Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.     

Low-molecular-weight components of olive oil mill waste-waters

A new lignan 1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(3-acetyl-4-hydroxy-5-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, the secoiridoid 2H-pyran-4-acetic acid,3-hydroxymethyl-2,3-dihydro-5-(methoxycarbonyl)-2-methyl-, methyl ester, the phenylglycoside 4-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-1,4-dihydroxy-2-methoxybenzene and the lactone 3-[1-(hydroxymethyl)-1-propenyl] delta-glutarolactone were isolated and identified on the basis of spectroscopic data including two-dimensional NMR, as components of olive oil mill waste-waters. The known aromatic compounds catechol, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, tyrosol, hydroxytyrosol, 2-(4-hydroxy-3-methoxy)phenylethanol, 2-(3,4-dihydroxy)phenyl-1,2-ethandiol, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 1-O-[2-(3,4-dihydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, 1-O-[2-(4-hydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, D(+)-erythro-1-(4-hydroxy-3-methoxy)-phenyl-1,2,3-propantriol, p-hydroxyphenethyl-beta-D-glucopyranoside,2(3,4-dihydroxyphenyl)ethanol 3beta-D-glucopyranoside, and 2(3,4-dihydroxyphenyl)ethanol 4beta-D-glucopyranoside were also confirmed as constituents of the waste-waters.     

Synonymous Codon Choices in the Extremely GC-Poor Genome of Plasmodium falciparum: Compositional Constraints and Translational Selection

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection. Received: 10 November 1998 / Accepted: 28 January 1999     

Surface mechanomyogram reflects the changes in the mechanical properties of muscle at fatigue

The contractile properties of muscle are usually investigated by analysing the force signal recorded during electrically elicited contractions. The electrically stimulated muscle shows surface oscillations that can be detected by an accelerometer; the acceleration signal is termed the surface mechanomyogram (MMG). In the study described here we compared, in the human tibialis anterior muscle, changes in the MMG and force signal characteristics before, and immediately after fatigue, as well as during 6 min of recovery, when changes in the contractile properties of muscle occur. Fatigue was induced by sustained electrical stimulation. The final aim was to evaluate the reliability of the MMG as a tool to follow the changes in the mechanical properties of muscle caused by fatigue. Because of fatigue, the parameters of the force peak, the peak rate of force production and the peak of the acceleration of force production (d2F/dt2) decreased, while the contraction time and the half-relaxation time (1/2-RT) increased. The MMG peak-to-peak (p-p) also decreased. The attenuation rate of the force oscillation amplitude and MMG p-p at increasing stimulation frequency was greater after fatigue. With the exception of 1/2-RT, all of the force and MMG parameters were restored within 2 min of recovery. A high correlation was found between MMG and d2F/dt2 in un-fatigued muscle and during recovery. In conclusion, the MMG reflects specific aspects of muscle mechanics and can be used to follow the changes in the contractile properties of muscle caused by localised muscle fatigue.     

Stereotactic ablative radiation therapy for spinal metastases: experience at a single Brazilian institution

BackgroundThis study aims to assess the clinical outcomes of patients with spine metastases who underwent stereotactic ablative radiation therapy (SABR) as part of their treatment. SABR has arisen as a contemporary treatment option for spinal metastasis patients with good prognoses.Materials and methodsBetween November 2010 and September 2018, Spinal SABR was performed in patients with metastatic disease in different settings: radical (SABR only), postoperative (after decompression and/or fixation surgery), and reirradiation. Local control (LC), pain control, overall survival (OS) and toxicities were reported.ResultsEighty-five patients (corresponding to 96 treatments) with spine metastases were included. The median age was 59 years (range, 23–91). In most SA BR (82.3%, n = 79) was performed as the first local spine treatment, while in 12 settings (12.5%), fixation and/or decompression surgery was performed prior to SABR. Two-year overall survival rate was 74.1%, and median survival was 19 months. The LC rate at 2 years was 72.3%. With regard to pain control, among 67 patients presenting with pain before SA BR, 83.3% had a complete response, 12.1% had a partial response, and 4.6% had progression. Vertebral compression fractures occurred in 10 patients (11.7%), of which 5 cases occurred in the reirradiation setting. Radiculopathy and myelopathy were not observed. No grade III or IV toxicities were seen.ConclusionThis is the first study presenting a Brazilian experience with spinal SA BR, and the results confirm its feasibility and safety. SABR was shown to produce good local and pain control rates with low rates of adverse events.     

