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Libraries and archives house a wide variety of documents made of materials of vegetal and animal origin: unbound papers and books, manuscripts and printed books, photographs (negative and positive), prints, maps, available to the public for reading needs, study and information. These materials are often subject to fluctuations in environmental and micro-environmental parameters. For this reason, it is essential to implement constant monitoring and control of environmental conditions and potential deteriogens in order to slow down deterioration processes. The monitoring of the microbial degradation of paper documents in the Historical Archives of Palermo Astronomical Observatory has revealed microorganisms (bacteria and fungi) that may be considered responsible for damaging the items examined, thus enabling an evaluation of the real risks and the proper methodologies to use to avoid future recolonization., Conservation Science in Cultural Heritage, Vol 15, No 2 (2015): Special Issue for Second Conference on Biology and Archaeobiology – from Knowledge to Preventive Conservation

Stone works of art located outdoors are exposed to natural deterioration, due to several physical, chemical and biological factors. Biological macro- and micro-systems (e.g. weeds, insects, algae, mosses, fungi and bacteria) may induce the biodeterioration of stone materials. In February 2014, the state of conservation of the Fountain of the two Dragons in Palermo was examined, during which time biological colonization in some areas of the fountain and statues, was also detected. The monument represents an excellent substrate for the development and growth of microrganisms and organisms that need a continuous supply of water. Direct solar irradiance promotes the development of photoautotrophic organisms, bryophyte flora, in addition to several microbial genera, responsible for pigmentation, discoloring, efflorescence. After autotrophic colonization, some organisms find an ideal habitat also during wintertime, including two species of gastropods and two species of Hemiptera, omnivorous, a species of Collembola, mostly fungivorous. This interdisciplinary study has enabled the detection and identification of different biological communities, providing information for a subsequent project of preventive conservation of the fountain and its surroundings., Conservation Science in Cultural Heritage, Vol 15, No 2 (2015): Special Issue for Second Conference on Biology and Archaeobiology – from Knowledge to Preventive Conservation

The biodeterioration of historic-artistic manufacts is related to several biological systems, including fungi and bacteria, whose metabolic activities and vegetative development have a direct consequence on the conservation of cultural assets. Generally, different chemical compounds are utilized as biocides in order to control biodeteriogens growth, but recently the attention has been focused on potential risks of their use towards human health (operators, visitors) and the environment. In order to develop alternative methods, various natural products have been tested, particularly to control the colonization by fungi and bacteria. In this study, antimicrobial activity of three different plant products, Tea tree essential oil, Calamintha nepeta and Allium sativum L. extracts, has been evaluated against Bacillus subtilis, Micrococcus luteus, Penicillium chrysogenum and Aspergillus spp. (previously isolated from colonized artworks) through three different in vitro antimicrobial assays (micro-dilution in microtiter plates, well plates diffusion and agar disc diffusion method). The bioassays show a different microbial susceptibility to plant extracts, establishing for each bacteria and fungi the Minimum Inhibitory Concentration (MIC) and defining the diameter of the growth inhibition area. This result supports the data reported in literature and shows an important potential suggestion for the possible use in the control of biodeterioration of cultural heritage, safe both for human health and environment.

The phylum of Cnidaria is one of the first branches in the tree of animal life to provide crucial insights on the evolution of immunity. Cnidarians are diblastic aquatic animals with radial symmetry and they are the simplest multicellular organisms that have reached the level of tissue organization. The renewed interest in the study of immunity in Cnidaria has led to additional information to the scenario of the first stages of immunity evolution revealing the cellular processes involved in symbiosis, in the regulation of homeostasis and in the fight against infections. We investigated the inflammatory response in Cnidarian following injection of various substances different in type and dimension, and observed clear, strong and specific reactions especially after injection of bacteria. The enzymes evaluation (protease, phosphatase and esterase), showing how the injection of different bacterial strains alters the expression of these enzymes suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. The Cnidaria phylum has evolved using biotoxins as defense or predation mechanisms for ensure survival in hostile and competitive environments such as the seas and oceans indeed the tissues and the mucus produced by cnidarians are involved in immune defense and contain a large variety of toxins such enzymes, potent pore forming toxins, and neurotoxins. They could also take advantage of the multi-functionality of some of their toxins. The bioactive molecules were characterized and purified by biological assays, acid extraction, HPLC purifications, mass spectroscopy and peptide synthesis. Here, we show the cnidarian bioactive molecules as antimicrobial peptides and enzymes in order to draw important applications in fields ranging from pharmacology to cultural heritage.

