Promotion of Resilience in Migrants: A Systematic Review of Study and Psychosocial Intervention.

This systematic review aimed to contribute to a better and more focused understanding of the link between the concept of resilience and psychosocial interventions in the migrant population. The research questions concerned the type of population involved, definition of resilience, methodological choices and which intervention programmes were targeted at migrants. In the 90 articles included, an heterogeneity in defining resilience or not well specified definition resulted. Different migratory experiences were not adequately considered in the selection of participants. Few resilience interventions on migrants were resulted. A lack of procedure's descriptions that keep in account specific migrants' life-experiences and efficacy's measures were highlighted.

[Quality of professional life of working with asylum seekers: compassion satisfaction, burnout and secondary traumatic stress in reception professionals]. / La qualità della vita professionale di chi lavora con i richiedenti asilo: Compassion Satisfaction, Burnout e Secondary Traumatic Stress negli operatori dell'accoglienza.

SUMMARY: In the last years, in Italy, Extraordinary Reception Centres have been set up to meet the primary and secondary needs of asylum seekers landing on the Mediterranean coast. After the opening of the ERCs, a new professional body, the reception professionals, has been formed in the national territory. According to the context and the goals of the ERC, at the beginning no specific training was required, so their professional profile derived from the different background. Considering their institutional task, such as facilitating the reception and the full care of asylum seekers, the reception professionals are daily involved in the relationship with the respondents and exposed to their traumatic stories or symptoms. In fact, asylum seekers are people who are often deeply traumatized by past experiences, by the journey, but also disoriented and unprepared for the complex experience of reception and integration, wich influence the climate and quality of professional life of reception professionals. As happen in helping professionals continuously exposed to stressful or traumatic events, also in the work of helping for and welcoming asylum seekers there is a high risk of developing the negative symptoms associated with Burnout and vicarious trauma. Although, in the last twenty years, the quality of professional life has been extensively studied in several areas, there are no studies that explore this issue among professionals in the field of reception. In this study, the questionnaire ProQOL 5 was submitted to the reception professionals of the Extraordinary Reception Centres of Parma and its province, actively involved in helping relationship with asylum seekers, with the aim of defining the state of psychosocial well-being and their quality of professional life. Although it has been shown that on average reception professionals report good satisfaction in carrying out their work, three profiles have emerged. The first group reports a higher level of Burnout, the second group a greater Compassion Satisfaction and the third group, instead, a higher level of Burnout and Secondary Traumatic Stress. The data obtained allow to partially fill a gap in the literature. Moreover, the results suggest the need of interventions of prevention and management of organizations, in order to promote the psychosocial well-being of this emerging professional body.

New Insights into Structural and Functional Roles of Indole-3-acetic acid (IAA): Changes in DNA Topology and Gene Expression in Bacteria.

: Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E.meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms.

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.

Structure and Properties of DNA Molecules Over The Full Range of Biologically Relevant Supercoiling States.

Topology affects physical and biological properties of DNA and impacts fundamental cellular processes, such as gene expression, genome replication, chromosome structure and segregation. In all organisms DNA topology is carefully modulated and the supercoiling degree of defined genome regions may change according to physiological and environmental conditions. Elucidation of structural properties of DNA molecules with different topology may thus help to better understand genome functions. Whereas a number of structural studies have been published on highly negatively supercoiled DNA molecules, only preliminary observations of highly positively supercoiled are available, and a description of DNA structural properties over the full range of supercoiling degree is lacking. Atomic Force Microscopy (AFM) is a powerful tool to study DNA structure at single molecule level. We here report a comprehensive analysis by AFM of DNA plasmid molecules with defined supercoiling degree, covering the full spectrum of biologically relevant topologies, under different observation conditions. Our data, supported by statistical and biochemical analyses, revealed striking differences in the behavior of positive and negative plasmid molecules.

Every OGT Is Illuminated by Fluorescent and Synchrotron Lights.

O6-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs) are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus, respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins.

In vivo and in vitro 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.

Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase.

BACKGROUND: Alkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. METHODS: As a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. RESULTS: Conformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. CONCLUSIONS: Our data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. GENERAL SIGNIFICANCE: A general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.

RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals.

