Mitochondrial non-syndromic sensorineural hearing loss: a clinical, audiological and pathological study from Italy, and revision of the literature

Over the last decade, a number of distinct mutations in the mtDNA (mitochondrial DNA) have been found to be associated with both syndromic and non-syndromic forms of hearing impairment. Their real incidence as a cause of deafness is poorly understood and generally underestimated. Among the known mtDNA mutations, the A1555G mutation in the 12S gene has been identified to be one of the most common genetic cause of deafness, and it has been described to be both associated to non-syndromic progressive SNHL (sensorineural hearing loss) and to aminoglycoside-induced SNHL. In the present study, we have investigated the presence of mtDNA alterations in patients affected by idiopathic non-syndromic SNHL, both familiar and sporadic, in order to evaluate the frequency of mtDNA alterations as a cause of deafness and to describe the audiological manifestations of mitochondrial non-syndromic SNHL. In agreement with previous studies, we found the A1555G mutation to be responsible for a relevant percentage (5.4%) of cases affected with isolated idiopathic sensorineural hearing impairment.     

Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences

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 alpha-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.     

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.     

A threonine synthase homolog from a mammalian genome

The genomes of several vertebrates contain two genes encoding proteins highly similar to threonine synthase (TS), even though the biosynthesis of l-threonine (l-Thr) is not known to occur in these animals. We report a bioinformatic analysis of the two TS-like genes, the recombinant expression of one murine TS homolog (mTSH2) and its initial biochemical characterization. Recombinant mTSH2 contained bound pyridoxal-5'-phosphate (PLP), but did not synthesize l-Thr. The enzyme did, however, bind O-phospho-homoserine (PHS; the actual TS substrate) and degraded it to alpha-ketobutyrate, phosphate, and ammonia-a known side reaction of microbial TSs. mTSH2 also degraded O-phospho-threonine (PThr) to alpha-ketobutyrate, showing that it can act as a catabolic phospho-lyase on both gamma- and beta-phosphorylated substrates. These findings suggest an unusual evolutionary origin for mTSH2, whereby an original TS enzyme became 'recycled' into a phospho-lyase upon dismissal, in metazoa, of the l-Thr biosynthetic pathway.     

A global assessment of forest surface albedo and its relationships with climate and atmospheric nitrogen deposition

We present a global assessment of the relationships between the short‐wave surface albedo of forests, derived from the MODIS satellite instrument product at 0.5° spatial resolution, with simulated atmospheric nitrogen deposition rates (N_dep), and climatic variables (mean annual temperature T_m and total annual precipitation P), compiled at the same spatial resolution. The analysis was performed on the following five forest plant functional types (PFTs): evergreen needle‐leaf forests (ENF); evergreen broad‐leaf forests (EBF); deciduous needle‐leaf forests (DNF); deciduous broad‐leaf forests (DBF); and mixed‐forests (MF). Generalized additive models (GAMs) were applied in the exploratory analysis to assess the functional nature of short‐wave surface albedo relations to environmental variables. The analysis showed evident correlations of albedo with environmental predictors when data were pooled across PFTs: T_m and N_dep displayed a positive relationship with forest albedo, while a negative relationship was detected with P. These correlations are primarily due to surface albedo differences between conifer and broad‐leaf species, and different species geographical distributions. However, the analysis performed within individual PFTs, strengthened by attempts to select ‘pure’ pixels in terms of species composition, showed significant correlations with annual precipitation and nitrogen deposition, pointing toward the potential effect of environmental variables on forest surface albedo at the ecosystem level. Overall, our global assessment emphasizes the importance of elucidating the ecological mechanisms that link environmental conditions and forest canopy properties for an improved parameterization of surface albedo in climate models.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.     

Crystal structure of the receptor-binding protein head domain from Lactococcus lactis phage bIL170

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry, is subject to lytic phage infections. In the first step of infection, phages recognize the host saccharidic receptor using their receptor binding protein (RBP). Here, we report the 2.30-A-resolution crystal structure of the RBP head domain from phage bIL170. The structure of the head monomer is remarkably close to those of other lactococcal phages, p2 and TP901-1, despite any sequence identity with them. The knowledge of the three-dimensional structures of three RBPs gives a better insight into the module exchanges which have occurred among phages.     

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.