinized tissue were recorded 12 months after surgical-orthodontic treatment, and the differences between the 2 sides were analyzed as primary outcomes. In addition, data for BIMC and PIMC were directly compared. In the BIMC group, probing depths were significantly higher for lateral incisors than for the untreated side (P = 0.044), and keratinized tissue values were significantly lower for canines than for the untreated side (P = 0.006). No significant differences were observed in the PIMC group. In BIMC, surgical-orthodontic treatment with open technique resulted in loss of periodontal keratinized tissue in the treated tooth and periodontal attachment loss in adjacent lateral incisors. However, the periodontal status of PIMC was not affected by surgical-orthodontic treatment with open technique.

Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process. Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process.

Genetic and environmental factors contribute to thyroid diseases. Although still debated, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to induce thyroid dysfunction in humans and rodents. The data here reported point out the contribution of the exposure window and genetic background in mediating the low-dose TCDD effects on thyroid. Indeed, early (from E0.5 to PND30) and low-dose (0,001 μg/kg/day) TCDD exposure reduced the circulating fT4 and altered the expression of thyroid specific transcripts. The role of genetic components was estimated monitoring the same markers in Pax8+/− and Nkx2-1+/− mice, susceptible to thyroid dysfunction, exposed to 0, 1 μg/kg/day TCDD from E15.5 to PND60. Haploinsufficiency of either Pax8 or Nkx2-1 genes exacerbated the effects of the exposure impairing the thyroid enriched mRNAs in sex dependent manner. Such effect was mediated by mechanisms involving the Nkx2-1/p53/p65/IĸBα pathway in vitro and in vivo. Foetal exposure to TCDD impaired both thyroid function and genes expression while thyroid development and differentiation did not appear significantly affected. In mouse, stronger effects were related to earlier exposure or specific genetic background such as either Pax8 or Nkx2-1 haploinsufficiency, both associated to hypothyroidism in humans. Furthermore, our data underline that long exposure time are needed to model in vitro and in vivo results.

We derive analytic approximations to the solutions of the Lane-Emden equation, a basic equation in Astrophysics that describes the Newtonian equilibrium structure of a self-gravitating polytropic fluid sphere. After recalling some basic results, we focus on the construction of rational approximations, discussing the limitations of previous attempts, and providing new accurate approximate solutions. © 2015 Elsevier B.V.

Accurate and robust short-term load forecasting plays a significant role in electric power operations. This paper proposes a variant of genetic programming, improved by incorporating semantic awareness in algorithm, to address a short term load forecasting problem. The objective is to automatically generate models that could effectively and reliably predict energy consumption. The presented results, obtained considering a particularly interesting case of the South Italy area, show that the proposed approach outperforms state of the art methods. Hence, the proposed approach reveals appropriate for the problem of forecasting electricity consumption. This study, besides providing an important contribution to the energy load forecasting, confirms the suitability of genetic programming improved with semantic methods in addressing complex real-life applications. © 2014 Elsevier B.V.

Air temperature is an effective predictor for electricity demand, especially during hot periods where the need of electric air conditioning can be high. This paper presents for the first time an assessment of the use of seasonal climate forecasts of temperature for medium-term electricity demand prediction. The retrospective seasonal climate forecasts provided by ECWMF (European Centre for Medium-Range Weather Forecasts) are used to forecast the June-July Italian electricity demand for the period 1990-2007.We find a relationship between summer (June-July) average temperature patterns over Europe and Italian electricity demand using both a linear and non-linear regression approach. With the aim to evaluate the potential usefulness of the information contained into the climate ensemble forecast, the analysis is extended considering a probabilistic approach.Results show that, especially in the Center-South of Italy, seasonal forecasts of temperature issued in May lead to a significant correlation coefficient of electricity demand greater than 0.6 for the summer period. The average correlation obtained from seasonal forecasts is 0.53 for the temperature predicted in May and 0.19 for the predictions issued in April for the linear model, while the non-linear approach leads to the coefficients of 0.62 and 0.36 respectively. For the probabilistic approach, seasonal forecasts exhibit a positive and significant skill-score in predicting the demand above/below the upper/lower tercile in many regions.This work is a significant progress in understanding the relationship between temperature and electricity demand. It is shown that much of the predictable electricity demand anomaly over Italy is connected with so-called heat-waves (i.e. long lasting positive temperature anomalies) over Europe. © 2014 Elsevier Ltd.

This study fills in knowledge gaps for the European air-conditioning (AC) market, which is fundamentally important to raising awareness about primary energy utilization. In contrast to space heating (SH) and domestic hot water (DHW) preparation, the European Union (EU) AC market is barely explored in scientific literature. While the focus of previous research has been on the residential sector, a shortfall of data for the services (wholesale and retail, offices, education, health, hotels and bars) exists. In this paper, data describing the actual space cooling (SC) market in Europe (quantity of SC units, equivalent full-load hours, installed capacities, seasonal energy efficiency values as well as cooled floor area per AC type and/or sector) is collected and explored using a bottom-up approach. Results indicate that SC is responsible for a significant portion of EU electricity consumption in households (nearly 5%) and even more in the service sector (~13%). Energy consumption for SC in the EU28 appears to be more than 140 TWh/y. The quantification of the European AC consumption shows a significant difference between the service and residential sectors: about 115 versus 25 TWh/y respectively. The SC market in Europe is characterized by a high potential for growth, especially in households., We would like to convey our deepest appreciation to the Horizon 2020 Hotmaps Project (Grant Agreement No. 723677), which provided the funding to carry out the present investigation. We are thankful to Pierre Iachetti (Eurac Research), who edited the entire text of the present investigation. Finally, our gratitude goes to Amy Segata (Eurac Research) for designing the graphs of this study.

Photovoltaic (PV) power forecasting has the potential to mitigate some of effects of resource variability caused by high solar power penetration into the electricity grid. Two main methods are currently used for PV power generation forecast: (i) a deterministic approach that uses physics-based models requiring detailed PV plant information and (ii) a data-driven approach based on statistical or stochastic machine learning techniques needing historical power measurements. The main goal of this work is to analyze the accuracy of these different approaches. Deterministic and stochastic models for dayahead PV generation forecast were developed, and a detailed error analysis was performed. Four years of site measurements were used to train and test the models. Numerical weather prediction (NWP) data generated by the weather research and forecasting (WRF) model were used as input. Additionally, a new parameter, the clear sky performance index, is defined. This index is equivalent to the clear sky index for PV power generation forecast, and it is here used in conjunction to the stochastic and persistence models. The stochastic model not only was able to correct NWP bias errors but it also provided a better irradiance transposition on the PV plane. The deterministic and stochastic models yield day-ahead forecast skills with respect to persistence of 35% and 39%, respectively. Copyright © 2017 by ASME.

The growing photovoltaic generation results in a stochastic variability of the electric demand that could compromise the stability of the grid, increase the amount of energy reserve and the energy imbalance cost. On regional scale, the estimation of the solar power generation from the real time environmental conditions and the solar power forecast is essential for Distribution System Operators, Transmission System Operator, energy traders, and Aggregators. In this context, a new upscaling method was developed and used for estimation and forecast of the photovoltaic distributed generation in a small area of Italy with high photovoltaic penetration. It was based on spatial clustering of the PV fleet and neural networks models that input satellite or numerical weather prediction data (centered on cluster centroids) to estimate or predict the regional solar generation. Two different approaches were investigated. The simplest and more accurate approach requires a low computational effort and very few input information should be provided by users. The power estimation model provided a RMSE of 3% of installed capacity. Intra-day forecast (from 1 to 4 h) obtained a RMSE of 5%–7% and a skill score with respect to the smart persistence from −8% to 33.6%. The one and two days ahead forecast achieved a RMSE of 7% and 7.5% and a skill score of 39.2% and 45.7%. The smoothing effect on cluster scale was also studied. It reduces the RMSE of power estimation of 33% and the RMSE of day-ahead forecast of 12% with respect to the mean single cluster value. Furthermore, a method to estimate the forecast error was also developed. It was based on an ensemble neural network model coupled with a probabilistic correction. It can provide a highly reliable computation of the prediction intervals. © 2017 Elsevier Ltd

Electricity demand forecasting is a critical task for energy management of power grids. Due to the wide use of refrigeration and residential air-conditioning devices, electricity demand in Italy is influenced by weather conditions, especially during summer. This paper performs daily load forecasting for Italy through statistical modeling with the aim of studying the influence of temperature. The actual capability of available weather forecasts to contribute in predicting electricity loads is evaluated by using weather data from numerical weather prediction (NWP) models. Time-series models have been used and compared with a naive predictor on working-days daily load during June and July in years 2003-2009 considering lead-times between one and five days. Results are analyzed both at the national level and at regional scale, using unprecedented historical load data provided by the Italian transmission grid manager. It is shown that the use of weather data provided by NWP models leads to performance improvements, especially for the hottest areas where the use of electricity is more heavily influenced by temperature. Furthermore, by observing the gap between load forecast models using reanalysis and operational forecast weather data we can obtain some clues about the limitations of the weather forecast models we used on specific geographic areas in Italy. © 2013 Elsevier B.V.

The temporal variance of soil moisture, vegetation and evapotranspiration over land has been recognized to be strongly connected to the temporal variance of precipitation. However, the feedbacks and couplings between these variables are still not well understood and quantified. Furthermore, soil moisture and vegetation processes are associated with a memory and therefore they may have important implications for predictability. In this study we apply a generalized linear method, specifically designed to assess the reciprocal forcing between connected fields, to the latest available observational data sets of global precipitation, evapotranspiration, vegetation and soil moisture content. For the first time a long global observational data set is used to investigate the spatial and temporal land variability and to characterize the relationships and feedbacks between land and precipitation. The variables considered show a significant coupling among each other. The analysis of the response of precipitation to soil moisture evidences a robust coupling between these two variables. In particular, the first two modes of variability in the precipitation forced by soil moisture appear to have a strong link with volcanic eruptions and El Niño–Southern Oscillation (ENSO) cycles, respectively, and these links are modulated by the effects of evapotranspiration and vegetation. It is suggested that vegetation state and soil moisture provide a biophysical memory of ENSO and major volcanic eruptions, revealed through delayed feedbacks on rainfall patterns. The third mode of variability reveals a trend very similar to the trend of the inter-hemispheric contrast in sea surface temperature (SST) and appears to be connected to greening/browning trends of vegetation over the last three decades.

The aim of the paper is to compare several data-driven models using different Numerical Weather Prediction (NWP) input data and then to build up an outperforming Multi-Model Ensemble (MME) and its prediction intervals. Statistic, stochastic and hybrid machine-learning algorithms were developed and the NWP data from IFS and WRF models were used as input. It was found that the same machine learning algorithm differs in performance using as input NWP data with comparable accuracy. This apparent inconsistency depends on the capability of the machine learning model to correct the bias error of the input data. The stochastic and the hybrid model using the same WRF input, as well as the stochastic and the non-linear statistic models using the same IFS input, produce very similar results. The MME resulting from the averaging of the best data-driven forecasts, improves the accuracy of the outperforming member of the ensemble, bringing the skill score from 42% to 46%. To reach this performance, the ensemble should include forecasts with similar accuracy but generated with the higher variety of different data-driven technology and NWP input. The new performance metrics defined in the paper help to explain the reasons behind the different models performance. © 2016 Elsevier Ltd.

Epidemiological and experimental data highlighted the thyroid-disrupting activity of bisphenol A (BPA). Although pivotal to identify the mechanisms of toxicity, direct low-dose BPA effects on thyrocytes have not been assessed. Here, we report the results of microarray experiments revealing that the transcriptome reacts dynamically to low-dose BPA exposure, adapting the changes in gene expression to the exposure duration. The response involves many genes, enriching specific pathways and biological functions mainly cell death/proliferation or DNA repair. Their expression is only slightly altered but, since they enrich specific pathways, this results in major effects as shown here for transcripts involved in the DNA repair pathway. Indeed, even though no phenotypic changes are induced by the treatment, we show that the exposure to BPA impairs the cell response to further stressors. We experimentally verify that prolonged exposure to low doses of BPA results in a delayed response to UV-C-induced DNA damage, due to impairment of p21-Tp53 axis, with the BPA-treated cells more prone to cell death and DNA damage accumulation. The present findings shed light on a possible mechanism by which BPA, not able to directly cause genetic damage at environmental dose, may exert an indirect genotoxic activity.

A group of 157 women with primary breast cancer (BC) were typed for HLA antigens, and gene frequencies were compared to those of 327 control healthy individuals. Diagnosis of BC was made for all patients on surgical mastectomy specimens; histologic grading, estrogen (ER) and progesterone (PgR) receptors were determined on all primary tumors. Typed antigens included the majority of the specificities controlled by the HLA-A, -B and -C loci, according to the 8th International Histocompatibility Testing Workshop recommendations. No significant discrepancy in their frequencies was found in the undivided sample as compared to controls. The analysis of HLA gene frequency was extended to subsets of patients identified by the following prognostic features: (a) age at tumor diagnosis (pre-menopause vs. post-menopause); (b) receptor status (presence vs. absence of ER and PgR); (c) mammary gland dysplasia (presence vs. absence); (d) histologic grade (grade 3 vs. grades 1 and 2 combined); (e) time to relapse before or after 24 months following mastectomy). A moderate deviation from normal of some genes was found in several subsets, often affecting only one of the antithetical subgroups (feature present vs. feature absent). In the instance of B5, the increase in frequency of the gene in one of the subset pair (ER + subjects) was balanced by a decrease of the same gene in the counterpart (ER-subjects). Increased frequencies were found for the B7 gene in the following prognostic groups: (a) lack of ER (0.08); (b) lack of PgR (0.09); (c) absence of mammary dysplasia (0.075); (d) histologic grade 3 (0.10); and (e) premenopause (0.12), the last two showing significant divergence from normal. When features (d) and (e) on the one hand and (a), (b), (d) and (e) on the other were combined, B7 reached frequencies of 0.18 (p less than 5 X 10(-4] and of 0.29 (p less than 5 X 10(-6], respectively.

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In the present study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of the Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5β. The corresponding protein, termed Hel112 by us, was produced in Escherichia coli in soluble form, purified to homogeneity and characterized. Gel-filtration chromatography and glycerol-gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterization of the two oligomeric species revealed that only the monomeric form has an ATP-dependent 3′–5′ DNA-helicase activity, whereas, unexpectedly, both the monomeric and dimeric forms possess DNA strand-annealing capability. The Hel112 monomeric form is able to unwind forked and 3′-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10−9M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200), recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma), renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO) mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman's capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3β precedes the development of the glomerulocystic phenotype. Downregulation of β-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3β. Alterations of β-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3β/β-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.

Load forecasting is a critical task for all the operations of power systems. Especially during hot seasons, the influence of weather on energy demand may be strong, principally due to the use of air conditioning and refrigeration. This paper investigates the application of Genetic Programming on day-ahead load forecasting, comparing it with Neural Networks, Neural Networks Ensembles and Model Trees. All the experimentations have been performed on real data collected from the Italian electric grid during the summer period. Results show the suitability of Genetic Programming in providing good solutions to this problem. The advantage of using Genetic Programming, with respect to the other methods, is its ability to produce solutions that explain data in an intuitively meaningful way and that could be easily interpreted by a human being. This fact allows the practitioner to gain a better understanding of the problem under exam and to analyze the interactions between the features that characterize it.

