Saccharomyces cerevisiae multifunctional protein RAP1 binds to a conserved sequence in the Polyoma virus enhancer and is responsible for its transcriptional activity in yeast cells

AbstractThe Polyoma virus enhancer (A + B domain) activates transcription in Saccharomyces cerevisiae when joined to appropriate yeast promoter elements. We demonstrate by DNase I footprints and inhibition of binding by specific antibody, that the yeast protein RAP1 binds to the B-domain of the Polyoma enhancer and, at least in some promoter contexts, is responsible for transcriptional activity of the enhancer B-domain in yeast. Close matches to a consensus RAP1-binding site are also present in other viral enhancers

Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms

A Novel Member of the Bacterial-Archaeal Regulator Family Is a Nonspecific DNA-binding Protein and Induces Positive Supercoiling

In hyperthermophilic Archaea genomic DNA is from relaxed to positively supercoiled in vivo because of the action of the enzyme reverse gyrase, and this peculiarity is believed to be related to stabilization of DNA against denaturation. We report the identification and characterization of Smj12, a novel protein of Sulfolobus solfataricus, which is homologous to members of the so-called Bacterial-Archaeal family of regulators, found in multiple copies in Eubacteria and Archaea. Whereas other members of the family are sequence-specific DNA- binding proteins and have been implicated in transcriptional regulation, Smj12 is a nonspecific DNA-binding protein that stabilizes the double helix and induces positive supercoiling. Smj12 is not abundant, suggesting that it is not a general architectural protein, but rather has a specialized function and/or localization. Smj12 is the first protein with the described features identified in Archaea and might participate in control of superhelicity during DNA transactions

Mental, physical and socio-economic status of adults living in spain during the late stages of the state of emergency caused by COVID-19

Research has shown that the confinement measures implemented to curb the spread of COVID-19 can have negative effects on people’s lives at multiple levels. The objective of this cross-sectional study was to better understand the mental, physical, and socio-economic status of adults living in Spain during the late stages of the state of emergency caused by COVID-19. Five hundred and forty-four individuals responded to an online survey between 3 June and 30 July 2020. They were asked to report data about their mental and physical health, financial situation, and satisfaction with the information received about the pandemic. Means, percentages, t-test, ANOVAs, and logistic regressions were computed. A third of the participants reported symptoms of anxiety, depression, and stress, and worries about their health and the future. Participants also described mild levels of fatigue and pain during lockdown (66%), and a reduction in household income (39%). Respondents that were female, younger, single, and with lower levels of education reported experiencing a greater impact of the COVID-19 pandemic. The data showed that the negative effects of lockdown were present in the late stages of the state of emergency. The findings can be used to contribute to the development of programs to prevent or mitigate the negative impact of confinement measures.Fundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase–helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein–protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways

The DNA Alkylguanine DNA Alkyltransferase-2 (AGT-2) Of Caenorhabditis Elegans Is Involved In Meiosis And Early Development Under Physiological Conditions

DNA alkylguanine DNA alkyltransferases (AGTs) are evolutionary conserved proteins that repair alkylation damage in DNA, counteracting the effects of agents inducing such lesions. Over the last years AGTs have raised considerable interest for both the peculiarity of their molecular mechanism and their relevance in cancer biology. AGT knock out mice show increased tumour incidence in response to alkylating agents, and over-expression of the human AGT protein in cancer cells is frequently associated with resistance to alkylating chemotherapy. While all data available point to a function of AGT proteins in the cell response to alkylation lesions, we report for the first time that one of the two AGT paralogs of the model organism C. elegans, called AGT-2, also plays unexpected roles in meiosis and early development under physiological conditions. Our data suggest a role for AGT-2 in conversion of homologous recombination intermediates into post-strand exchange products in meiosis, and show that agt-2 gene down-regulation, or treatment of animals with an AGT inhibitor results in increased number of germ cells that are incompatible with producing viable offspring and are eliminated by apoptosis. These results suggest possible functions for AGTs in cell processes distinct from repair of alkylating damage

Selected papers from the 15th and 16th international conference on Computational Intelligence Methods for Bioinformatics and Biostatistics

