A family of protein-deglutamylating enzymes associated with neurodegeneration.

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.

IgG3 donor-specific antibodies with a proinflammatory glycosylation profile may be associated with the risk of antibody-mediated rejection after kidney transplantation.

The pathogenicity of de novo donor-specific antibodies (dnDSA) varies according to their characteristics. While their MFI, complement-fixing ability, and IgG3 subclass are associated with ABMR occurrence and graft loss, they are not fully predictive of outcomes. We investigated the role of the Fc glycosylation of IgG3 dnDSA in ABMR occurrence using mass spectrometry after isolation by single HLA antigen beads. Between 2014 and 2018, we enrolled 54 patients who developed dnDSA (ABMR- n = 24; ABMR+ n = 30) in two French transplant centers. Fucosylation, galactosylation, GlcNAc bisection, and sialylation of IgG3 dnDSA were compared between ABMR+ and ABMR- patients. IgG3 dnDSA from ABMR+ patients exhibited significantly lower sialylation (7.5% vs. 10.5%, p < .001) and higher GlcNAc bisection (20.6% vs. 17.4%, p = .008). Fucosylation and galactosylation were similar in both groups. DSA glycosylation was not correlated with DSA MFI. In a multivariate analysis, low IgG3 sialylation, high IgG3%, time from transplantation to kidney biopsy, and tacrolimus-free regimen were independent predictive factors of ABMR. We conclude that a proinflammatory glycosylation profile of IgG3 dnDSA is associated with a risk of ABMR occurrence. Further studies are needed to confirm the clinical interest of DSA glycosylation and to clarify its role in determining the risk of ABMR and graft survival.

Chemical Characterization and Biological Activity of the Essential Oil from Araucaria brasiliensis Collected in Ecuador.

The purpose of this study was to determine the chemical composition, physical properties, enantiomeric composition and cholinesterase inhibitory activity of the essential oil (EO) steam-distilled from the leaves of the plant Araucaria brasiliensis Loud. collected in Ecuador. The chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS) analysis on two capillary GC columns (DB5-ms and HP-INNOWax). Thirty-three compounds were identified in the EO; the main compounds were beyerene (26.08%), kaurene (24.86%), myrcene (11.02%), α-pinene (9.99%) and 5,15-rosadiene (5.87%). Diterpene hydrocarbons (65.41%), followed by monoterpene hydrocarbons (21.11%), were the most representative components of the EO. Enantioselective analysis of the EO showed four pairs of enantiomeric compounds, α-pinene, camphene, γ-muurolene and δ-cadinene. In an in vitro assay, the EO showed moderate inhibitory activity towards the enzyme butyrylcholinesterase (BuChE) (95.7 µg/mL), while it was inactive towards acetylcholinesterase (AChE) (225.3 µg/mL). Further in vivo studies are needed to confirm the anticholinesterase potential of the EO.

Proteasome 19S RP and translation preinitiation complexes are secreted within exosomes upon serum starvation.

The aim of our study was to investigate the impact of macroautophagy on exosome secretion. Exosomes are small membrane vesicles released in the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. They were initially discovered as a way to remodel the reticulocyte plasma membrane before entering the blood circulation (Current Opinion in Hematology 2010, 17:177-183) and are now essentially studied as mediators of intercellular communication. Using iTRAQ proteomics, we compared the protein composition of purified exosomes secreted by cells impaired or not for macroautophagy by Atg5 depletion, during serum starvation conditions or complete medium culture. We show that the absence of serum modifies exosomal content, especially inducing secretion of two cytoplasmic protein complexes, namely proteasomal 19S regulatory particle (RP) and components of noncanonical translation preinitiation complex (PIC). This process is enhanced when autophagy is impaired by Atg5 depletion. Moreover, we show that the proteasome 20S core particle (CP) is released in the extracellular space. However, in striking contrast to what seen for its 19S RP regulator, release is independent of the exosomal vesicles, Atg5 expression and cell culture conditions. Exosome secretion can thus be considered as a cell process that participates in and reflects cell homeostasis, and care must be taken when studying potential extracellular function of exosomes due to the possible copurification of proteasome 20S CP.

