Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains.     

A theoretical study of the<Emphasis Type="Italic"> cis</Emphasis>-dihydroxylation mechanism in naphthalene 1,2-dioxygenase

The catalytic mechanism of naphthalene 1,2-dioxygenase has been investigated by means of hybrid density functional theory. This Rieske-type enzyme, which contains an active site hosting a mononuclear non-heme iron(II) complex, uses dioxygen and two electrons provided by NADH to carry out the cis-dihydroxylation of naphthalene. Since a (hydro)peroxo-iron(III) moiety has been proposed to be involved in the catalytic cycle, it was probed whether and how this species is capable of cis-dihydroxylation of the aromatic substrate. Different oxidation and protonation states of the Fe–O₂ complex were studied on the basis of the crystal structure of the enzyme with oxygen bound side-on to iron. It was found that feasible reaction pathways require a protonated peroxo ligand, Fe^III–OOH; the deprotonated species, the peroxo-iron(III) complex, was found to be inert toward naphthalene. Among the different chemical patterns which have been explored, the most accessible one involves an epoxide intermediate, which may subsequently evolve toward an arene cation, and finally to the cis-diol. The possibility that an iron(V)-oxo species is formed prior to substrate hydroxylation was also examined, but found to implicate a rather high energy barrier. In contrast, a reasonably low barrier might lead to a high-valent iron-oxo species [i.e. iron(IV)-oxo] if a second external electron is supplied to the mononuclear iron center before dioxygenation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0537-0     

Candida albicans isolates with different genomic backgrounds display a differential response to macrophage infection

Few human pathogens possess the ability exhibited by Candida albicans to colonize and cause symptomatic infections at different body sites. The host immune system is the major factor determining whether this opportunistic yeast behaves as a commensal or as a pathogen, since C. albicans strains appear capable of expressing similar virulence factors in response to specific body-district cues. This report provides evidence showing that C. albicans isolates with diverse genomic backgrounds (b and c karyotypes) differently modulate their pathogenic potential when assayed in cocultures with human monocytic derived macrophages (THP-1 cells). Striking differences were observed in the ability to undergo bud-hypha transition, a relevant C. albicans virulence factor, between b and c karyotypes (P<0.0001) upon their internalization by macrophages. All c types were able to develop hyphal forms, resist intracellular killing, replicate, and escape from macrophages. The b type isolates, which were shown to be more efficiently ingested by THP-1 cells than the c type strains (P=0.013), were susceptible to intracellular killing and predominantly found as blastoconidia inside macrophages. Despite their different intracellular disposition, both b and c type isolates were equally able to undergo morphogenesis and to express NRG1 and HWP1 genes, markers of the bud-hypha transition program, during in vitro propagation. Since macrophages play a critical role in the host resistance to C. albicans, the different response of b and c isolates to macrophage infection suggests that the c type strains are better suited to behave as a more virulent strain cluster.     

Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: is aniA essential for meningococcal survival?

The present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolate-derived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.     

NMR assignments of the DNA binding domain of Ml4 protein from Mesorhizobium loti

Ml4 protein from Mesorhizobium loti has a 58% sequence identity with the Ros protein from Agrobacterium tumefaciens that contains a prokaryotic Cys₂His₂ zinc finger domain. Interestingly, Ml4 is a zinc-lacking protein that does not contain the Cys₂His₂ motif and is able to bind the Ros DNA target sequence with high affinity. Here we report the ¹H, ¹⁵N and ¹³C NMR assignments of the Ml4 protein DNA binding domain (residue 52–151), as an important step toward elucidating at a molecular level how this prokaryotic domain can overcome the metal requirement for proper folding and DNA-binding activity.     

Automated audio recording as a means of surveying tinamous (Tinamidae) in the Peruvian Amazon

1. The use of machine learning technologies to process large quantities of remotely collected audio data is a powerful emerging research tool in ecology and conservation.
2. We applied these methods to a field study of tinamou (Tinamidae) biology in Madre de Dios, Peru, a region expected to have high levels of interspecies competition and niche partitioning as a result of high tinamou alpha diversity. We used autonomous recording units to gather environmental audio over a period of several months at lowland rainforest sites in the Los Amigos Conservation Concession and developed a Convolutional Neural Network‐based data processing pipeline to detect tinamou vocalizations in the dataset.
3. The classified acoustic event data are comparable to similar metrics derived from an ongoing camera trapping survey at the same site, and it should be possible to combine the two datasets for future explorations of the target species'' niche space parameters.
4. Here, we provide an overview of the methodology used in the data collection and processing pipeline, offer general suggestions for processing large amounts of environmental audio data, and demonstrate how data collected in this manner can be used to answer questions about bird biology.

     

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.     

Hsp90‐mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual‐specificity tyrosine‐phosphorylation‐regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.