Funding Information: CIBB 2019 was held at the Department of Human and Social Sciences of the University of Bergamo, Italy, from the 4th to the 6th of September 2019 []. The organization of this edition of CIBB was supported by the Department of Informatics, Systems and Communication of the University of Milano-Bicocca, Italy, and by the Institute of Biomedical Technologies of the National Research Council, Italy. Besides the papers focused on computational intelligence methods applied to open problems of bioinformatics and biostatistics, the works submitted to CIBB 2019 dealt with algebraic and computational methods to study RNA behaviour, intelligence methods for molecular characterization and dynamics in translational medicine, modeling and simulation methods for computational biology and systems medicine, and machine learning in healthcare informatics and medical biology. A supplement published in BMC Medical Informatics and Decision Making journal [] collected three revised and extended papers focused on the latter topic.publishersversionpublishe

Clinical efficacy of an ultrasound-guided bilateral rectus sheath block for umbilical hernia repair in calves: A prospective randomized trial

Introduction: Surgical umbilical hernia repair is a frequent procedure in newborn calves, requiring mandatory pain management. This study aimed to develop an ultrasound-guided rectus sheath block (RSB) and to evaluate its clinical efficacy in calves undergoing umbilical herniorrhaphy under general field anesthesia. Methods: Gross and ultrasound anatomy of the ventral abdomen and the diffusion of a new methylene blue solution after injection within the rectus sheath were described in seven fresh calf cadavers. Then, fourteen calves undergoing elective herniorrhaphy were randomly assigned to receive either bilateral ultrasound-guided RSB with 0.3 mL/kg of bupivacaine 0.25% and 0.15 µg/kg of dexmedetomidine or 0.3 mL/kg of 0.9% NaCl (control). Intraoperative data included cardiopulmonary variables and anesthetic requirements. Postoperative data included pain scores, sedation scores and peri-incisional mechanical threshold assessed by force algometry at specific time points after anesthetic recovery. Treatments were compared using Wilcoxon rank-sum, Student's t-test, and Cox proportional hazard model as appropriate. Mixed effect linear models on rank, with random effect calf; fixed effects time, treatment, and their interaction were used to compare pain scores and mechanical thresholds over time. Significance was set at p = 0.05. Results and Discussion: Calves receiving RSB recorded lower pain scores between 45 – 120 minutes (p < 0.05) and at 240 min after recovery (p = 0.02). And they recorded higher mechanical thresholds between 45 and 120 min after surgery (p < 0.05). Ultrasound-guided RSB provided effective perioperative analgesia in calves undergoing herniorrhaphy under field conditions

<i>In vivo </i>and <i>in vitro</i> protein imaging in thermophilic archaea by exploiting a novel protein tag

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms

Metabolic Changes Associated with Different Levels of Energy Deficits in Mediterranean Buffaloes during the Early Lactation Stage: Type and Role of the Main Lipid Fractions Involved

Cell function and energy redistribution are influenced by lipid classes (phospholipids (PLs), free fatty acids (FFAs), triglycerides (TGs), and cholesterol esters (CEs)). The aim of this study was to investigate metabolic alterations that are related to changes in lipid classes according to different levels of energy deficits in early lactating Mediterranean buffaloes (MBs). Sixty-three MBs were enrolled at the beginning of lactation using an observational study with a cross-sectional experimental design. Serum β-hydroxybutyrate (BHB) levels were used to group the animals into a healthy group (Group H; n = 38; BHB < 0.70 mmol/L) and hyperketonemia risk group (Group K; n = 25; BHB ≥ 0.70 mmol/L). Statistical analysis was performed using a linear model that included the effect of the group and body condition score to assess differences in fatty acid (FA) concentrations. A total of 40 plasma FAs were assessed in each lipid class. Among the FAs, eight PLs, seven FFAs, four TGs, and four CEs increased according to BHB levels, while three FFAs, three TGs, and one CE decreased. The changes among lipid class profiles suggested the influence of inflammatory response, liver metabolism, and the state of body lipid reserves. In addition, the possible similarities of buffaloes at risk of hyperketonemia with ketotic cows suggest the necessity of further investigations in these ruminants