N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging.

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS: The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS: The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION: We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

Evolution of C(4) phosphoenolpyruvate carboxykinase in grasses, from genotype to phenotype.

C(4) photosynthesis is an adaptation over the classical C(3) pathway that has evolved multiple times independently. These convergences are accompanied by strong variations among the independent C(4) lineages. The decarboxylating enzyme used to release CO(2) around Rubisco particularly differs between C(4) species, a criterion used to distinguish three distinct biochemical C(4) subtypes. The phosphoenolpyruvate carboxykinase (PCK) serves as a primary decarboxylase in a minority of C(4) species. This enzyme is also present in C(3) plants, where it is responsible for nonphotosynthetic functions. The genetic changes responsible for the evolution of C(4)-specific PCK are still unidentified. Using phylogenetic analyses on PCK sequences isolated from C(3) and C(4) grasses, this study aimed at resolving the evolutionary history of C(4)-specific PCK enzymes. Four independent evolutions of C(4)-PCK were shown to be driven by positive selection, and nine C(4)-adaptive sites underwent parallel genetic changes in different C(4) lineages. C(4)-adaptive residues were also observed in C(4) species from the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) subtype and particularly in all taxa where a PCK shuttle was previously suggested to complement the NADP-ME pathway. Acquisitions of C(4)-specific PCKs were mapped on a species tree, which revealed that the PCK subtype probably appeared at the base of the Chloridoideae subfamily and was then recurrently lost and secondarily reacquired at least three times. Linking the genotype to subtype phenotype shed new lights on the evolutionary transitions between the different C(4) subtypes.

F+0 renography in neonates and infants younger than 6 months: an accurate method to diagnose severe obstructive uropathy.

We studied the response to F+0 renography and the relative and absolute individual kidney function in neonates and < 6-mo-old infants before and after surgery for unilateral ureteropelvic junction obstruction (UJO). METHODS: The results obtained at diagnosis and after pyeloplasty for 9 children (8 boys, 1 girl; age range, 0.8-5.9 mo; mean age +/- SD, 2.4 +/- 1.5 mo) with proven unilateral UJO (i.e., affected kidney [AK]) and an unremarkable contralateral kidney (i.e., normal kidney [NK]) were evaluated and compared with a control group of 10 children (6 boys, 4 girls; age range, 0.8-2.8 mo; mean age, 1.5 +/- 0.7 mo) selected because of symmetric renal function, absence of vesicoureteral reflux or infection, and an initially dilated but not obstructed renal pelvis as proven by follow-up. Renography was performed for 20 min after injection of (123)I-hippuran (OIH) (0.5-1.0 MBq/kg) immediately followed by furosemide (1 mg/kg). The relative and absolute renal functions and the response to furosemide were measured on background-subtracted and depth-corrected renograms. The response to furosemide was quantified by an elimination index (EI), defined as the ratio of the 3- to 20-min activities: An EI > or = 3 was considered definitively normal and an EI < or = 1 definitively abnormal. If EI was equivocal (1 < EI < 3), the response to gravity-assisted drainage was used to differentiate AKs from NKs. Absolute separate renal function was measured by an accumulation index (AI), defined as the percentage of (123)I-OIH (%ID) extracted by the kidney 30-90 s after maximal cardiac activity. RESULTS: All AKs had definitively abnormal EIs at diagnosis (mean, 0.56 +/- 0.12) and were significantly lower than the EIs of the NKs (mean, 3.24 +/- 1.88) and of the 20 control kidneys (mean, 3.81 +/- 1.97; P < 0.001). The EIs of the AKs significantly improved (mean, 2.81 +/- 0.64; P < 0.05) after pyeloplasty. At diagnosis, the AIs of the AKs were significantly lower (mean, 6.31 +/- 2.33 %ID) than the AIs of the NKs (mean, 9.43 +/- 1.12 %ID) and of the control kidneys (mean, 9.05 +/- 1.17 %ID; P < 0.05). The AIs of the AKs increased at follow-up (mean, 7.81 +/- 2.23 %ID) but remained lower than those of the NKs (mean, 10.75 +/- 1.35 %ID; P < 0.05). CONCLUSION: In neonates and infants younger than 6 mo, (123)I-OIH renography with early furosemide injection (F+0) allowed us to reliably diagnose AKs and to determine if parenchymal function was normal or impaired and if it improved after surgery.

Vertical Transmission of Pneumocystis jirovecii in Humans

This study is part of the project “Pneumocystis Pathogenomics: Unravelling the Colonization-to-Disease Shift,” a Coordination Action supported by the European Commission (ERANET PathoGenoMics). This study was partially supported by the Spanish Ministry of Health (FIS 03/1743). M.A.M.-C. and C.d.l.H. were supported by the Spanish Ministry of Health (FIS CP-04/217 and FIS CM-04/146).

Manuel pour l'analyse des substances organiques / par J. Liebig,... ; traduit de l'allemand par A.-J.-L. Jourdan. Suivi de l'examen critique des procédés et des résultats de l'analyse des corps organisés / par F. V. Raspail,...

Comprend : Examen critique des procédés et des résultats de l'analyse des corps organisés

Lactate quartile concentration and prognosis in severe sepsis and septic shock.

Introduct ion The Surviving Sepsis Campaign (SSC) indicates that a lactate (LT) concentration greater than 4ımmol/l indicates early resuscitation bundles. However, several recent studies have suggested that LT values lower than 4ımmol/l may be a prognostic marker of adverse outcome. The aim of this study was to identify clinical and analytical prognostic parameters in severe sepsis (SS) or septic shock (ShS) according to quartiles of blood LT concentration. Methods A cohort study was designed in a polyvalent ICU. We studied demographic, clinical and analytical parameters in 148 critically ill adults, within 24ıhours from SS or ShS onset according to SSC criteria. We tested for diı erences in baseline characteristics by lactate interval using a KruskalıWallis test for continuous data or a chi-square test for categorical data and reported the median and interquartile ranges; SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 148 consecutive episodes of SS (16%) or ShS (84%). The median age was 64 (interquartile range, 48.7 to 71)ıyears; male: 60%. The main sources of infection were respiratory tract 38% and intra-abdomen 45%; 70.7% had medical pathology. Mortality at 28ıdays was 22.7%. Quartiles of blood LT concentration were quartile 1 (Q1): 1.87ımmol/l or less, quartile 2 (Q2): 1.88 to 2.69ımmol/l, quartile 3 (Q3): 2.7 to 4.06ımmol/l, and quartile 4 (Q4): 4.07ımmol/l or greater (Tableı1). The median LT concentrations of each quartile were 1.43 (Q1), 2.2 (Q2), 3.34 (Q3), and 5.1 (Q4) mmol/l (Pı<0.001). The diı erences between these quartiles were that the patients in Q1 had signiı cantly lower APACHE II scores (Pı=ı0.04), SOFA score (Pı=ı0.024), number of organ failures (NOF) (Pı<0.001) and ICU mortality (Pı=ı0.028), compared with patients in Q2, Q3 and Q4. Patients in Q1 had signiı cantly higher cholesterol (Pı=ı0.06) and lower procalcitonin (Pı=ı0.05) at enrolment. At the extremes, patients in Q1 had decreased 28-day mortality (Pı=ı0.023) and, patients in Q4 had increased 28-day mortality, compared with the other quartiles of patients (Pı=ı0.009). Interestingly, patients in Q2 had signiı cant increased mortality compared with patients in Q1 (Pı=ı0.043), whereas the patients in Q2 had no signiı cant diı erence in 28-day mortality compared with patients in Q3. Conclusion Adverse outcomes and several potential risk factors, including organ failure, are signiı cantly associated with higher quartiles of LT concentrations. It may be useful to revise the cutoı value of lactate according to the SSC (4 mmol/l).

Large-scale analysis of orthologs and paralogs under covarion-like and constant-but-different models of amino acid evolution.

Functional divergence between homologous proteins is expected to affect amino acid sequences in two main ways, which can be considered as proxies of biochemical divergence: a "covarion-like" pattern of correlated changes in evolutionary rates, and switches in conserved residues ("conserved but different"). Although these patterns have been used in case studies, a large-scale analysis is needed to estimate their frequency and distribution. We use a phylogenomic framework of animal genes to answer three questions: 1) What is the prevalence of such patterns? 2) Can we link such patterns at the amino acid level with selection inferred at the codon level? 3) Are patterns different between paralogs and orthologs? We find that covarion-like patterns are more frequently detected than "constant but different," but that only the latter are correlated with signal for positive selection. Finally, there is no obvious difference in patterns between orthologs and paralogs.