An integrated approach for the characterization of bioaerosol was employed in different sites (that include hypogeal and semi-confined areas), characterized by great cultural/artistic interest besides peculiar architectural structures, thermo- hygrometric and lighting parameters. These typologies of indoor environments preserve several artworks like mural paintings, stone-works, paper or parchments that are susceptible of microbial colonization. The presence of fungal spores and low air change can induce both potentially effects to human health (users/operators) or biodegradation of historical-artistic manufacts. We perform bioaerosol sampling by a portable sampler (Sartorius MD8), equipped with gelatin filters and non-invasive sampling (Nylon membrane or sterile swab) is carried out on works of art surface. Microbial consortia is revealed and characterized by Optical, Scanning Electron and Confocal Laser Scanning Microscopy (OM, SEM, CLSM), in vitro culture and molecular analysis (PCR, sequencing, sequence analysis). The inter-disciplinary approach applied in this study, represents a valuable contribution for the proper planning of both direct and/or indirect biological growth control and for the conservative restoration procedure (1, 2). This work was supported by Research Project It@cha - Italian Technologies for Advanced application in Cultural Heritage Assets, grant PON 01_00625 “Ricerca e Competitività” 2007-2013. (1) Palla F. et al. (2006) Characterization of bacterial community in indoor environment. Heritage, Weathering and Conservation, 1: 361-365. (2) Palla F. et al. (2010) Microscopy and molecular biology techniques for the study biocenosis diversity in semi-confined environments. Conservation Science in Cultural Heritage, 10: 185-194.

In this study molecular tools are applied to reveal and identify bacterial colonization in waterlogged wood to assessing the changes induced in anatomical structure, previously observed by Optical and Scanning Electron Microscopy (1). The results obtained by observation of wooden thin sections (OM), shown the presence of black and dark-brown areas and mineral concretions. The SEM analysis revealed a specific cell walls alteration, attributable to bacterial activity, other than abundant pyrite framboids (FeS2). The presence of sulfur compounds in archaeological waterlogged wood can indicate both long-term burial in anoxic environment and colonization by sulfate-reducing bacteria. Molecular methods allow us extract microbial genomic DNA from wood samples and in vitro amplify (PCR) bacteria DNA target sequences (16S, ITS-rRNA) (2). Through sequences analysis of PCR products cellulosolytic and ligninolytic bacteria, such as Pseudomonas, Cellulomonas, Xanthomonas and Bacillus spp, have been revealed. Moreover the presence of Marinobacter sp. and Desulforudis audaxviator, respectively iron-oxidizing and sulfate-reducing bacteria, are identify. We hypothesize that this investigation approach, can be applied to a variety of wooden artifacts of archaeological findings for both characterization of microbial colonization in order to understanding the main degradation phenomena, indispensable for a correct conservation strategies. (1) Safa A. et al. (2012) Using SEM in monitoring changes in archaeological wood: A review. Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) (2) Palla, F., (2012) Analytical techniques: analysis of microbial colonization. In Science and Conservation in Museum Collections, B. Fabbri (ed), Nardini, Firenze. 14, 459-470.

S32. Wooden artifacts: from wood identification to dating and conservation/restoration strategies DNA analysis as tool for identification of bacteria in archaeological waterlogged wood Palla, Franco; University of Palermo; Palermo; Italy Barresi, Giovanna; University of Palermo; Palermo; Italy Di Carlo, Enza; University of Palermo; Palermo; Italy In this work molecular techniques were applied in order to integrate the results obtained by Optical (OM) and Scanning Electron (SEM) Microscopy, to understanding and assessing the changes in the anatomical structure of archaeological waterlogged wood (Pinus sp.) induced by bacteria colonization. Observation of wooden thin sections by OM showed the presence of black and dark-brown areas (must probably due to sulphur compound) and mineral concretions. The SEM micrographs revealed a specific cell wall alteration, attributable to bacterial activity and abundant pyrite framboids (as single structure or clustered). The presence of sulfur compounds in archaeological waterlogged wood, indicate both long-term burial in anoxic environment and colonization by sulfate-reducing bacteria. Molecular biology investigation was performed through ad hoc protocols by direct DNA extraction from wood samples and in vitro amplification of bacteria DNA target sequences (16S, ITS regions- rRNA). The results reveal and identify bacterial genus as Pseudomonas, Cellulomonas, Xanthomonas and Bacillus that, as reported in the related scientific literature, are the most common cellulosolytic and ligninolytic bacteria. Moreover were also revealed the presence of Marinobacter sp. and Desulforudis audaxviator, respectively iron - oxidizing and sulfate - reducing bacteria. The investigation protocol set up in this work can be applied to a range of wooden artifacts of archaeological findings for both identification of bacteria colonization shed some light on the degradation phenomena, indispensable for correct conservation and restoration strategies. Corresponding author: Palla, Franco

