Toward a personalized response approach in sepsis 4.0 / Hacia una estrategia de respuesta personalizada en sepsis 4.0

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Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria.

Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm(− 2) (0.08 pmol cm(− 2)). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL(− 1)) over a large linear working range (10­10(6) CFU mL(− 1)). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.

Ribonucleotide reductase (RNR) from Chlamydia trachomatis is a class I RNR composed of proteins R1 and R2. In protein R2, the tyrosine residue harboring the radical that is necessary for catalysis in other class I RNRs is replaced by a phenylalanine. Active C. trachomatis RNR instead uses the Fe(III)-Fe(IV) state of the iron cluster in R2 as an initiator of catalysis. The paramagnetic Fe(III)-Fe(IV) state, identified by (57)Fe substitution, becomes electron spin resonance detectable in samples that are frozen during conditions of ongoing catalysis. Its amount depends on the conditions for catalysis, such as incubation temperature and the R1/R2 ratio. The results link induction of the Fe(III)-Fe(IV) state with enzyme activity of chlamydial RNR. Based on these observations, a reaction scheme is proposed for the iron site. This scheme includes (i) an activation cycle involving reduction and an oxygen reaction in R2 and (ii) a catalysis cycle involving substrate binding and turnover in R1.

The open reading frame present in the nrdEF cluster of Corynebacterium ammoniagenes is a transcriptional regulator belonging to the GntR family.

In this work we have identified the cagntR gene, present between the nrdE and nrdF genes of Corynebacterium ammoniagenes, as a transcriptional regulator belonging to the GntR family. This gene encodes a transcriptional factor actively transcribed in the opposite direction relative to the nrdHIEF operon. It is expressed in a cell-culture-dependent fashion and, although the members of this family have been reported to regulate transcription of genes found within their vicinity, we have shown that cagntR is not involved in the transcriptional regulation of either nrdE or nrdF. The role of this regulator, however, remains unknown.

Applications of ovarian tissue transplantation in experimental biology and medicine.

Nowadays, high-dose chemotherapy and radiotherapy treatments for cancer are more effective but can severely affect the ovarian follicular store, compromising the fertility of surviving young patients. A promising alternative to prevent fertility loss in these patients is the cryopreservation and transplantation of ovarian tissue. Slices of animal and human ovarian tissue have been shown to survive the cryopreservation process. After transplantation, follicular development and restoration of hormone secretion have been observed in animal and human studies. This review addresses recent developments on ovarian tissue transplantation in animals and humans. We also illustrate the indications and technical difficulties of the procedure and the ethical issues that should be considered.

Corynebacterium ammoniagenes class Ib ribonucleotide reductase: transcriptional regulation of an atypical genomic organization in the nrd cluster.

Ribonucleotide reductases (RNRs) are a family of complex enzymes that play an essential role in all organisms because they catalyse de novo synthesis of deoxyribonucleotides required for DNA replication and repair. Three different classes of RNR have been described according to their metal cofactors and organic radicals. Class Ib RNR is encoded in four different genes (nrdH, nrdI, nrdE and nrdF) organized in an operon. The authors previously cloned and sequenced the genes encoding the active class Ib RNR of Corynebacterium ammoniagenes and showed that these genes are clustered in an atypical nrdEF region, which differs from that of other known class Ib enzymes because of an intergenic sequence (1171 bp) present between nrdE and nrdF. This study investigated the transcriptional organization and regulation of this nrd region by RT-PCR. Three different and independent mRNA were found (nrdHIE, nrdF and an ORF present in the intergenic region), each one being transcribed from its own promoter and being essential for normal growth. The ratio of nrdF to nrdHIE mRNA was 9.1, as determined by competitive RT-PCR; the expression of both nrdHIE and nrdF was found to be dependent on the culture growth phase, and was induced in the presence of hydroxyurea, manganese and hydrogen peroxide. This is believed to be the first direct evidence for a manganese-dependent transcriptional regulation of nrd genes. These results suggest a common and coordinated regulation of the different nrd genes, despite their being transcribed from independent promoters.