Photovoltaic (PV) power production increased drastically in Europe throughout the last years. About the 6% of electricity in Italy comes from PV and for an efficient management of the power grid an accurate and reliable forecasting of production would be needed. Starting from a dataset of electricity production of 65 Italian solar plants for the years 2011-2012 we investigate the possibility to forecast daily production from one to ten days of lead time without using on site measurements. Our study is divided in two parts: an assessment of the predictability of meteorological variables using weather forecasts and an analysis on the application of data-driven modelling in predicting solar power production. We calibrate a SVM model using available observations and then we force the same model with the predicted variables from weather forecasts with a lead time from one to ten days. As expected, solar power production is strongly influenced by cloudiness and clear sky, in fact we observe that while during summer we obtain a general error under the 10% (slightly lower in south Italy), during winter the error is abundantly above the 20%., Submitted to Renewable Energy

The eukaryotic GINS complex has an essential role in the initiation and elongation phases of genome duplication. It is composed of four paralogous subunits--Sld5, Psf1, Psf2 and Psf3--which are ubiquitous and evolutionarily conserved in eukaryotic organisms. Here, we report the biochemical characterization of the human GINS complex (hGINS). The four hGINS subunits were coexpressed in Escherichia coli in a highly soluble form and purified as a complex. hGINS was shown to interact directly with the heterodimeric human DNA primase, by using either surface plasmon resonance measurements or by immunoprecipitation experiments carried out with anti-hGINS antibodies. The DNA polymerase alpha-primase synthetic activity was specifically stimulated by hGINS on various primed DNA templates. The significance of these findings is discussed in view of the molecular dynamics at the human replication fork.

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

The analysis of deformation and damage mechanisms induced by shallow tunnelling on masonry structures is carried out using an integrated, geotechnical and structural, numerical approach based on two-dimensional finite-element analyses. The masonry construction, schematised as a block structure with periodic texture, is regarded at a macroscopic scale as a homogenised anisotropic medium. The overall mechanical properties display anisotropy and singularities in the yield surface, arising from the discrete nature of the block structure and the geometrical arrangement of the blocks. The soil is modelled by means of a linear elastic-perfectly plastic model. The numerical analyses are performed assuming plane strain and plane stress conditions for the soil and the masonry structure, respectively. A displacement-controlled technique is adopted to simulate the tunnel construction, which produces settlement troughs in agreement with the empirical Gaussian predictions at different volume losses under free-field conditions. In order to test the numerical approach, a preliminary set of parametric analyses is carried out considering a simple masonry wall, characterised by different geometrical and mechanical properties, founded on a clayey deposit. Then, the case study of the Felice aqueduct in Rome (Italy), undercrossed by two tunnels of a new metro line, is considered. Significant differences are observed between the uncoupled analysis, where displacements predicted under free-field conditions are simply applied at the foundation level of the structure, and the interaction-based one, the latter being characterised by a reduced amount of tensile plastic strain. Numerical results in terms of vertical displacements at the ground level and on the structure are found to be in good agreement with monitoring data, thus validating the numerical model for this class of soil–structure interaction problems.

We derive accurate analytic approximations to the solution of the isothermal Lane-Emden equation, a basic equation in Astrophysics that describes the Newtonian equilibrium structure of self-gravitating, isothermal fluid spheres. The solutions we obtain, using analytic arguments and rational approximations, have simple forms, and are accurate over a radial extent that is much larger than that covered by conventional series expansions around the origin. Our best approximation has a maximum error on density of 0.04 % at 10 core radii, and is still within 1 % from an accurate numerical solution at a radius three times larger. © 2014 Springer Science+Business Media Dordrecht.

In the energy sector climate information can play a strategic role, particularly in hot spot regions such as the Mediterranean region. In order to limit GHG emissions, within the European Union framework there will be opportunities for trading renewable energy quotas among member states and to gain credit for electricity imported from renewable sources in countries outside the European Union. This framework will trigger new strong interaction between climate information providers and energy sector stakeholders. The basic assumption of this perspective, of course, is that a prediction of climate on multi-decadal time scales is attainable. © 2013 Elsevier Inc. All rights reserved.

Background The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene. Results We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it. Conclusions Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.

Background Chondroitin sulphate is a complex polysaccharide having important structural and protective functions in animal tissues. Extracted from animals, this compound is used as a human anti-inflammatory drug. Among bacteria, Escherichia coli K4 produces a capsule containing a non-sulphate chondroitin and its development may provide an efficient and cheap fermentative production of the polysaccharide. Results A random N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis was performed on E. coli K4 to isolate mutants showing an increased production of chondroitin. Several mutants were isolated, one of which, here named VZ15, produced about 80% more chondroitin than the wild type E. coli. We found that the mutant has a missense mutation in the codon 313 of kfoC, the gene encoding chondroitin polymerase (K4CP), with a change from arginine to glutamine. A docking analysis to explain the increased productivity of the K4CP enzyme is presented. Conclusion The enhanced chondroitin production by the E. coli K4 mutant reported here shows the validity of the strain improvement strategy for more cost-friendly fermentative processes in the production of this pharmaceutically important but so-far expensive polysaccharide.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1-268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269-686); and the C-terminal domain (C-ter, residues 504-686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the alpha-helices forming the "fingers" sub-domain (alpha-helices R, R' and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but - in contrast to mice homozygous for a null K-ras allele - they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis. © 2005 European Molecular Biology Organization.

The cotG and cotH genes of Bacillus subtilis encode two previously characterized spore coat proteins. The two genes are adjacent on the chromosome and divergently transcribed by sigma(K), a sporulation-specific sigma factor of the RNA polymerase. We report evidence that the cotH promoter maps 812 bp upstream of the beginning of its coding region and that the divergent cotG gene is entirely contained between the promoter and the coding part of cotH. A bioinformatic analysis of all entirely sequenced prokaryotic genomes showed that such chromosomal organization is not common in spore-forming bacilli. Indeed, CotG is present only in B. subtilis, B. amyloliquefaciens, and B. atrophaeus and in two Geobacillus strains. When present, cotG always encodes a modular protein composed of tandem repeats and is always close to but divergently transcribed with respect to cotH. Bioinformatic and phylogenic data suggest that such genomic organizations have a common evolutionary origin and that the modular structure of the extant cotG genes is the outcome of multiple rounds of gene elongation events of an ancestral minigene

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.

Objective— Tissue factor (TF) is normally expressed at low levels in the media of blood vessels, but it is readily induced after vessel injury. It is not known whether vascular damage per se or thrombus formation is responsible for this phenomenon. Methods and Results— Cyclic flow variations (CFVs), attributable to recurrent thrombus formation, were induced in stenotic rabbit carotid arteries with endothelial injury. CFVs were observed for 30 minutes and 2, 4, and 8 hours in different groups of animals. Another group of rabbits pretreated with hirudin before inducing arterial damage to inhibit thrombus formation was observed for 8 hours. Arterial sections were immunostained for TF. Undamaged arteries served as controls. In additional rabbits, in situ hybridization experiments were performed. No TF expression was observed in the media of control vessels, whereas a progressive increase in TF mRNA and protein expression was observed in carotid arteries as CFVs progressed. No increase in TF expression was observed in animals pretreated with hirudin. In vitro experiments demonstrated that TF mRNA is induced in smooth muscle cells stimulated with activated platelets as well as with some platelet-derived mediators. Conclusions— This phenomenon may contribute to sustain intravascular thrombus formation after the initial thrombogenic stimulus.