An integrated approach was applied for the characterization of the biological airborne particulate in four Sicilian sites with peculiar structure, thermo-hygrometric and lighting parameters. We focalized the attention on: ,) Crypt in the Cathedral Treasury Museum (Palermo), an underground environment (lithic and stone artifact), daily visited by tourist and characterized by a reduced indoor-outdoor exchange; it) the Saints Cave in Licodia Eubea (Catania), a semi-confined environment (fresco), strongly influenced by the surrounding countryside and by a continuous airflow; iit) the Diocesan Historic Archive in Palermo, (documentary funds, IX-XXsec.), characterized by low air change rate and reduced frequency of fruitors; iv) the Sibilla Antrum (Trapani)a hypogeal environment (frescoes), where biological particulate can be vehicle by visitors and sometimes related to the presence of rodent pest. Non-invasive sampling was carried out on surfaces by Nylon membrane or by sterile swab. A portable sampler equipped with gelatin filters was utilized for aerosol. Microbial consortia colonizing both the works of art surfaces and the aerosol of conservation/exposure environments, were revealed and characterized by optical and electron microscopy (OM, SEM, CLSM), in vitro culture and molecular analysis.Combining biological results (Fig.1) with environment parameters data, acquired by HOBO U12 (temperature / relative humidity/ lighting) data-Ioggers (equipped with EEPROM memory), we define a protocol to prevent artifacts biodeterioration and to evaluate the potential health risk for visitors and operators, according to a preventive conservation strategy.

A significant association has been recently shown between the expression of neutrophil gelatinase-associated lipocalin (NGAL) in tumors and its urinary levels. Thus NGAL urinary detection has been proposed as a method for the early diagnosis of brain tumors. In view of this, the objective of this study was to investigate whether NGAL expression differs according to brain tumor type or in primary vs. metastatic brain neolasias. 42 surgically resected formalin fixed and paraffin embedded neoplasias, including 15 cases of brain metastasis and 27 cases of primary central nervous system (CNS) tumors (11 meningiomas; 1 pilocytic astrocytoma, 2 diffuse astrocytomas, 2 oligoastrocytomas, 2 oligodendrogliomas, 1 anaplastic oligoastrocytoma, 7 glioblastomas, 1 ependymoma) were submitted to the immunohistochemical procedure. Sections were incubated overnight with the primary antibody against NGAL. NGAL staining was found in all the analyzed glioblastomas and in the anaplastic oligoastrocytoma. No NGAL immuno-expression was evidenced in all the other cases. A statistically significant correlation was demonstrated between NGAL presence and high proliferation index in the primary tumors. In conclusion, our findings suggest that NGAL expression is restricted to high grade gliomas among primary brain tumors, and that brain metastases do not express this protein. Considering the correlation between NGAL expression in tumors and its urinary levels, if our observations will be further validated, NGAL urinary detection might be used as an additional tool in the pre-surgical definition of brain lesions involving difficult differential diagnosis.

The focus of this study was the identification of bacterial colonies in waterlogged wood fragments from the rostrum of a excellent workmanship, that is very likely one of the wrecks attributed to Sextus Pompey fleet (36 BC) and discovered in Acqualadroni, Messina, Sicily, Italy (2008). The wood samples were analyzed by light and Scanning Electron Microscopy (SEM), in vitro culture and molecular technique (DNA base techniques). The results, focused on bacterial consortia, allowed us to reveal the presence of Pseudomonas sp., Sphingomonas sp., Xanthomonas sp. besides Marinobacter sp. and Desulforudis audaxviator. A prompt and accurate characterization of bacterial colonization represents one of the preliminary step in preservation/restoration projects, especially for waterlogged wood since the metabolic activity of specific bacteria induce and accelerate the deterioration processes. Although it is reported in a case study, this multiple approach is useful for reveal and identify bacterial colonizing both organic and inorganic artifacts.

In the last decades molecular biology has provided innovative approaches in order to set up specific protocols for the conservation and restoration of cultural assets. In this study, which falls within the so called field of Blue-biotechnology, new bio-reactive peptides isolated from marine invertebrate organisrns (Cnidaria and Molluscs) were tested aim to bio-cleaning (proteolytic- peptides) the surfaces or to control (antimicrobial-peptides) the colonization of historic-artistic manufacts by fungi or bacteria. Particularly, the proteolytic-peptides showed hydrolytic activity, specific for animal-glue, in a range of temperatures of 4-37°C; than acting without heating the surface, by a contro Iled procedure and avoiding damages to the constitutive materials, ali criteria required for conservation /restoration procedures. These hydrolases allow to carry out the removal of the protein layers in a controlled way, acting at the same temperature of the environment where the objects are restored (19-25.5 0C). lnstead, the antirnicrobial-peptides have been tested on a plethora of fungi and bacteria colonies and a specific activity, against Micrococcus spp., have been showed. We are testing the antimicrobial-peptides as a "biologica! biocide" in order to protect both organic and inorganic substrates. We hypothesize that the use of these molecules will give an important contribution to the development of innovative and efficient technologies concerning bio-cleaning and antimicrobial growth protocols, according to conservative restoration procedures.