The anaerobic ribonucleotide reductase from Lactococcus lactis. Interactions between the two proteins NrdD and NrdG.

Deoxyribonucleotide synthesis by anaerobic class III ribonucleotide reductases requires two proteins, NrdD and NrdG. NrdD contains catalytic and allosteric sites and, in its active form, a stable glycyl radical. This radical is generated by NrdG with its [4Fe-4S](+) cluster and S-adenosylmethionine. We now find that NrdD and NrdG from Lactobacillus lactis anaerobically form a tight alpha(2)beta(2) complex, suggesting that radical generation by NrdG and radical transfer to the specific glycine residue of NrdD occurs within the complex. Activated NrdD was separated from NrdG by anaerobic affinity chromatography on dATP-Sepharose without loss of its glycyl radical. NrdD alone then catalyzed the reduction of CTP with formate as the electron donor and ATP as the allosteric effector. The reaction required Mg(2+) and was stimulated by K(+) but not by dithiothreitol. Thus NrdD is the actual reductase, and NrdG is an activase, making class III reductases highly similar to pyruvate formate lyase and its activase and suggesting a common root for the two anaerobic enzymes during early evolution. Our results further support the contention that ribonucleotide reduction during transition from an RNA world to a DNA world started with a class III-like enzyme from which other reductases evolved when oxygen appeared on earth.

Occurrence of multiple ribonucleotide reductase classes in gamma-proteobacteria species.

Ribonucleotide reductase (RNR) is central to de novo synthesis of deoxyribonucleotides and essential for all living cells. Three classes have been described; class I is oxygen dependent and represented by two subclasses, Ia (NrdAB) and Ib (NrdEF); class II (NrdJ) is indifferent to oxygen; and class III (NrdDG) is oxygen sensitive. More than one class can be found in an organism, reflecting the oxygen status of its environment. We have investigated, by using PCR and Southern blot, the occurrence of the different classes among species of the gamma-Proteobacteria. Class III are present in all species tested, but the presence of the other classes varies. Some species contain one unique additional enzyme, class Ia, Ib, or II, whereas others contain two additional enzymes, class Ia and Ib, or class Ia and II.

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein.

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.

The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis. Catalytic properties and allosteric regulation of the pure enzyme system.

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes.

Three separate classes of ribonucleotide reductases exist in nature. They differ widely in protein structure. Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria. Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes. Surprisingly, the genomic sequence of Pseudomonas aeruginosa contains sequences for each of the three classes. Here, we show by DNA hybridization that other species of Pseudomonas also contain the genes for three classes. Extracts from P. aeruginosa and P. stutzeri grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity. Unexpectedly, class I activity increased greatly during microaerobic conditions. The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form. The catalytic properties of all enzymes are similar to those of other bacterial reductases. However, the Pseudomonas class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding Escherichia coli enzyme but similar to the mouse enzyme. In similarity searches, the amino acid sequence of the class I enzyme of P. aeruginosa was more related to that of eukaryotes than to that of E. coli or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene. The results raise many questions concerning the physiological function and evolution of the three classes in Pseudomonas species.

The manganese-containing ribonucleotide reductase of Corynebacterium ammoniagenes is a class Ib enzyme.

Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mn-containing RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

nrdD and nrdG genes are essential for strict anaerobic growth of Escherichia coli.

In Escherichia coli ribonucleotide reduction is catalyzed by two separate enzymes during aerobic and anaerobic growth. The aerobic enzyme is coded by the nrdAB genes, the anaerobic enzyme by nrdDG. We now show that knock-out mutants of either nrdD or nrdG cannot grow during strict anaerobiosis, achieved by inclusion of sodium sulfide in the medium. Interestingly, these mutants grow well under microaerophilic conditions by overproducing the aerobic enzyme. Under such conditions wild-type bacteria turn off nrdAB and switch on nrdDG.

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.