Contactin-1 is a novel target antigen in membranous nephropathy associated with chronic inflammatory demyelinating polyneuropathy.

Primary membranous nephropathy (MN) is an autoimmune glomerular disease in which autoantibodies are directed against podocyte proteins. In about 80% of cases the main targeted antigen is the phospholipase A2 receptor 1 (PLA2R1). Anti-PLA2R1 antibodies are mainly immunoglobulin G type 4 (IgG4). However, the antigenic target remains to be defined in 20% of cases. MN can be associated with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system where a common antigenic target has yet to be identified. To ascertain a possible novel target antigen, we analyzed kidney biopsies from five patients positive for anti-contactin 1 antibodies and presenting with MN combined with chronic inflammatory demyelinating polyneuropathy. Eluted IgG from biopsy sections against contactin 1 and nerve tissue were screened. Western blot revealed contactin 1 expression in normal kidney glomeruli. Confocal microscopic analysis showed the presence and colocalization of contactin 1 and IgG4 on the glomerular basement membrane of these patients. Glomerular contactin 1 was absent in patients with anti-PLA2R1-associated MN or membranous lupus nephritis or a healthy control. The eluted IgG from contactin 1-positive biopsy sections but not the IgG eluted from patients with PLA2R1 MN bound contactin 1 with the main eluted subclass IgG4. Eluted IgG could bind paranodal tissue (myelinated axon) and colocalized with commercial anti-contactin 1 antibody. Thus, contactin 1 is a novel common antigenic target in MN associated with chronic inflammatory demyelinating polyneuropathy. However, the precise pathophysiology remains to be elucidated.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

Chemical Composition and Anticholinesterase Activity of the Essential Oil of Leaves and Flowers from the Ecuadorian Plant Lepechinia paniculata (Kunth) Epling.

This work aimed to study the chemical composition, cholinesterase inhibitory activity, and enantiomeric analysis of the essential oil from the aerial parts (leaves and flowers) of the plant Lepechinia paniculata (Kunth) Epling from Ecuador. The essential oil (EO) was obtained through steam distillation. The chemical composition of the oil was evaluated by gas chromatography, coupled to mass spectrometry (GC-MS) and a flame ionization detector (GC-FID). The analyses led to the identification of 69 compounds in total, of which 40 were found in the leaves and 29 were found in the flowers of the plant. The major components found in the oil were 1,8-Cineole, ß-Pinene, δ-3-Carene, α-Pinene, (E)-Caryophyllene, Guaiol, and ß-Phellandrene. Flower essential oil showed interesting selective inhibitory activity against both enzymes AChE (28.2 ± 1.8 2 µg/mL) and BuChE (28.8 ± 1.5 µg/mL). By contrast, the EO of the leaves showed moderate mean inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), with IC50 values of 38.2 ± 2.9 µg/mL and 47.4 ± 2.3 µg/mL, respectively.

A Novel Chemical Profile of a Selective In Vitro Cholinergic Essential Oil from Clinopodium taxifolium (Kunth) Govaerts (Lamiaceae), a Native Andean Species of Ecuador.

A novel chemical profile essential oil, distilled from the aerial parts of Clinopodium taxifolium (Kunth) Govaerts (Lamiaceae), was analysed by Gas Chromatography-Mass Spectrometry (GC-MS, qualitative analysis) and Gas Chromatography with Flame Ionization Detector (GC-FID, quantitative analysis), with both polar and non-polar stationary phase columns. The chemical composition mostly consisted of sesquiterpenes and sesquiterpenoids (>70%), the main ones being (E)-ß-caryophyllene (17.8%), α-copaene (10.5%), ß-bourbonene (9.9%), δ-cadinene (6.6%), cis-cadina-1(6),4-diene (6.4%) and germacrene D (4.9%), with the non-polar column. The essential oil was then submitted to enantioselective GC analysis, with a diethyl-tert-butyldimethylsilyl-ß-cyclodextrin diluted in PS-086 chiral selector, resulting in the following enantiomeric excesses for the chiral components: (1R,5S)-(-)-α-thujene (67.8%), (1R,5R)-(+)-α-pinene (85.5%), (1S,5S)-(-)-ß-pinene (90.0%), (1S,5S)-(-)-sabinene (12.3%), (S)-(-)-limonene (88.1%), (S)-(+)-linalool (32.7%), (R)-(-)-terpinen-4-ol (9.3%), (S)-(-)-α-terpineol (71.2%) and (S)-(-)-germacrene D (89.0%). The inhibition activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of C. taxifolium essential oil was then tested, resulting in selective activity against BChE with an IC50 value of 31.3 ± 3.0 µg/mL (positive control: donepezil, IC50 = 3.6 µg/mL).

New imidazoquinoxaline derivatives: Synthesis, biological evaluation on melanoma, effect on tubulin polymerization and structure-activity relationships.

Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122µM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin.

Exploring Southern Ecuador's Traditional Medicine: Biological Screening of Plant Extracts and Metabolites.

Ecuador stands as a nation inheriting a profound ancestral legacy in the utilization of medicinal plants, reflective of the rich biodiversity embraced by various ethnic groups. Despite this heritage, many of these therapeutic resources remain insufficiently explored concerning their toxicity and potential pharmacological effects. This study focused on a comprehensive evaluation of cytotoxicity and the potential subcellular targets within various extracts and nine isolated metabolites from carefully selected medicinal plants. Assessing their impact on the breast cancer cell line (MCF7), we subsequently examined the most active fractions for effects on the cell cycle, microtubule network, centrosome duplication, γH2AX foci, and E-cadherin. The investigated crude extracts and isolated compounds from Ecuadorian medicinal plants demonstrated cytotoxic effects, influencing diverse cellular pathways. These findings lend credence to the traditional uses of Ecuadorian medicinal plants, which have served diverse therapeutic purposes. Moreover, they beckon the exploration of the specific chemicals, whether in isolation or combination, responsible for these observed activities.

Constituents and Selective BuChE Inhibitory Activity of the Essential Oil from Hypericum aciculare Kunth.

A potential source of new inhibitors of cholinesterase enzymes are certain compounds of natural plant origin; therefore, in the study described herein we have determined the chemical composition and the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of the essential oil (EO) steam distilled from aerial parts of Hypericum aciculare, which was collected in southern Ecuador. The oil qualitative and quantitative composition was determined by GC-FID and GC-MS using a non-polar and a polar chromatographic column. A total of fifty-three constituents were identified, that accounted for about 98% of the EO content. The hydrocarbon n-nonane (16.4-28.7%) and the aldehyde n-decanal (20.7-23.1%) were the predominant oil constituents. In addition, the EO showed significant inhibition of BuChE (IC50 = 28.3 ± 2.7 µg/mL) and moderate activity towards AChE (IC50 = 82.1 ± 12.1 µg/mL). Thus, the EO from H. aciculare aerial parts is an interesting candidate to investigate the mechanism of selective ChE inhibition by the two ChE enzymes with the aim to discover potential targets to control the progression of the Alzheimer's disease (AD).

Constituents, Enantiomeric Content, and ChE Inhibitory Activity of the Essential Oil from Hypericum laricifolium Juss. Aerial Parts Collected in Ecuador.

The physical properties, chemical composition, enantiomer distribution, and cholinesterase (ChE) inhibitory activity were determined for a steam-distilled essential oil (EO), with a yield of 0.15 ± 0.05 % (w/w), from H. laricifolium aerial parts, collected in southern Ecuador. The oil qualitative and quantitative analyses were performed by GC-EIMS and GC-FID techniques, using two capillary columns containing a non-polar 5%-phenyl-methylpolysiloxane and a polar polyethylene glycol stationary phase, respectively. The main constituents (>10%) detected on the two columns were, respectively, limonene (24.29, 23.16%), (E)-ß-ocimene (21.89, 27.15%), and (Z)-ß-ocimene (12.88, 16.03%). The EO enantioselective analysis was carried out using a column based on 2,3-diethyl-6-tert-butyldimethylsilyl-ß-cyclodextrin. Two mixtures of chiral monoterpenes were detected containing (1R,5R)-(+)-α-pinene (ee = 83.68%), and (S)-(-)-limonene (ee = 88.30%) as the major enantiomers. This finding led to some hypotheses about the existence in the plant of two enantioselective biosynthetic pathways. Finally, the EO exhibited selective inhibitory effects in vitro against butyrylcholinesterase (BuChE) (IC50 = 36.80 ± 2.40 µg/mL), which were about three times greater than against acetylcholinesterase (IC50 = 106.10 ± 20.20). Thus, the EO from Ecuadorian H. laricifolium is an interesting candidate for investigating the mechanism of the selective inhibition of BuChE and for discovering novel drugs to manage the progression of Alzheimer's disease.

Targeting the splicing isoforms of spleen tyrosine kinase affects the viability of colorectal cancer cells.

Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. Our goal was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Our analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. We also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For our functional studies, we used short hairpin RNA tools to target the expression of Syk in CRC cells bearing the activating K-Ras (G13D) mutation. Our results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, we report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in our group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo.

Selective BuChE Inhibitory Activity, Chemical Composition, and Enantiomeric Content of the Essential Oil from Salvia leucantha Cav. Collected in Ecuador.

The essential oil (EO) of Salvia leucantha Cav. was isolated by steam distillation of the aerial parts collected in the South of Ecuador. Its physical properties were evaluated and the chemical composition of the oil was determined by GC-MS and GC-FID analyses using two chromatographic columns, DB-5ms and HP-INNOWax. Six major compounds were identified, namely, the sesquiterpenes 6.9-guaiadiene (19.14%), (E)-caryophyllene (16.80%), germacrene D (10.22%), (E)-ß-farnesene (10.00%), and bicyclogermacrene (7.52%), and the monoterpenoid bornyl acetate (14.74%). Furthermore, four pairs of enantiomers were determined by enantioselective GC-MS of the essential oil. (-)-germacrene D and (+)-α-pinene showed the highest enantiomeric excess (ee%). In an in vitro assay, the essential oil demonstrated an interesting inhibitory activity of the enzyme butyrylcholinesterase (BuChE), with an IC50 = 32.60 µg/mL, which is the highest determined for a Salvia species. In contrast, the oil was weakly active against acetylcholinesterase (AChE) with an IC50 > 250 µg/mL.

Cyclophilin A as negative regulator of apoptosis by sequestering cytochrome c.

The release of cytochrome c from the mitochondrial intermembrane space is a decisive event in programmed cell death. Once in the cytoplasm, cytochrome c is involved in the formation of the macromolecular complex termed apoptosome, which activates procaspase-9 which in turn activates downstream procaspase-3. There are increasing evidence indicating that cyclophilin A is highly expressed in many tumors and cell lines where it exerts an anti-apoptotic function. In brain tissue, which over-expresses constitutively cyclophilin A, we found mixed dimers composed of cyclophilin A and cytochrome c. In a cell-free system we observed that pure cyclophilin A inhibited cytochrome c-dependent procaspase-3 activation. Moreover, we detected cyclophilin A-cytochrome c complexes within the cytoplasm of HCT116 cells following staurosporine-induced apoptosis. Our results strongly support that, in tumor cells, cyclophilin A is able to inhibit procaspase-3 activation by sequestering cytochrome c.

Matrix-assisted laser desorption/ionization imaging mass spectrometry of oxaliplatin derivatives in heated intraoperative chemotherapy (HIPEC)-like treated rat kidney.

Oxaliplatin [1,2-diaminocyclohexane (dach)-Pt complex] is a platinum anticancer drug which is mainly used in the treatment of advanced colorectal cancer, particularly in Heated Intraoperative Chemotherapy (HIPEC) for the treatment of colorectal peritoneal carcinomatosis. In order to better understand the penetration of oxaliplatin in treated tissues we performed a direct imaging of tissue sections from HIPEC-like treated rat kidney using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This procedure allowed the detection and localization of oxaliplatin and its metabolites, the monocysteine and monomethionine complexes, in kidney sections. Specifically, oxaliplatin and its metabolites were localized exclusively in the kidney cortex, suggesting that it did not penetrate deeply into the organ. Based on these results, an imaging analysis of human tumors collected after HIPEC is currently in progress to assess the distribution of oxaliplatin and/or metabolites with the aim of defining clinical conditions to improve drug penetration.

Chemical composition and selective BuChE inhibitory activity of the essential oils from aromatic plants used to prepare the traditional Ecuadorian beverage horchata lojana.

ETHNOBOTANICAL AND ETHNOMEDICINAL RELEVANCE: In southern Ecuador, horchata lojana is a popular aromatic and refreshing beverage that is prepared from an aqueous infusion of different mixtures of local medicinal and aromatic plants. The drink is considered a traditional anti-inflammatory agent and brain tonic; due these properties, it has been drunk since Colonial Times. Several pharmacological studies have evaluated the effects of horchata aqueous infusion. However, the aromatic profile and the contribution of the volatile components to the biological activity of the drink have not been investigated so far. For these reasons, we have determined the chemical composition of the essential oils (EOs) distilled from five mixtures of aromatic plants commonly used for the preparation of this traditional drink. Moreover, to support the curative properties of the aromatic plants, the anticholinesterase activity of the EOs was examined. MATERIAL AND METHODS: Different bunches of fresh mixed medicinal and aromatic plants, called tongos, are sold at local markets in the province of Loja for the preparation of different types of horchata. In this research we have purchased plant bunches sold at five popular markets of Loja province. Subsequently, aromatic plants in each bunch were separated from medicinal plants and were then hydrodistilled to give the corresponding EOs. Subsequently, the chemical composition of each EO was determined by GC-MS/GC-FID techniques, whereas the cholinesterase inhibitory activity in vitro was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. AIMS OF THE STUDY: i) to contribute to the chemical and pharmacological study of the aroma components of the traditional Ecuadorian drink horchata lojana; ii) to identify botanically the mixtures of aromatic plants used to make the drink; iii) to establish, on the basis of the chemical composition of the EOs, the compounds mainly responsible for the characteristic beverage flavor; iv) to establish the possible existence of an aromatic pattern characteristic of each horchata preparation; v) to test the anticholinesterase activity of the EOs against AChE and BuChE in order to support the traditional consume of the drink as an effective brain tonic. RESULTS: A total of 23 botanical families and 32 species of plants used for the preparation of five different variants of the traditional horchata lojana beverage, have been identified. Fourteen aromatic species were determined to be responsible for the characteristic flavor of the drink. All the analyzed EOs belong to the monoterpene type. A total of 88 compounds have been identified in the different EOs, twenty-four of which are common components of the oils. CONCLUSIONS: According to the main components of the EOs distilled from the five groups of horchata lojana plants, four aromatic profiles have been defined: (i) neral + geranial + carvone, (ii) neral + geranial + myrcene; (iii) geranial + methyl eugenol + isomenthone + neral + citronellol; (iv) (E)-anethole + geranial + pulegone. Moreover, according to the literature, several aromatic plants and individual EOs components exhibit a wide range of biological activities. This finding as well as the significant BuChE inhibitory activity exhibited in vitro by the EOs give scientific support to the use of identified aromatic plants in the traditional preparation of horchata, that is considered a natural analgesic and anti-inflammatory remedy, and an effective brain tonic.

Distribution of de novo Donor-Specific Antibody Subclasses Quantified by Mass Spectrometry: High IgG3 Proportion Is Associated With Antibody-Mediated Rejection Occurrence and Severity.

Donor-specific antibodies (DSAs) are the main risk factor for antibody-mediated rejection (ABMR) and graft loss but could have variable pathogenicity according to their IgG subclass composition. Luminex-based test might lack sensitivity for the detection of IgG subclasses and this test does not allow quantifying the relative abundance of each IgG subclass. We investigated the precise repartition of each DSA subclass and their role in ABMR occurrence and severity, using an innovative mass spectrometry-based method. Between 2014 and 2018, we enrolled 69 patients who developed de novo DSA (n = 29 without ABMR, and n = 40 with ABMR) in two transplant centers. All IgG subclasses were detected in every samples tested: 62.7% were IgG1, 26.6% were IgG2, 6.6% were IgG3, and 4.2% were IgG4. The IgG3 proportion was significantly higher in the ABMR+ compared to the ABMR- group (8.4% vs. 5.6%, p = 0.003). The proportion of IgG1, IgG2, and IgG4 of DSA was similar between the two groups. Higher IgG3 level was associated with higher C4d deposition, higher microvascular inflammation scores, and glomerular filtration rate decline >25%. IgG3 proportion was not correlated with DSA MFI. Multivariate analysis showed that proteinuria and high level of IgG3 DSA were the only two factors independently associated with ABMR. In conclusion, de novo DSA are always composed of the four IgG subclasses, but in different proportions. High IgG3 proportion is associated with ABMR occurrence and severity and with poorer outcome, independently of DSA MFI.

Selection and mass spectrometry characterization of peptides targeting semiconductor surfaces.

We report on elaboration of 12-mer peptides that reveal specific recognition for the following semiconductor (SC) surfaces: GaAs(100), InAs(100), GaN(0001), ZnSe(100), ZnTe(100), GaAs(111)A, GaSb(100), CdSe(100). A M13 bacteriophage library was used to screen 10(9) different 12-mer peptides against these substrates to finally isolate, in maximum six amplification cycles, peptides that bind to the target surfaces. The specific peptides for the InAs and ZnSe surfaces were obtained. Contrary, for the other SC surfaces several peptides with high affinities have been isolated. Aiming for a better specificity, when the phage display has been conducted through six cycles, the screening procedure got dominated by a phage present in the M13 bacteriophage library and the SVSVGMKPSPRP peptide has been selected for different SCs. The high amplification potential of this phage has been observed previously with different targets. Thus, precaution should be undertaken in defining adhesion peptides with the phage display technique and real affinity of the obtained biolinkers should be studied with other methods. We employed mass spectrometry (MALDI-TOF/TOF) to demonstrate the preferential attachment (or not) of the SVSVGMKPSPRP peptide to the different SC surfaces. This allows us to define a realistic selection of the expressed peptides presenting affinity for the studied eight SC surfaces. We demonstrate that with increasing the dielectric constants of the employed solvents, adhesion of the SVSVGMKPSPRP peptide onto GaN(0001) is hindered.

Chemical, Enantioselective, and Sensory Analysis of a Cholinesterase Inhibitor Essential Oil from Coreopsis triloba S.F. Blake (Asteraceae).

The fresh leaves of Coreopsis triloba S.F. Blake, collected at Cerro Villonaco in Loja, Ecuador, were investigated with respect to their essential oil (EO). The chemical composition was determined qualitatively through gas chromatography coupled with mass spectrometry (GC-MS) and quantitatively by gas chromatography coupled with flame ionization (GC-FID), using relative response factors (RRF) based on the enthalpy of combustion. The essential oil contained between 92.5% and 93.4% of monoterpene hydrocarbons, with (E)-ß-ocimene being the main component (35.2-35.9%), followed by ß-phellandrene (24.6-25.0%), α-pinene (15.3-15.9%), myrcene (10.9-11.0%), sabinene (2.2-2.4%), (Z)-ß-ocimene (1.5%), and germacrene D (1.2-1.3%). The enantiomeric distribution of α-pinene, ß-pinene, limonene, and germacrene D was also determined. The main components responsible for the aroma were identified through aroma extract dilution analysis (AEDA), a gas chromatography-olfactometry (GC-O) based technique, being α-pinene, ß-pinene (0.6%), terpinolene (0.1%), α-copaene (0.1-0.3%), ß-phellandrene, and (E)-4,8-dimethyl-1,3,7-nonatriene (0.1-0.2%) the main olfactory constituents according to the decreasing factor of dilution (FD) order. The biological tests showed IC50 inhibition values of 42.2 and 6.8 µg/mL for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively.