The use of multiplex PCR for the diagnosis of viral severe acute respiratory infection in children: a high rate of co-detection during the winter season.

Respiratory tract infection is a major cause of hospitalization in children. Although most such infections are viral in origin, it is difficult to differentiate bacterial and viral infections, as the clinical symptoms are similar. Multiplex polymerase chain reaction (PCR) methods allow testing for multiple pathogens simultaneously and are, therefore, gaining interest. This prospective case-control study was conducted from October 2013 to February 2014. Nasopharyngeal (NP) and oropharyngeal (throat) swabs were obtained from children admitted with severe acute respiratory infection (SARI) at a tertiary hospital. A control group of 40 asymptomatic children was included. Testing for 16 viruses was done by real-time multiplex PCR. Multiplex PCR detected a viral pathogen in 159/177 (89.9 %) patients admitted with SARI. There was a high rate of co-infection (46.9 %). Dual detections were observed in 64 (36.2 %), triple detections in 17 (9.6 %), and quadruple detections in 2 (1.1 %) of 177 samples. Seventy-eight patients required intensive care unit (ICU) admission, of whom 28 (35.8 %) had co-infection with multiple viruses. AdV, HBoV, HRV, HEV, and HCoV-OC43 were also detected among asymptomatic children. This study confirms the high rate of detection of viral nucleic acids by multiplex PCR among hospitalized children admitted with SARI, as well as the high rate of co-detection of multiple viruses. AdV, HBoV, HRV, HEV, and HCoV-OC43 were also detected in asymptomatic children, resulting in challenges in clinical interpretation. Studies are required to provide quantitative conclusions that will facilitate clinical interpretation and application of the results in the clinical setting.

Myrosinase hydrolysates of Brassica oleraceae L. var. italica reduce the risk of colon cancer.

By means of liquid chromatography-electrospray ionization (LC-ESI) mass spectrometry two glucosinolates, glucoiberin and 3-hydroxy,4(α-L-rhamnopyranosyloxy) benzyl glucosinolate, were identified in the aqueous extract of Brassica oleraceae L var. italica. Further, two compounds were isolated after enzymatic hydrolysis of the aqueous extract by myrosinase, one of them was identified as 4-vinyl-3-pyrazolidinone. The second compound (sulphoraphane) 1-isothiocyanate-4-methyl-sulphinyl butane, converted to the most stable form of thiourea (sulphoraphane thiourea). The crude extract (80% alcohol extract) of broccoli florets was examined for cytotoxic activity against different human cancer cell lines, it showed good inhibition of colon cancer (IC(50) 3.88 µg/mL). On the other hand each of the successive extracts (petroleum ether, chloroform, ethyl acetate and ethanol) showed no significant cytotoxic activity. When myrosinase hydrolysate was tested for cytotoxic activity on the colon cancer cell line it showed very high activity - 95% lethality up to 0.78 µg/mL.

Association between HLA-E *0101 homozygosity and recurrent miscarriage in Egyptian women.

The objective was to investigate the frequency of human leucocyte antigen (HLA)-E alleles in Egyptian women with and without recurrent miscarriage (RM) to evaluate their role on the maintenance of pregnancy. A case-control study was adopted. HLA-E gene polymorphism typing was carried out by restriction fragment length polymorphism for 108 women with RM and 120 fertile female controls. The frequency of HLA-E *0101 allele was higher in patients with RM and HLA-E*0103 allele was higher in fertile controls, and the difference was statistically significant (P=0.003, P(c)=0.006). HLA-E*0101/0101 genotype was the most frequent genotype in patients (45.4%), followed by HLA-E*0101/0103 (44.4%) and finally HLA-E*0103/0103 genotype (10.2%). The difference in the frequency of HLA-E*0101/0101 homozygous genotype in patients with RM compared with that in the fertile controls was statistically significant (OR=2.02, 95% CI=1.13-3.62, P=0.011, P(c)=0.033). We found an increased frequency of homozygosity for HLA-E*0101 in Egyptian women with RM. HLA-E*0101 homozygosity may thus be a risk factor for RM.

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.

Diagnosis of chronic camel trypanosomosis by detection of the antibody of trypanosome tyrosine aminotransferase.

Sera from animals with acute and chronic T. evansi infections were examined directly for trypanosome tyrosine aminotransferase activity and indirectly for the ability of these area from mice and camels with high parasitaemias contained significant levels of trypanosome tyrosine aminotransferase activity. In contrast the chronic sera from both mice and camels did not contain significant tyrosine aminotransferase activity but the chronic sera were able to neutralize the enzyme activity in trypanosome homogenates. In addition to the sera from other pathological conditions did not neutralize the enzyme activity. It is suggested that the inhibitory factor in the chronic sera is antibody. The potential use of the direct enzyme assay, and the indirect neutralization assay as diagnostic tools are discussed. Finally, the use of these assays to distinguish between early (acute) and late (chronic) infections are also suggested.

The 'natural' hybrid haemoglobin from mule. Interrelationships with its parent haemoglobins from horse and donkey.

The equilibrium O2-binding properties of the hybrid haemoglobin (Hb) present in vivo in erythrocytes from mule and of its parent Hbs from horse and donkey were compared with special reference to the effect of heterotropic ligands such as Cl-, D-glycerate 2,3-bisphosphate (DPG) and inositol hexakisphosphate. All these Hbs display a decreased effect by polyphosphates, confirming that what has been observed for horse Hb [Giardina, Brix, Clementi, Scatena, Nicoletti, Cicchetti, Argentin & Condò (1990) Biochem. J. 266, 897-900] is common to other equine species, at least from a qualitative standpoint. However, different quantitative aspects can be detected, which can be accounted for by a different role for the two types of chain in characterizing the binding free energy for the various heterotropic effectors. In particular, it is shown that the binding mode of DPG and inositol hexakisphosphate displays different features since long-range effects can be observed clearly for inositol hexakisphosphate but not for DPG. In general terms, in spite of a different intrinsic O2 affinity, the modulation of functional properties by third ligands leads these Hbs to behave, under physiological conditions, similarly to human HbA. It might represent an interesting example of how different species with similar functional needs find different ways to produce a similar functional behaviour.

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.

Temperature modulation of bovine hemoglobins.

The functional properties of hemoglobin from Egyptian water buffalo have been characterized as a function of pH, temperature and chloride concentration. Alongside overall similarities shared with ox and Arctic ruminant hemoglobins, hemoglobin from buffalo shows significant differences with respect to the effect of temperature. The results obtained may suggest that the limited effect of temperature on oxygen binding recently reported for ox hemoglobin could be regarded as an interesting case of a reminiscence of a past glacial age.

Hemoglobins from bats (Myotis myotis and Rousettus aegyptiacus): a possible example of molecular adaptation to different physiological requirements.

The functional properties of the hemoglobin systems from two different species of bat i.e. Rousettus aegyptiacus and Myotis myotis have been studied as a function of chloride, polyphosphates, pH and temperature. Apart from overall similarities shared with most mammalian hemoglobins, the two systems show significant differences with respect to the effect of chloride and temperature sensitivity. These findings have been related to the different physiological needs of the two species.

Arctic adaptation in reindeer. The energy saving of a hemoglobin.

Previous results [(1988) Arct. Med. Res. 47, 83-88] have shown that hemoglobin from reindeer is characterized by a low overall heat of oxygenation. This particular aspect has been investigated further in a series of precise oxygen equilibrium experiments. The results obtained show a peculiar dependence of the temperature effect on the fractional saturation of hemoglobin with oxygen, which could be regarded as a very interesting case of molecular adaptation to extreme environmental conditions.

Arctic life adaptation--I. The function of reindeer hemoglobin.

1. The functional properties of hemoglobin from the reindeer (Rangifer tarandus tarandus L.) are characterized as a function of pH, temperature and organic phosphate concentration. 2. Alongside overall similarities shared with most vertebrate hemoglobins, hemoglobin from the reindeer shows significant differences with respect to the effect of both organic phosphates and chloride anions. 3. The limited effect of temperature on oxygen binding (delta H = -4 kcal/mol O2) could be regarded as an interesting case of molecular adaptation to extreme environmental conditions.

Arctic life adaptation--II. The function of musk ox (Ovibos muschatos) hemoglobin.

1. The hemoglobin system from musk ox (Ovibos muschatos) has been characterized from the functional point of view with special regard to the effect of organic phosphates and temperature. 2. The results are similar to those previously obtained in the case of reindeer and confirm that hemoglobins from arctic animals may display very low enthalpy change for the reaction with oxygen. 3. This finding is considered an example of molecular adaptation of respiratory pigments to extreme environmental conditions.