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3ß differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3ß proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3ß-polyribosome association requires TDRD3, which directly interacts with Top3ß and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.

A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA- alkyl-transferase by protein engineering.

In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of proteins (SNAP-tag™ technology) was proposed as a valid alternative to classical protein-tags (green fluorescent proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms. Here, we introduce an engineered variant of the thermostable alkylguanine-DNA-alkyl-transferase from the Archaea Sulfolobus solfataricus (SsOGT-H5), which displays a catalytic efficiency comparable to the SNAP-tag™ protein, but showing high intrinsic stability typical of proteins from this organism. The successful heterologous expression obtained in a thermophilic model organism makes SsOGT-H5 a valid candidate as protein-tag for organisms living in extreme environments.

NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing.

The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing.

Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins.

Chromatin structure and dynamics in hot environments: architectural proteins and DNA topoisomerases of thermophilic archaea.

In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity.

Genome stability: recent insights in the topoisomerase reverse gyrase and thermophilic DNA alkyltransferase.

Repair and defence of genome integrity from endogenous and environmental hazard is a primary need for all organisms. Natural selection has driven the evolution of multiple cell pathways to deal with different DNA damaging agents. Failure of such processes can hamper cell functions and induce inheritable mutations, which in humans may cause cancerogenicity or certain genetic syndromes, and ultimately cell death. A special case is that of hyperthermophilic bacteria and archaea, flourishing at temperatures higher than 80 °C, conditions that favor genome instability and thus call for specific, highly efficient or peculiar mechanisms to keep their genome intact and functional. Over the last few years, numerous studies have been performed on the activity, function, regulation, physical and functional interaction of enzymes and proteins from hyperthermophilic microorganisms that are able to bind, repair, bypass damaged DNA, or modify its structure or conformation. The present review is focused on two enzymes that act on DNA catalyzing unique reactions: reverse gyrase and DNA alkyltransferase. Although both enzymes belong to evolutionary highly conserved protein families present in organisms of the three domains (Eucarya, Bacteria and Archaea), recently characterized members from hyperthermophilic archaea show both common and peculiar features.

The reverse gyrase from Pyrobaculum calidifontis, a novel extremely thermophilic DNA topoisomerase endowed with DNA unwinding and annealing activities.

Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.

Biochemical and structural studies of the Mycobacterium tuberculosis O6-methylguanine methyltransferase and mutated variants.

Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the host's infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O(6)-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O(6)-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G · C-to-A · T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology.

Synergic and opposing activities of thermophilic RecQ-like helicase and topoisomerase 3 proteins in Holliday junction processing and replication fork stabilization.

RecQ family helicases and topoisomerase 3 enzymes form evolutionary conserved complexes that play essential functions in DNA replication, recombination, and repair, and in vitro, show coordinate activities on model recombination and replication intermediates. Malfunctioning of these complexes in humans is associated with genomic instability and cancer-prone syndromes. Although both RecQ-like and topoisomerase 3 enzymes are present in archaea, only a few of them have been studied, and no information about their functional interaction is available. We tested the combined activities of the RecQ-like helicase, Hel112, and the topoisomerase 3, SsTop3, from the thermophilic archaeon Sulfolobus solfataricus. Hel112 showed coordinate DNA unwinding and annealing activities, a feature shared by eukaryotic RecQ homologs, which resulted in processing of synthetic Holliday junctions and stabilization of model replication forks. SsTop3 catalyzed DNA relaxation and annealing. When assayed in combination, SsTop3 inhibited the Hel112 helicase activity on Holliday junctions and stimulated formation and stabilization of such structures. In contrast, Hel112 did not affect the SsTop3 DNA relaxation activity. RecQ-topoisomerase 3 complexes show structural similarity with the thermophile-specific enzyme reverse gyrase, which catalyzes positive supercoiling of DNA and was suggested to play a role in genome stability at high temperature. Despite such similarity and the high temperature of reaction, the SsTop3-Hel112 complex does not induce positive supercoiling and is thus likely to play different roles. We propose that the interplay between Hel112 and SsTop3 might regulate the equilibrium between recombination and anti-recombination activities at replication forks.

Activity and regulation of archaeal DNA alkyltransferase: conserved protein involved in repair of DNA alkylation damage.

Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability.