Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy.

Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α-synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in α-synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in α-synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo , providing key evidence to support the hypothesis that α-synuclein adopts unique strains in patients. Here, to further interrogate α-synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate α-synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of α-synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro , demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α-synuclein misfolding. Overall, our studies use a structure-based approach to investigate α-synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

Improvement of New Dianionic Ionic Liquids vs Monoanionic in Solubility of Poorly Water-Soluble Drugs.

New ionic liquids (ILs) based on dianionic phosphonate anions and ammonium cations were prepared and characterized. They were used as excipients to increase the water solubility of two oral drugs, piroxicam and ibuprofen, that are slightly soluble in water. An increment in solubility of 300-fold was achieved for ibuprofen when compared with pure water, with only 0.25 mol% of IL in water. Interestingly, this was achieved with the less toxic dianionic ionic liquid [N4 1 2OH 2OH]2 [C2H5PO3], which presents an IC50 of 120 mM (≈0.25 mol%). On the other hand, piroxicam showed an increase of 480-fold for the same dianionic ionic liquid, with the same ionic liquid percentage. In contrast, for monoanionic ionic liquids, the effect was not so pronounced, and only a 10-fold was obtained, in the presence of 0.3 mol% of IL. The lipophilicity (logP) of drugs decreased in the presence of these ILs. Cytotoxicity profile of these ILs was determined and they did not show a significant impact towards healthy fibroblasts. The cytotoxicity of ibuprofen and piroxicam was also determined, and cellular viability almost did not change when ionic liquid was in the presence of 1 mM of oral drug.

A conserved coccidian gene is involved in Toxoplasma sensitivity to the anti-apicomplexan compound, tartrolon E.

New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.

Simultaneous Separation of Antioxidants and Carbohydrates From Food Wastes Using Aqueous Biphasic Systems Formed by Cholinium-Derived Ionic Liquids.

The food industry produces significant amounts of waste, many of them rich in valuable compounds that could be recovered and reused in the framework of circular economy. The development of sustainable and cost-effective technologies to recover these value added compounds will contribute to a significant decrease of the environmental footprint and economic burden of this industry sector. Accordingly, in this work, aqueous biphasic systems (ABS) composed of cholinium-derived bistriflimide ionic liquids (ILs) and carbohydrates were investigated as an alternative process to simultaneously separate and recover antioxidants and carbohydrates from food waste. Aiming at improving the biocompatible character of the studied ILs and proposed process, cholinium-derived bistriflimide ILs were chosen, which were properly designed by playing with the cation alkyl side chain and the number of functional groups attached to the cation to be able to create ABS with carbohydrates. These ILs were characterized by cytotoxicity assays toward human intestinal epithelial cells (Caco-2 cell line), demonstrating to have a significantly lower toxicity than other well-known and commonly used fluorinated ILs. The capability of these ILs to form ABS with a series of carbohydrates, namely monosaccharides, disaccharides and polyols, was then appraised by the determination of the respective ternary liquid-liquid phase diagrams at 25°C. The studied ABS were finally used to separate carbohydrates and antioxidants from real food waste samples, using an expired vanilla pudding as an example. With the studied systems, the separation of the two products occurs in one-step, where carbohydrates are enriched in the carbohydrate-rich phase and antioxidants are mainly present in the IL-rich phase. Extraction efficiencies of carbohydrates ranging between 89 and 92% to the carbohydrate-rich phase, and antioxidant relative activities ranging between 65 and 75% in the IL-rich phase were obtained. Furthermore, antioxidants from the IL-rich phase were recovered by solid-phase extraction, and the IL was recycled for two more times with no losses on the ABS separation performance. Overall, the obtained results show that the investigated ABS are promising platforms to simultaneously separate carbohydrates and antioxidants from real food waste samples, and could be used in further related applications foreseeing industrial food waste valorization.

Enhancement of water solubility of poorly water-soluble drugs by new biocompatible N-acetyl amino acid N-alkyl cholinium-based ionic liquids.

The major challenge of the pharmaceutical industry is to find potential solvents for poorly water-soluble drug molecules. Ionic liquids (ILs) have attracted this industry as (co-) solvents due to their unique physicochemical and biological properties. Herein, a straightforward approach for the enhancement of the water solubility of paracetamol and sodium diclofenac is presented, using new biocompatible N-acetyl amino acid N-alkyl cholinium-based ionic liquids as co-solvents (0.2-1 mol%). These new ionic liquids were able to increase the water solubility of these drugs up to four times that in pure water or in an inorganic salt solution. In the presence of these ILs, the drugs lipophilicity (log P was not significantly changed for paracetamol, but for sodium diclofenac it was possible to decrease significantly its lipophilicity. Concerning cytotoxicity in human dermal fibroblasts it was observed that ILs did not show a significant toxicity, and were able to improve cell viability compared with the respective precursors.

Integrating Fenton's process and ion exchange for olive mill wastewater treatment and iron recovery.

A novel integrated methodology involving Fenton's process followed by ion exchange (IE) was proposed for the treatment of olive mill wastewater. Fenton's process was optimized and it was able to remove up to 81% of chemical oxygen demand when pH 3.5, reaction time 1 h, [Fe2+] = 50 mg L-1 and [Fe2+]/[H2O2] = 0.002 were applied. In spite of the potential of this treatment approach, final iron removal from the liquid typically entails pH increase and iron sludge production. The integration of an IE procedure using Lewatit TP 207 resin was found to be able to overcome this important environmental shortcoming. The resin showed higher affinity toward Fe3+ than to Fe2+. However, the iron removal efficiency of an effluent coming from Fenton's was independent of the type of the initial iron used in the process. The presence of organic matter had no significant effect over the resin iron removal efficiency. Even if some efficiency decrease was observed when a high initial iron load was applied, the adsorbent mass quantity can be easily adapted to reach the desired iron removal. The use of IE is an interesting industrial approach able to surpass Fenton's peroxidation drawback and will surely boost its full-scale application in the treatment of bio-refractory effluents.

A contribution to the rational design of Ru(CO)3Cl2L complexes for in vivo delivery of CO.

A few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related complexes with the formula [Ru(CO)3Cl2L] (L = DMSO (3), L-H3CSO(CH2)2CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)2CH(NH2)CO2H (6b); 3-NC5H4(CH2)2SO3Na (7); 4-NC5H4(CH2)2SO3Na (8); PTA (9); DAPTA (10); H3CS(CH2)2CH(OH)CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWN) shows the addition of Ru(II)(CO)(H2O)4 at the His15 binding site. Soakings with 7(4UWN) produced the metallacarboxylate [Ru(COOH)(CO)(H2O)3](+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO(-) on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.

Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics.

The complex fac-[Mo(CO)(3)(histidinate)]Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO.

Hydrogen activation by high-valent oxo-molybdenum(VI) and -rhenium(VII) and -(V) compounds.

The high-valent oxo-molybdenum(VI) and -rhenium(VII) and -(V) derivatives MoO2Cl2, ReCH3O3 (MTO) and ReIO2(PPh3)2 catalyze the selective hydrogenation of alkynes to alkenes at 80 degrees C under 40 atm of pressure. The reduction of sulfoxides to sulfides has also been performed by oxo-rhenium and -molybdenum complexes using hydrogen as a reducing agent. Activation of hydrogen by MoO2Cl2 and MoO2(S2CNEt2)2 was shown by means of DFT calculations to proceed by H-H addition to the Mo=O bond, followed by hydride migration to yield a water complex.

Oral health status of 207 head and neck cancer patients before, during and after radiotherapy.

The aim of this retrospective study was to describe the oral health status of patients before, during, and after radiotherapy (RT) for the treatment of head and neck cancer (HNC). Before RT, the following data was collected: presence of unrecoverable teeth, residual roots, unerupted teeth, use of dentures, periodontal alterations, caries, candidiasis, and xerostomia. Mucositis, candidiasis, and xerostomia were evaluated during RT. Patients continued to be followed after RT for evaluation of mucositis, candidiasis, xerostomia, radiation caries, and osteoradionecrosis. For statistical analysis, 95% confidence intervals (CI) were determined using sample size, population, and percentages. Before RT, 120 (57.9%) patients presented with alterations in the oral cavity namely, 85 (41.0%) with periodontal disease, 44 (21.2%) with residual roots, 25 (12.0%) with caries, 15 (7.2%) with candidiasis, and 12 (5.8%) had an unerupted tooth present. Xerostomia was a complaint of 19 patients (9.1%). Restorations were indicated for 33 patients (15.9%), whereas extraction was indicated for 104 (50.2%) patients. During RT, mucositis was found in 80 (61.7%) patients, candidiasis in 60 (45.8%), and xerostomia was a complaint of 82 patients (62.6%). After RT, mucositis persisted in 21 patients (19.2%), candidiasis was identified in 23 patients (21.1%), and xerostomia was reported by 58 patients (53.2%). Radiation caries developed in 12 patients (11.0%), whereas six patients (5.5%) developed osteoradionecrosis. The demographic profile herein presented will be useful as baseline data to provide additional epidemiological information and to determine future measures for prevention and treatment of RT-induced complications and sequelae.

Direct and remarkably efficient conversion of methane into acetic acid catalyzed by amavadine and related vanadium complexes. A synthetic and a theoretical DFT mechanistic study.

Vanadium(IV or V) complexes with N,O- or O,O-ligands, i.e., [VO{N(CH2CH2O)3}], Ca[V(HIDPA)2] (synthetic amavadine), Ca[V(HIDA)2], or [Bu4N]2[V(HIDA)2] [HIDPA, HIDA = basic form of 2,2'-(hydroxyimino)dipropionic or -diacetic acid, respectively], [VO(CF3SO3)2], Ba[VO(nta)(H2O)]2 (nta = nitrilotriacetate), [VO(ada)(H2O)] (ada = N-2-acetamidoiminodiacetate), [VO(Hheida)(H2O)] (Hheida = 2-hydroxyethyliminodiacetate), [VO(bicine)] [bicine = basic form of N,N-bis(2-hydroxyethyl)glycine], and [VO(dipic)(OCH2CH3)] (dipic = pyridine-2,6-dicarboxylate), are catalyst precursors for the efficient single-pot conversion of methane into acetic acid, in trifluoroacetic acid (TFA) under moderate conditions, using peroxodisulfate as oxidant. Effects on the yields and TONs of various factors are reported. TFA acts as a carbonylating agent and CO is an inhibitor for some systems, although for others there is an optimum CO pressure. The most effective catalysts (as amavadine) bear triethanolaminate or (hydroxyimino)dicarboxylates and lead, in a single batch, to CH3COOH yields > 50% (based on CH4) or remarkably high TONs up to 5.6 x 103. The catalyst can remain active upon multiple recycling of its solution. Carboxylation proceeds via free radical mechanisms (CH3* can be trapped by CBrCl3), and theoretical calculations disclose a particularly favorable process involving the sequential formation of CH3*, CH3CO*, and CH3COO* which, upon H-abstraction (from TFA or CH4), yields acetic acid. The CH3COO* radical is formed by oxygenation of CH3CO* by a peroxo-V complex via a V{eta1-OOC(O)CH3} intermediate. Less favorable processes involve the oxidation of CH3CO* by the protonated (hydroperoxo) form of that peroxo-V complex or by peroxodisulfate. The calculations also indicate that (i) peroxodisulfate behaves as a source of sulfate radicals which are methane H-abstractors, as a peroxidative and oxidizing agent for vanadium, and as an oxidizing and coupling agent for CH3CO* and that (ii) TFA is involved in the formation of CH3COOH (by carbonylating CH3*, acting as an H-source to CH3COO*, and enhancing on protonation the oxidizing power of a peroxo-VV complex) and of CF3COOCH3 (minor product in the absence of CO).

Catalyzing aldehyde hydrosilylation with a molybdenum(VI) complex: a density functional theory study.

[MoCl(2)O(2)] catalyzes the hydrosilylation reaction of aldehydes and ketones, as well as the reduction of other related groups, in apparent contrast to its known behavior as an oxidation catalyst. In this work, the mechanism of this reaction is studied by means of density functional theory calculations using the B3LYP functional complemented by experimental data. We found that the most favorable pathway to the first step, the Si--H activation, is a [2+2] addition to the Mo=O bond, in agreement with previous and related work. The stable intermediate that results is a distorted-square-pyramidal hydride complex. In the following step, the aldehyde approaches this species and coordinates weakly through the oxygen atom. Two alternative pathways can be envisaged: the classical reduction, in which a hydrogen atom migrates to the carbon atom to form an alkoxide, which then proceeds to generate the final silyl ether, or a concerted mechanism involving migration of a hydrogen atom to a carbon atom and of a silyl group to an oxygen atom to generate the silyl ether weakly bound to the molybdenum atom. In this Mo(VI) system, the gas-phase free energies of activation for both approaches are very similar, but if solvent effects are taken into account and HSiMe(3) is used as a source of silicon, the classical mechanism is favored. Several unexpected results led us to search for still another route, namely a radical path. The energy involved in this and the classical pathway are similar, which suggests that hydrosilylation of aldehydes and ketones catalyzed by [MoCl(2)O(2)] in acetonitrile may follow a radical pathway, in agreement with experimental results.

Dioxomolybdenum(VI) complexes as catalysts for the hydrosilylation of aldehydes and ketones.

The dioxomolybdenum(VI) complexes [MoO2Cl2] (1), [MoO2(acac)2] (2), [MoO2(S2CNEt2)2] (3), [CpMoO2Cl] (4), [MoO2(mes)2] (5) and the polymeric organotin-oxomolybdates [(R3Sn)2MoO4] [R = n-Bu (6), t-Bu (7), Me (8)] were examined as catalysts for the hydrosilylation of aldehydes and ketones with dimethylphenylsilane. Of these, [MoO2Cl2] (1) was the most efficient catalyst, affording quantitative yields of the corresponding silylated ethers at room temperature in acetonitrile. Complexes 2, 4-8 also catalyzed the same reaction but required heating at 80 degrees C and longer reaction times compared with 1. Compound 3 is inactive. The wide scope of molybdenum oxide-mediated hydrosilylation was established with a variety of aldehydes and ketones. Counter intuitively, the activity of is 1 highest in NCMe. In the absence of a carbonyl substrate, [MoO2Cl2(NCBu(t))] (10) reacts with HSiMe2Ph affording [MoO(OSiMe2Ph)Cl2]2 (11) which has been fully characterized by NMR and IR spectroscopy, elemental analyses and mass spectrometry. Addition of radical scavengers strongly slows down the [MoO2Cl2]-based hydrosilylation suggesting the intermediacy of oxygen-centered radicals.

Atençäo médica à gestaçäo e ao parto de mäes adolescentes / Medical care for teenage mother during pregnancy and at delivery

Estudou-se em Ribeirao Preto, SP, Brasil, no período de 1§ de junho de 1978 a 31 de maio de 1979, 98% do universo de nascidos vivos, totalizando 8878 crianças nascidas de parto único, sendo 6750 procedentes de Ribeirao Preto. Observou-se um aumento da proporçao de maes adolescentes em Ribeirao Preto (14,1%) quando comparada com estudo realizado 10 anos antes (11,7%). A idade materna menor de 20 anos esteve associada com os indicadores mais desfavoráveis para a saúde perinatal, no que diz respeito à atençao médica à gestaçao e ao parto. Essa situaçao ficou mais evidente entre as maes adolescentes de classes sociais menos favorescidas, revelando que o grupo de maes adolescentes nao é homogêneo, mas apresenta diferentes proporçoes de risco para a saúde perinatal de acordo com as fraçoes de classe social que exitem no seu interior