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1.  Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase 
Many agricultural and industrial food by-products are rich in cellulose and xylan. Their enzymatic degradation into monosaccharides is seen as a basis for the production of biofuels and bio-based chemicals. Lytic polysaccharide monooxygenases (LPMOs) constitute a group of recently discovered enzymes, classified as the auxiliary activity subgroups AA9, AA10, AA11 and AA13 in the CAZy database. LPMOs cleave cellulose, chitin, starch and β-(1 → 4)-linked substituted and non-substituted glucosyl units of hemicellulose under formation of oxidized gluco-oligosaccharides.
Here, we demonstrate a new LPMO, obtained from Myceliophthora thermophila C1 (MtLPMO9A). This enzyme cleaves β-(1 → 4)-xylosyl bonds in xylan under formation of oxidized xylo-oligosaccharides, while it simultaneously cleaves β-(1 → 4)-glucosyl bonds in cellulose under formation of oxidized gluco-oligosaccharides. In particular, MtLPMO9A benefits from the strong interaction between low substituted linear xylan and cellulose. MtLPMO9A shows a strong synergistic effect with endoglucanase I (EGI) with a 16-fold higher release of detected oligosaccharides, compared to the oligosaccharides release of MtLPMO9A and EGI alone.
Now, for the first time, we demonstrate the activity of a lytic polysaccharide monooxygenase (MtLPMO9A) that shows oxidative cleavage of xylan in addition to cellulose. The ability of MtLPMO9A to cleave these rigid regions provides a new paradigm in the understanding of the degradation of xylan-coated cellulose. In addition, MtLPMO9A acts in strong synergism with endoglucanase I. The mode of action of MtLPMO9A is considered to be important for loosening the rigid xylan–cellulose polysaccharide matrix in plant biomass, enabling increased accessibility to the matrix for hydrolytic enzymes. This discovery provides new insights into how to boost plant biomass degradation by enzyme cocktails for biorefinery applications.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0284-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4504452  PMID: 26185526
Biorefinery; LPMO; Cellulose; Endoglucanase; Myceliophthora thermophila C1; Xylan
2.  Tolerability of a Fully Maturated Cheese in Cow’s Milk Allergic Children: Biochemical, Immunochemical, and Clinical Aspects 
PLoS ONE  2012;7(7):e40945.
From patients’ reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow’s milk (CM) allergic patients.
Objective and Methods
To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), β-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards.
The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly αS1-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG.
58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.
PMCID: PMC3400663  PMID: 22829901
3.  Letter from the Editors 
Artificial DNA, PNA & XNA  2012;3(2):29-30.
We are happy to publish this special issue dedicated to Prof. Rosangela Marchelli. This issue not only celebrates her long-standing scientific activity on occasion of her significant anniversary, but it is meant to recognize her contribution to Bioorganic Chemistry in the field of Artificial DNA, and in particular of Peptide Nucleic Acids.
PMCID: PMC3429528  PMID: 22777061
4.  PNA bearing 5-azidomethyluracil 
Artificial DNA, PNA & XNA  2012;3(2):53-62.
Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition.
PMCID: PMC3429531  PMID: 22772040
modified uracil; peptide nucleic acids; PNA; solid-phase modification; click reaction; orthogonal protection
5.  Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays 
Artificial DNA, PNA & XNA  2012;3(2):63-72.
PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.
PMCID: PMC3429532  PMID: 22772038
PNA; olive oil; hazelnut oil; SNP; cultivar identification; DNA fingerprinting
6.  Vaccination of Lactating Dairy Cows for the Prevention of Aflatoxin B1 Carry Over in Milk 
PLoS ONE  2011;6(10):e26777.
The potential of anaflatoxin B1 (AnAFB1) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB1-KLH) in controlling the carry over of the aflatoxin B1 (AFB1) metabolite aflatoxin M1 (AFM1) in cow milk is reported. AFB1 is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB1 is AFB1 chemically modified as AFB1-1(O-carboxymethyl) oxime. In comparison to AFB1, AnAFB1 has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB1-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB1 IgG antibodies (Abs) which were cross reactive with AFB1, AFG1, and AFG2. The elicited anti-AFB1 Abs were able to hinder the secretion of AFM1 into the milk of cows continuously fed with AFB1. Vaccination of lactating animals with conjugated AnAFB1 may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.
PMCID: PMC3203903  PMID: 22053212
7.  A pyrenyl-PNA probe for DNA and RNA recognition 
Artificial DNA, PNA & XNA  2010;1(2):83-89.
The design and the synthesis of a PNA oligomer containing a pyrenyl residue in the backbone were performed. PNA sequence was chosen complementary to a “G rich” target sequence involved in G-quadruplex formation. The pyrenyl unit replaced a nucleobase in the middle of the PNA through covalent linkage to the backbone by a carboxymethyl unit. A systematic study on the binding properties of this probe towards DNA and RNA complementary strands was carried out by UV and fluorescence spectroscopy. UV melting curves indicated that the PNA probe binds more tightly to RNA rather than to DNA. Thermodynamic data obtained by Van't Hoff fitting of the melting curves indicated that, in the case of RNA, a more favorable interaction occurs between the pyrenyl unit and the RNA nucleobases, leading to a very favorable enthalpic contribution.
The fluorescence analysis showed specific quenching of the pyrene emission associated to the formation of the full-match PNA-DNA or PNA-RNA duplexes. Again, this behavior was more evident in the case of RNA, consistently with the stronger interaction of the pyrenyl unit with the complementary strand. In order to study the sequence specificity of the pyrenyl-PNA probe (pyr-PNA), recognition experiments on mismatched DNA and RNA sequences were also performed.
PMCID: PMC3116571  PMID: 21686243
peptide nucleic acid; pyrene; DNA; RNA; fluorescence
8.  Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences 
Nucleic Acids Research  2008;36(5):1464-1471.
Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this ‘double-duplex invasion’, a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the α-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G–C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.
PMCID: PMC2275137  PMID: 18203747
9.  Cellular Uptakes, Biostabilities and Anti-miR-210 Activities of Chiral Arginine-PNAs in Leukaemic K562 Cells 
Chembiochem  2012;13(9):1327-1337.
A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R8-conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 m urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding.
PMCID: PMC3401907  PMID: 22639449
cell permeation; cellular differentiation; chiral PNA; microRNA; peptide nucleic acids; RNA

Results 1-9 (9)