Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

The role of glycosylation in non-productive adsorption of cellulase to lignin isolated from pretreated corn stover.

Glycosylation, a general post-translational modification for fungal cellulase, has been shown to affect cellulase binding to its substrate. However, the exact impact of glycosylation on cellulase-lignin interaction remain unclear. Here, we demonstrated that the lignin isolated from tetrahydrofuran-pretreated corn stover exhibits strong adsorption capability to cellulase due to its negatively charged and porous structure. For the cellulases with varying glycosylation levels, the less-glycosylated protein showed high adsorption capability to lignin, and that trend was observed for the main cellulase components secreted by Penicillium oxilicum, including endoglucanase PoCel5B, cellobiohydrolase PoCel7A-2, and ß-glucosidase PoBgl1. Additionally, N-glycan sites and motifs were examined using mass spectrometry, and protein structures with N-glycans were constructed, where PoBgl1 and PoCel7A-2 contained 13 and 1 glycosylated sites respectively. The results of molecular dynamics simulations indicated that the N-glycans impacted on the solvent-accessible surface area and secondary structure of protein, and the binding conformation of lignin fragment on cellulase, resulting in a decrease in binding energy (14 kcal/mol for PoBgl1 and 13 kcal/mol for PoCel7A-2), particularly for van der Waals and electrostatic interaction. Those findings suggested that glycosylation negatively impacted the lignin-cellulase interaction, providing a theoretical basis for the rational engineering of enzymes to reduce lignin-enzyme interaction.

High-affinity water-soluble system for efficient naringinase immobilization in polyvinyl alcohol-dimethyl sulfoxide lens-shaped particles.

Polyvinyl alcohol (PVA) is a water-soluble, biocompatible and biodegradable synthetic polymer whose application in the immobilization of biological agents for use in biocatalysis has shown promising results. This study aimed to investigate and optimize the immobilization of naringinase from Penicillium decumbens in PVA networks, targeting for the hydrolysis of naringin. Variables such as the most suitable cross-linker, catalyst, inorganic salt, co-solvents and solidification process were identified as key issues for PVA-based methods to form lens-shaped particles, while retaining high enzyme activity and stability. Major improvements were established for better and more reproducible immobilization conditions, namely, by designing a new immobilization apparatus to produce uniform lens-shaped particles. The common problems of PVA-based entrapment were significantly mitigated, through the use of selected cross-linker, glutaraldehyde (GA), and co-solvent, dimethyl sulfoxide (DMSO), which decreased the toxicity of the immobilization process and allowed the control of membrane porosity, respectively. The relevance of DMSO and GA and their interaction and effect on the swelling ratio, encapsulation efficiency and residual activity of PVA biocatalysts were established. The immobilization of naringinase in PVA under a DMSO concentration of 60%, cross-linked with 1% GA, and particle lens size of 3.5-4.0 mm, width of 100-300 µm and average particle volume of 12.5 ± 0.92 µL, allowed an encapsulation efficiency of 98.6% and an average residual activity of 87% ± 3.6%. The kinetic characterization of the immobilized naringinase showed no changes in pH profile, whereas hydrolytic activity increased up to 60 °C. Immobilization in PVA/DMSO/GA lens-shaped particles enhanced the storage stability of naringinase. Moreover, these naringinase bio-immobilizates retained a conversion rate higher than 78% after 23 runs.

Characterization of the family GH54 alpha-L-arabinofuranosidases in Penicillium funiculosum, including a novel protein bearing a cellulose-binding domain.

