Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

The role of procalcitonin in neonatal intensive care unit patients with candidemia

Candidemia is a major infectious complication in neonatal patients. The isolation of yeasts from blood is still the ??gold standard?? for its diagnosis, but other laboratory markers (i.e., circulating antigens) have been studied with varying specificities and sensitivities. The aim of this study was to evaluate the role of procalcitonin for the diagnosis of candidemia in neonatal patients at high risk. To verify if the use of different commercial methods can highlight dissimilar results of sensitivity and/or specificity, the determination of procalcitonin serum levels was estimated by two systems. Overall, 90 patients from a Neonatal Intensive Care Units were enrolled, of whom six developed Candida bloodstream infection. Four of six infants with candidemia had slight increase of procalcitonin values (0.5?C1?ng/mL). Only one baby showed very high levels but he had fungal and bacterial sepsis at the same time, while no elevation was observed in the sixth patient. No statistically significant difference was observed between two different methods at the time of monitoring (p?>?0.643). Both methods showed a sensitivity of 83.3?% at diagnosis, while the specificity was 73.8 and 63.1?% by methods A and B, respectively. In the light of the low sensibility and specificity of this assay, we can assume that the determination of procalcitonin would not seem to play a significant role in the diagnosis of fungal infection in neonatal patients.     

Mortality,Morbidity, and Developmental Outcomes in Infants Born to Women Who Received Either Mefloquine or Sulfadoxine-Pyrimethamine as Intermittent Preventive Treatment of Malaria in Pregnancy: A Cohort Study

Background

Little is known about the effects of intermittent preventive treatment of malaria in pregnancy (IPTp) on the health of sub-Saharan African infants. We have evaluated the safety of IPTp with mefloquine (MQ) compared to sulfadoxine-pyrimethamine (SP) for important infant health and developmental outcomes.

Methods and Findings

In the context of a multicenter randomized controlled trial evaluating the safety and efficacy of IPTp with MQ compared to SP in pregnancy carried out in four sub-Saharan countries (Mozambique, Benin, Gabon, and Tanzania), 4,247 newborns, 2,815 born to women who received MQ and 1,432 born to women who received SP for IPTp, were followed up until 12 mo of age. Anthropometric parameters and psychomotor development were assessed at 1, 9, and 12 mo of age, and the incidence of malaria, anemia, hospital admissions, outpatient visits, and mortality were determined until 12 mo of age. No significant differences were found in the proportion of infants with stunting, underweight, wasting, and severe acute malnutrition at 1, 9, and 12 mo of age between infants born to women who were on IPTp with MQ versus SP. Except for three items evaluated at 9 mo of age, no significant differences were observed in the psychomotor development milestones assessed. Incidence of malaria, anemia, hospital admissions, outpatient visits, and mortality were similar between the two groups. Information on the outcomes at 12 mo of age was unavailable in 26% of the infants, 761 (27%) from the MQ group and 377 (26%) from the SP group. Reasons for not completing the study were death (4% of total study population), study withdrawal (6%), migration (8%), and loss to follow-up (9%).

Conclusions

No significant differences were found between IPTp with MQ and SP administered in pregnancy on infant mortality, morbidity, and nutritional outcomes. The poorer performance on certain psychomotor development milestones at 9 mo of age in children born to women in the MQ group compared to those in the SP group may deserve further studies.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00811421","term_id":"NCT00811421"}}NCT00811421     

A Protein Disulfide Isomerase Controls Neuronal Migration through Regulation of Wnt Secretion

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Classification of Rhinoentomophthoromycosis into Atypical,Early, Intermediate,and Late Disease: A Proposal

Background

Rhinoentomophthoromycosis, or rhino-facial conidiobolomycosis, is a rare, grossly disfiguring disease due to an infection with entomophthoralean fungi. We report a case of rhinoentomophthoromycosis from Gabon and suggest a staging system, which provides information on the prognosis and duration of antifungal therapy.

Methods

We present a case of rhinoentomophthoromycosis including the histopathology, mycology, and course of disease. For the suggested staging system, all cases on confirmed rhinoentomophthoromycosis published in the literature without language restriction were eligible. Exclusion criteria were missing data on (i) duration of disease before correct diagnosis, (ii) outcome, and (iii) confirmation of entomophthoralean fungus infection by histopathology and/or mycology. We classified cases into atypical (orbital cellulitis, severe pain, fever, dissemination), early, intermediate, and late disease based on the duration of symptoms before diagnosis. The outcome was evaluated for each stage of disease.

Findings

The literature search of the Medpilot database was conducted on January 13, 2014, (updated on January 18, 2015). The search yielded 8,333 results including 198 cases from 117 papers; of these, 145 met our inclusion criteria and were included in the final analysis. Median duration of treatment was 4, 3, 4, and 5 months in atypical, early, intermediate, and late disease, respectively. Cure rates were clearly associated with stage of disease and were 57%, 100%, 82%, and 43% in atypical, early, intermediate, and late disease, respectively.

Conclusion

We suggest a clinical staging system that underlines the benefit of early case detection and may guide the duration of antifungal treatment. The scientific value of this classification is its capacity to structure and harmonize the clinical and research approach towards rhinoentomophthoromycosis.     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Influence of thymosin on E-rosette formation of lymphoid cells in leukemic and nonleukemic children.

The influence of two thymosin and two spleen control preparations on the E-rosette formation of peripheral blood lymphocytes and leukemic cells from non-leukemic children with depressed T cell values and leukemic children was investigated. Both thymosin preparations but also one of the control preparations induced a significant increase in the mean percentage of E-rosette forming PBL in the non-leukemic children with depressed T cell values. Thymosin and spleen preparations, however, did not convert the non-erythrocyte binding blasts to erythrocyte binding cells in either common or pre-T-ALL.     

Mansonella,including a Potential New Species,as Common Parasites in Children in Gabon

Background

Like other tropical African countries, Gabon is afflicted by many parasitic diseases, including filariases such as loiasis and mansonellosis. This study aimed to assess the prevalence of these two filarial diseases in febrile and afebrile children using quantitative real-time PCR and standard PCR assays coupled with sequencing.

Methodology/Principal Findings

DNA from blood specimens of 1,418 Gabonese children (1,258 febrile and 160 afebrile) were analyzed. Overall, filarial DNA was detected in 95 (6.7%) children, including 67 positive for M. perstans (4.7%), which was the most common. M. perstans was detected in 61/1,258 febrile children (4.8%) and 6/160 afebrile children (3.8%, P = 0.6). Its prevalence increased statistically with age: 3.5%, 7.7% and 10.6% in children aged ≤5, 6–10 and 11–15 years, respectively. M. perstans prevalence was significantly higher in Koulamoutou and Lastourville (12% and 10.5%, respectively) than in Franceville and Fougamou (2.6% and 2.4%, respectively). Loa loa was detected in seven febrile children including one co-infection with M. perstans. Finally, 21 filarial DNA positive were negative for M. perstans and Loa loa, but ITS sequencing could be performed for 12 and allowed the identification of a potential new species of Mansonella provisionally called “DEUX”. Mansonella sp. “DEUX” was detected only in febrile children.

Conclusions/Significance

Further study should be performed to characterize Mansonella sp. “DEUX” and evaluate the clinical significance of mansonellosis in humans.