Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost-benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.     

Studies on the interaction between hyaluronan and a rat colon cancer cell line

Binding studies with 125I-Tyr labelled hyaluronan (HA) on a cultured rat colon cancer cell line were performed to characterize the association of HA to tumour cells in vitro. Results show a specific and saturable binding (Kd= 1.36nM) which indicates the presence of an HA binding receptor on the tumour cells. There is a specific constant increase of cell-associated HA over time, which indicates that HA is specifically taken up by the cells through endocytosis. The binding of 125I-Tyr labelled HA was more effectively inhibited by unlabelled HA of high MW in relation to low MW species of the polysaccharide indicating that the receptor binds HA of high MW with greater affinity than low MW species. In competition experiments, the HA-binding could not be inhibited by other polysaccharides such as chondroitin sulphate and heparin. Nor could ligands for scavenger receptors and antibodies directed towards ICAM-1, CD 44 and RHAMM (Receptor for HA Mediated Motility) significantly inhibit the association of HA to tumour cells.     

Amphibian Declines in Brazil: An Overview1

Population declines have previously been reported for at least 31 amphibian species in Brazil, in the families Leptodactylidae (19), Hylidae (7), Centrolenidae (2), Dendrobatidae (2), and Bufonidae (1). In five Brazilian museum collections, we found no entries of new records dating back to at least 15 yr ago for 13 of these species. We suggest that these taxa be studied in more detail to verify their status and to generate basic ecological data. Museum data indicate that the remaining species have been recently found in areas of reported crashes, or elsewhere. Several apparent declines in Brazil can be associated with habitat loss, interspecific interactions, natural fluctuations, or lack of intensive sampling. Personal observations and field data also indicate possible declines in the states of Paraná and Ceará as well as in highlands within the Cerrado biome, in the state of Minas Gerais. Records suggest declines of montane and stream‐associated populations of Brazilian amphibians in apparently pristine habitats. Field work is necessary to confirm these cases and to examine whether factors associated with similar extinctions in other parts of the globe—such as pathogens and climate change—are also related to local disappearances. To clarify pending questions and perhaps circumvent new cases, it is important to invest in short‐ and long‐term field studies, and in the maintenance and expansion of museum collections.     

Enantiomeric LC separation of calcium antagonists on protein-based chiral stationary phases

Three chiral calcium antagonist drugs, bepridil and two dihydropyridine derivatives (nicardipine and REC 15/2375), have been successfully separated within short retention times using either the α₁-acid glycoprotein chiral stationary phase (Chiral AGP) or the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component (propan-2-ol, acetonitrile, ethanol) in order to modulate the retention properties of each system. The influence of pH and percentage of organic modifier on retention, selectivity, resolution, and column performance are discussed for bepridil analyzed on Chiral AGP and for the two dihydropyridines (nicardipine and REC 15/2375) analyzed on Ultron ES-OVM stationary phases. © 1993 Wiley-Liss, Inc.     

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.     

Palm live aboveground biomass in the riparian zones of a forest in Central Amazonia

Aboveground biomass estimates in the Amazon region remain uncertain, partly due to extrapolations based mainly on samples collected in upland terrains of terra-firme forests. Most biomass estimates were focused on dicotyledonous trees or included other plant groups as a category of trees. Palms dominate areas that represent 20% of the Brazilian Amazon. However, their contribution to biomass estimates and the variation within riparian zones remain poorly documented. We estimated the biomass of palms larger than 1–cm diameter at breast height (1.3 m aboveground) in riparian plots (n = 40); investigated the potential bias caused by the use of dicotyledonous- or family- rather than species-level equations for biomass estimation; compared palm biomass between riparian and non-riparian plots (n = 72); and evaluated the effects of soil, topography, and stream characteristics in riparian plots on palm biomass. Mean palm biomass in riparian zones estimated with species-level equations (27.50 ± 12.94 Mg/ha, range: 3.32–63.27 Mg/ha) was three times greater than biomass estimated with a family-level equation (9.04 ± 4.29 Mg/ha, range: 1.51–21.25 Mg/ha) and was greater than mean biomass estimated with a pantropical equation (20.46 ± 9.29 Mg/ha, range: 3.67–47.99 Mg/ha). Mean palm biomass in riparian zones was four times greater than in non-riparian zones. Palm biomass was high in flatter areas with poorly drained soils, but lower around streams with higher discharge. Inclusion of palms can contribute to reducing the uncertainties in biomass estimates in Amazonian forests. Recognition of the importance of riparian zones may improve conservation policies. Abstract in Portuguese is available with online material.     

A longitudinal study of free leptin index in pre-eclamptic pregnancies

The ratio between circulating levels of leptin and soluble leptin receptor (sOB-R), the free leptin index (FLI), is used as a marker of leptin resistance. Therefore, the aim of our study was to investigate the FLI in mild pre-eclamptic pregnancies in a nested case–control study within a prospective observational study. Circulating levels of leptin and sOB-R levels rise significantly during pregnancy in healthy (p < 0.05) (n = 46) and pre-eclamptic pregnancies (p < 0.05) (n = 20). Serum levels of leptin were significantly higher in pre-eclamptic compared to healthy pregnancies at second and third trimesters of pregnancy (p < 0.05). Additionally, serum levels of sOB-R were significantly lower in pre-eclamptic pregnancies during the second and third trimesters of pregnancy compared to healthy pregnancies (p < 0.05). Moreover, we found that FLI did not vary significantly during pregnancy in healthy women (p > 0.05), while it increases in pre-eclamptic pregnancies (p < 0.05). Indeed, FLI was significantly higher at second and third trimesters of pregnancy in pre-eclamptic compared to healthy pregnancies (p < 0.05). In addition, FLI was significantly higher in the luteal phase compared with the follicular phase of the menstrual cycle in eumenorrheic women (p < 0.05). Receiver operating characteristic (ROC) curve analysis revealed the ability of leptin (AUC = 0.72) and FLI (AUC = 0.67) as a reliable predictor for mild pre-eclampsia during the second trimester of pregnancy. In conclusion, our findings show that FLI were significantly increased in mild pre-eclamptic pregnancies and allowed us to hypothesize that this rise might alter leptin bioavailability and bioactivity which might lead to the sympathetic hyperactivity and the hypertensive disorders during pregnancy.     

Effects of blocking CD24 and CD47 ‘don't eat me’ signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia

Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) with a poor prognosis, at high risk of relapse after conventional treatment. MCL-associated tumour microenvironment (TME) is characterized by M2-like tumour-associated macrophages (TAMs), able to interact with cancer cells, providing tumour survival and resistance to immuno-chemotherapy. Likewise, monocyte-derived nurse-like cells (NLCs) present M2-like profile and provide proliferation signals to chronic lymphocytic leukaemia (CLL), a B-cell malignancy sharing with MCL some biological and phenotypic features. Antibodies against TAMs targeted CD47, a ‘don't eat me’ signal (DEMs) able to quench phagocytosis by TAMs within TME, with clinical effectiveness when combined with Rituximab in pretreated NHL. Recently, CD24 was found as valid DEMs in solid cancer. Since CD24 is expressed during B-cell differentiation, we investigated and identified consistent CD24 in MCL, CLL and primary human samples. Phagocytosis increased when M2-like macrophages were co-cultured with cancer cells, particularly in the case of paired DEMs blockade (i.e. anti-CD24 + anti-CD47) combined with Rituximab. Similarly, unstimulated CLL patients-derived NLCs provided increased phagocytosis when DEMs blockade occurred. Since high levels of CD24 were associated with worse survival in both MCL and CLL, anti-CD24-induced phagocytosis could be considered for future clinical use, particularly in association with other agents such as Rituximab.