The soil deuteromycete Penicillium funiculosum is characterized by its remarkable capacity to produce a wide variety of cellulolytic and hemicellulolytic enzymes. In the course of the genome sequencing of this industrial fungus, four different genes encoding glycosyl hydrolase family 54 (GH54)22 alpha-L-arabinofuranosidases were identified. Three of them termed PfabfB1, PfabfB3, and PfabfB4 were highly similar, encoding proteins of 507, 508, and 505 amino acids, respectively. They exhibited structural features typical of GH54 enzymes, including an N-terminal catalytic domain connected to a C-terminal arabinose-binding domain (ABD). The fourth gene termed PfafbB2 codes for an unusual 400 amino acid length GH54 alpha-L: -arabinofuranosidase, in which the ABD was replaced by a fungal cellulose-binding domain (fCBD). This domain was shown to be functional since it allowed this protein to be retained onto microcrystalline cellulose, and the fusion of this CBD to the C-terminal end of PfAbfB1 allowed this protein to bind to cellulose. Expression analysis of the four PfabfB genes during an industrial-like process fermentation on complex carbohydrates revealed that PfafB2 was expressed more than 20,000-fold, while PfabfB3 and PfabfB4 were increased moderately at the end of the fermentation. In contrast, the transcript levels of PfabfB1 remained unchanged throughout the process. This new type of GH54 alpha-arabinofuranosidase encoded by PfabfB2 showed enzymatic properties slightly different to those of other GH54 enzymes characterized so far, including a higher thermostability, an optimum pH, and temperature of 2.6 and 50 degrees C, instead of 3.5 and 60 degrees C as found for PfAbfB1. Nonetheless, like other GH54 alpha-arabinofuranosidases, PfAbfB2 was able to release arabinose from various sources of branched arabinoxylan and arabinan.

Penctrimertone, a bioactive citrinin dimer from the endophytic fungus Penicillium sp. T2-11.

Penctrimertone (1), a novel citrinin dimer bearing a 6/6/6/6 tetracyclic ring scaffold, along with two known compounds xerucitrinic acid A (2) and citrinin (3) were isolated from the endophytic fungus Penicillium sp. T2-11. Their structures were unequivocally established by a comprehensive interpretation of the spectroscopic data, with the stereochemistry for 1 was defined by a combination of TDDFT-ECD calculations and the DP4+ probability analysis based on NMR chemical shift calculations. Bioassays revealed that compound 1 exhibited noticeable antimicrobial activities and moderate cytotoxicity. A plausible biosynthetic pathway of 1 was also proposed.

FISH and Calcofluor staining techniques to detect in situ filamentous fungal biofilms in water.

Filamentous fungi are a ubiquitous and diverse group of eukaryotic organisms and may contribute, along with bacteria, yeasts, protozoa and viruses, to the formation of biofilms in water distribution systems. However, fungal involvement in biofilms has not been demonstrated unambiguously. Furthermore, these fungi may be responsible for the production of tastes, odours and mycotoxins in drinking water making their early detection important. The detection of fme these problems a combination of two fluorescent techniques for direct detection was tested: (a) Fluorescence In Situ Hybridization (FISH) employing the universal rRNA probe EUK516, labelled with the red Cy3, followed by (b) staining with Calcofluor White MR2 fluorescent dye which stains fungal cell walls blue. Pure cultures of Penicillium brevicompactum were used to establish the methods followed by separate experiments with real water biofilm samples in PVC-C and cast iron coupons. FISH demonstrated eukaryotic microrganisms after approximately 5 h while the calcofluor method revealed chitinous filamentous structures in less than one hour. When the two methods were combined, additional resolution was obtained from the images of filamentous walls (blue) with intact protoplasm (red). In conclusion, FISH and Calcofluor staining provide rapid, direct and unambiguous information on the involvement of ff in biofilms which form in water.

Targeting of mannosylated liposome incorporated benzyl derivative of Penicillium nigricans derived compound MT81 to reticuloendothelial systems for the treatment of visceral leishmaniasis.

The antileishmanial property of a Benzyl derivative of a new antibiotic MT81 (Bz2MT81), isolated and purified from a fungal strain of Penicillium nigricans NRRL 917 was tested in free, liposome intercalated and mannose coated liposome intercalated forms in vivo against visceral leishmaniasis in hamsters. Mannose grafted liposome intercalated Bz2MT81 eliminated intracellular amastigotes of Leishmania donovani within splenic macrophages more efficiently than the liposome intercalated Bz2MT81 or free Bz2MT81. At a dose equivalent to 7.5 microg/Kg body weight when injected subcutaneously (s.c) in mannose grafted liposome intercalated form for 15 days in an interval of three days, the splenic parasitic load decreased to the extent of 79.1% of the total parasite present in infected control animals. Whereas, an identical amount (7.5 mug/Kg body weight) of Bz2MT81 in free or liposome intercalated form was found less effective in controlling the parasite in spleen (in free Bz2MT81 form, suppression of parasitic load is 49.8% and in liposome intercalated form, it is 55.1%). Both mannosylated liposomes and Bz2MT81 were noted non-toxic to the host peritoneal macrophages. Histological examinations of spleen and liver, kidney function tests (SGPT, alkaline phosphatase, creatinine and urea in blood plasma) showed that the toxicity of Bz2MT81 was reduced up to normal level when mannose grafted liposomal Bz2MT81 were administered.

Characterization of the wound-induced material in Citrus paradisi fruit peel by carbon-13 CP-MAS solid state NMR spectroscopy.

Grapefruit, Citrus paradisi, were injured, inoculated with Penicillium digitatum and incubated under conditions favourable for the accumulation of defence related material. Histochemical examination revealed that tissues adjacent to inoculated injuries contained phloroglucinol-HCl (PG-HCl) reactive material. Solvent washed cell wall preparations of intact and injured-inoculated peel were further purified using a mixture of cell wall degrading enzymes. Samples from injured inoculated tissue contained PG-HCl reactive globular material in addition to the fragments of xylem and cuticle found in controls. The principal chemical moieties of the material that accumulates in grapefruit injuries during wound-healing were studied by solid state 13C cross-polarization magic angle spinning NMR. A complete assignment of the NMR signals was made. From the analysis evidence was found that cellulose and hemicellulose are the biopolymers present in the intact peel samples, in addition, relevant quantities of cutin were found in the residues of enzyme digest. The NMR difference spectrum intact- wounded peels showed resonances which were attributed to all major functional groups of the aromatic-aliphatic suberin polyester of new material produced by the wounds. Information on the latter polyester was obtained by analyzing the T(1)rho (1H) relaxation.

Nitrogen source optimization for cellulase production by Penicillium funiculosum, using a sequential experimental design methodology and the desirability function.

The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.

Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures.

The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of 16 mycotoxins possibly related to the 'Sick Building Syndrome' on filters and in fungal cultures is described. Fungi-surface sampling as regards the 'Sick Building Syndrome' preferably happens by scraping off fungal material and vacuuming onto cellulose filters. Therefore, these two media were used as samples. They were spiked with nivalenol, deoxynivalenol, zearalenone, diacetoxyscirpenol, T-2 toxin, verrucarol, verrucarin A, neosolaniol, sterigmatocystin, roridin A, ochratoxin A, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2, which can be produced by isolates from fungi-damaged buildings. Deepoxy-deoxynivalenol was used as internal standard. Samples were extracted with organic solvents and the different mycotoxins were separated by high-performance liquid chromatography (HPLC) using a C18 reversed-phase SunFire analytical column and a mobile phase of variable mixtures of ammonium acetate (10 mM) and sodium acetate (20 microM) in water (solvent A) and in methanol (solvent B). The samples were run on-line with a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electrospray ionisation mode using multiple reaction monitoring (MRM). The detection limits of the procedure varied from 50 to 0.009 pg/microL for filter samples and from 75 to 0.04 pg/microL for fungal culture samples. As the method includes few and non-labourious sample treatment steps, it should allow for a high throughput of samples.

Partitioning of xylanolitic complex from Penicillium janthinellum by an aqueous two-phase system.

This work evaluates the influences of five parameters (pH, PEG molecular mass, PEG concentration, concentration of buffer K2HPO4-KH2PO4 and NaCl concentration) on xylanolitic complex partitioning produced by P. janthinellum in aqueous two-phase systems, using a 2(5) factorial experimental design. A mathematical model to quantify the influence of these parameters was attained and statistically tested. The optimum point for total protein extraction was obtained under the following conditions: pH 7.0, PEG 10 000, 3.67% PEG, 10% potassium phosphate and 12.4% NaCl. The partition coefficient (K) value experimentally obtained was 5.25 and that predicted by the model was 5.89.