Structuring functional groups of aquatic insects along the resistance/resilience axis when facing water flow changes

Understanding how differences in intensity and frequency of hydrological disturbances affect the resistance and resilience of aquatic organisms is key to manage aquatic systems in a fast‐changing world. Some aquatic insects have strategies that improve the permanence (resistance), while others use strategies that favor recolonization (resilience). Therefore, we carried out a manipulative experiment to understand the influence of functional characteristics of aquatic insects in their permanence and recolonization against hydrological disturbances in streams in the biodiversity hotspot of the Cerrado of Brazil. We placed 200 artificial substrates in five streams and submitted them to changing water flow regimes that differed both in frequency and intensity, and we observed the response of the aquatic community for 39 days. We used a hierarchical Bayesian approach to estimate the probabilities of permanence and recolonization of each life strategy group (nine groups). We observed that the most intense changes in the water flow tended to affect the permanence of almost all groups, but the intensity of this effect reduced over time. On the other hand, less frequent disturbances, regardless of intensity, tended to reduce the permanence of most groups of aquatic insects over time. The different effects of disturbance intensity may have been related to a greater recolonization capacity of some groups. The results we present are worrisome in a scenario of reduced riparian vegetation around streams and with the expectation of precipitation becoming more concentrated in shorter periods of time due to climate change in the Cerrado hotspot, reducing the occurrence of many groups of aquatic insects in their habitat, particularly those with traits associated with resistance against hydrological disturbance.     

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.     

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M β-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried β-sheet. The two mutations located in this β-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M β-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.     

Impact of COVID-19 on the curative treatment of prostate cancer: a national cross-sectional study

BackgroundCOVID-19 pandemic affected access to cancer treatment worldwide. However, there is a lack of data about the impact in developing countries. The objective was to evaluate COVID-19 impact on curative prostate cancer (Pca) treatment in Brazil.Materials and methodsWith data extracted from the Brazilian Ministry of Health database, the Non-COVID and COVID periods were analyzed to compare the absolute number of radical prostatectomy (RP) and radiotherapy (RT) executed in the country and regions.ResultsWith data from 50,169 Pca patients (Non-COVID = 28,106 cases and COVID = 22,063) treated with RP or RT in Brazil, a significant decline in patients receiving RT or RP (−6.043 cases; p = 0.0001) was detected. Both treatment procedures (RT or PR) were reduced in all five Brazilian regions comparing the Non-COVID and COVID periods. Overall, there was a reduction on RP and RT procedures in 92% (24/25) and 76% (19/25) of the evaluated states, respectively. Comparing the variation of RT and RP per state between COVID and Non-COVID period, there is a significant difference (−18.6% vs. −29%, p = 0.03) with a higher negative impact on the RP group. The RT and RP variation had no significant relationship with the incidence of COVID cases in the states. Limitations include the non-evaluation of treatment combinations, the impact of hypofractionated radiotherapy, and other factors influencing the treatment choice.ConclusionsDuring the COVID-19 pandemic, the curative treatment with RP and RT of Pca was abruptly limited and affected. However, the number of RP was more impacted than RT during the COVID period.     

Dystroglycan is associated to the disulfide isomerase ERp57

Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and β-DG, connected through the α-DG C-terminal domain and the β-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the β-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with β-DG in the ER and at the plasma membrane of 293-Ebna cells. The β-DG-ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. β-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and β-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated.