Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

Infrared spectroscopy of serum as a potential diagnostic screening approach for naturally occurring canine osteoarthritis associated with cranial cruciate ligament rupture.

OBJECTIVE: To evaluate infrared (IR) spectroscopy of serum as a screening tool to differentiate dogs affected by naturally occurring osteoarthritis (OA) associated with cranial cruciate ligament rupture (CrCLR) and controls. METHOD: 104 adult dogs with CrCLR (affected group) and 50 adult control dogs were recruited for this prospective observational study. Serum samples were collected preoperatively from CrCLR dogs and from a subset of these dogs at 4-, and 12-week post-surgical intervention to stabilize the affected stifles. Serum was collected once from control dogs. Dry films were made from serum samples, and IR absorbance spectra acquired. Data preprocessing, principal component analysis and multivariate analysis of covariance were performed to separate samples from the two groups, and to evaluate temporal differences. Weighted logistic regression with L1 regularization method was used to develop a predictive model. Model performance based on an independent test set was evaluated. RESULTS: Spectral data analysis revealed significant separation between the sera of CrCLR and control dogs (P < 0.0001), but not amongst different time points in the OA group. The sensitivity, specificity, AUC and accuracy of the test set were 84.62%, 96.15%, 93.20% and 92.31% respectively. CONCLUSIONS: These findings confirm the potential of IR-spectroscopy of serum with chemometrics methods to differentiate controls from dogs with OA associated with CrCLR. This is the first step in development of an economic, and comparatively simple IR-based screening serum test for OA. Utility of this tool as a clinical screening and diagnostic test requires further investigation and validation.

Spirocyclic sulfonamides with carbonic anhydrase inhibitory and anti-neuropathic pain activity.

A novel series of 4-oxo-spirochromane bearing primary sulfonamide group were synthetized as Carbonic Anhydrase inhibitors (CAIs) and tested for their management of neuropathic pain. Indeed, CAs have been recently validated as novel therapeutic targets in neuropathic pain. All compounds, here reported, showed strong activity against hCA II and hCA VII with KI values in the low or sub-nanomolar range. Two compounds (6d and 6l) showed good neuropathic pain attenuating effects and longer duration than drug reference acetazolamide in an animal model of oxaliplatin induced neuropathy.

Discovery of new organoselenium compounds as antileishmanial agents.

We report new organoselenium compounds bearing the sulfonamide moiety as effective inhibitors of the ß-isoform of Carbonic Anhydrase from the unicellular parasitic protozoan L. donovani chagasi. All derivatives were evaluated in vitro for their leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells. Compounds 3e-g showed their activity in the low micromolar range with IC50 values spanning from 0.72 to 0.81 µM and selectivity indexes (SI) > 8 (for 3g SI > 30), thus much higher than those observed for the reference drugs miltefosine and edelfosine. This is the first study which reports new selenoderivatives with promising leishmanicidal properties and acting as Carbonic Anhydrase inhibitors too thus paving the way to the development of innovative agents for the treatment of neglected diseases such as leishmaniasis.

Angiogenic and Restorative Abilities of Human Mesenchymal Stem Cells Were Reduced Following Treatment With Serum From Diabetes Mellitus Type 2 Patients.

This experiment investigated the impact of serum from patients with type 2 diabetes mellitus on the angiogenic behavior of human mesenchymal stem cells in vitro. Changes in the level of Ang-1, Ang-2, cell migration, and trans-differentiation into pericytes and endothelial lineage were monitored after 7 days. The interaction of mesenchymal stem cells with endothelial cells were evaluated using surface plasmon resonance technique. Paracrine restorative effect of diabetic stem cells was tested on pancreatic ß cells. Compared to data from FBS and normal serum, diabetic serum reduced the stem cell survival and chemotaxis toward VEGF and SDF-1α (P < 0.05). Diabetic condition were found to decline cell migration rate and the activity of MMP-2 and -9 (P < 0.05). The down-regulation of VEGFR-2 and CXCR-4 was observed with an increase in the level of miR-1-3p and miR-15b-5p at the same time. The paracrine angiogenic potential of diabetic stem cells was disturbed via the changes in the dynamic of Ang-1, Ang-2, and VEGF. Surface plasmon resonance analysis showed that diabetes could induce an aberrant increase in the interaction of stem cells with endothelial cells. After treatment with diabetic serum, the expression of VE-cadherin and NG2 and ability for uptake of Dil-Ac-LDL were reduced (P < 0.01). Conditioned media prepared from diabetic stem cells were unable to decrease fatty acid accumulation in ß-cells (P < 0.05). The level of insulin secreted by ß-cells was not affected after exposure to supernatant from diabetic or non-diabetic mesenchymal stem cells. Data suggest diabetes could decrease angiogenic and restorative effect of stem cells in vitro. J. Cell. Biochem. 119: 524-535, 2018. © 2017 Wiley Periodicals, Inc.

Symmetric molecules with 1,4-triazole moieties as potent inhibitors of tumour-associated lactate dehydrogenase-A.

A series of symmetric molecules incorporating aryl or pyridyl moieties as central core and 1,4-substituted triazoles as a side bridge was synthesised. The new compounds were investigated as lactate dehydro-genase (LDH, EC 1.1.1.27) inhibitors. The cancer associated LDHA isoform was inhibited with IC50 = 117-174 µM. Seven compounds exhibited better LDHA inhibition (IC50 117-136 µM) compared to known LDH inhibitor - galloflavin (IC50 157 µM).

Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma.

A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (KIs of 81.0-3084 nM), being highly effective as hCA II (KIs in the range of 0.25-10.8 nM), IX (KIs of 3.7-50.4 nM) and XII (KIs of 0.60-52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated.

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties.

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (KIs of 28.5-2954nM), being highly effective as hCA II (KIs in the range of 0.62-12.4nM) and XII (KIs of 0.54-7.11nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.

Benzenesulfonamides incorporating bulky aromatic/heterocyclic tails with potent carbonic anhydrase inhibitory activity.

Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strategies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in their molecules, and were investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four isoforms, with KIs of 7.6-322nM against hCA I, of 0.06-85.4nM against hCA II; of 6.7-152nM against hCA IX and of 0.49-237nM against hCA XII; respectively. However no relevant isoform-selective behavior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis were the most inhibited ones. The structure-activity relationship for inhibiting the four CAs with these derivatives is discussed in detail.

Inhibition studies of quinazoline-sulfonamide derivatives against the γ-CA (PgiCA) from the pathogenic bacterium, Porphyromonas gingivalis.

Carbonic anhydrases (CAs, EC 4.2.1.1) began to be investigated in detail in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many bacteria CAs are essential for the life cycle of the organism. The presence of CAs in pathogenic bacteria allows the development of anti-infectives with a new mechanism of action, less explored to date. Here, novel quinazoline derivatives crowned with sulfonamide functionality at position-2 were tested for their ability to inhibit the bacterial γ-CA (PgiCA), identified in the genome of Porphyromonas gingivalis. Six compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme γ-PgCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic human isoform II (hCAII). The best γ-PgCA inhibitor was compound 8c, with a K(I) of 3.53 nM and selectivity ratio of 24.5 and 24.8 against hCA I and hCA II, respectively. Many of these new compounds showed a high selectivity for bacterial enzyme respect to the mammalian CA isoforms (hCAI and hCAII). These results suggest that sulfonamides with quinazoline scaffold could be considered as suitable candidates for further derivatization to better understand the role of bacterial CAs in pathogenesis.

Inhibition of human carbonic anhydrase isozymes I, II, IX and XII with a new series of sulfonamides incorporating aroylhydrazone-, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenylmethylene)-1,3,4-thiadiazol-3(2H)-yl moieties.

A series of benzenesulfonamides incorporating aroylhydrazone, piperidinyl, sulfone, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties was investigated as inhibitors of four α-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h) isoforms hCA I, II (cytosolic, offtarget enzymes) and hCA IX and XII (transmembrane, tumor-associated isoforms). Low nanomolar activity was observed against hCA II (KIs of 0.56-17.1 nM) with these sulfonamides, whereas the slow cytosolic isoform hCA I was less inhibited by these compounds (KIs of 86.4 nM-32.8 µM). Most of these sulfonamides significantly inhibited CA IX, with KIs in the range of 4.5-47.0 nM, although some of the derivatives incorporating bulkier bicyclic moieties, as well as 2-thienyl fragments, showed a weaker activity against this isoform (KIs in the range 50.1-553 nM). All the investigated compounds also inhibited CA XII with KIs in the range 0.85-376 nM. The best inhibitors were those incorporating bulky [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl moieties and 1,3,4-thiadiazol-3(2H)-yl groups.

Quinazoline-sulfonamides with potent inhibitory activity against the α-carbonic anhydrase from Vibrio cholerae.

Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by simple, eco-friendly procedures. These compounds were tested for their ability to inhibit the α-carbonic anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human α-CA isoforms, hCA I and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II. The best VchCA inhibitor had a KI of 2.7 nM. Many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectivity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better understand the role of bacterial CAs in pathogenesis.

Inhibition of carbonic anhydrases from the extremophilic bacteria Sulfurihydrogenibium yellostonense (SspCA) and S. azorense (SazCA) with a new series of sulfonamides incorporating aroylhydrazone-, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenylmethylene)-1,3,4-thiadiazol-3(2H)-yl moieties.

A series of new sulfonamides was prepared starting from 2-oxo-N'-(4-sulfamoylphenyl)-propanehydrazonoyl chloride, a sulfanilamide derivative, which was reacted with aroylhydrazides, amines, or thiols. A library of derivatives incorporating aroylhydrazone, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties was thus synthesized. The new compounds were investigated as inhibitors of four α-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h) isoforms hCA I and II, and the bacterial ones recently isolated from the extremophilic bacteria Sulfurihydrogenibium yellostonense (SspCA) and Sulfurihydrogenibium azorense (SazCA). Low nanomolar activity was observed against hCA II (KIs of 0.56-17.1 nM) whereas hCA I was less inhibited by these compounds (K(I)s of 86.4 nM-32.8 µM). The bacterial CAs were also effectively inhibited by these derivatives (K(I)s in the range of 0.77-234 nM against SazCA, and of 6.2-89.1 against SspCA, respectively), with several low nanomolar/subnanomolar inhibitors detected against both of them. As SspCA and SazCA are among the most thermostable and catalytically active CAs, it is of interest to find modulators of their activity for potential biotechnologic applications.

5-(Adamantan-1-yl)-N-methyl-1,3,4-thia-diazol-2-amine.

In the title compound, C13H19N3S, the methyl-amine substituent is coplanar with the thia-diazole ring to which it is attached [C-N-C-S torsion angle = 175.9 (2)°] and the amine H atom is syn to the thia-diazole S atom. Supra-molecular chains along [101], sustained by N-H⋯N hydrogen bonding, feature in the crystal packing.

3-(Adamantan-1-yl)-4-[(E)-(2,6-di-fluoro-benzyl-idene)amino]-1-[(4-ethyl-piperazin-1-yl)meth-yl]-4,5-di-hydro-1H-1,2,4-triazole-5-thione.

In the title compound, C26H34F2N6S, the triazole ring is linked to a benzene ring via an imine bond [N=C = 1.255 (2) Å; conformation: E], with a dihedral angle of 25.21 (11)° between the rings. The 4-ethyl-piperazinyl residue is folded away from the thione-S atom. In the crystal, helical supra-molecular chains propagating along [010] and sustained by weak C-S⋯π(triazole) inter-actions occur [S⋯centroid distance = 3.2872 (10) Å]. Links between these chains are of the type benzene-C-H⋯N(imine) and π-π [between centrosymmetrically related benzene rings with an inter-centroid distance of 3.9241 (15) Å] and result in a three-dimensional architecture.

3-(Adamantan-1-yl)-4-methyl-1-({4-[3-(tri-fluoro-meth-yl)phen-yl]piperazin-1-yl}meth-yl)-4,5-di-hydro-1H-1,2,4-triazole-5-thione.

In the title compound, C25H32F3N5S, two independent mol-ecules comprise the asymmetric unit and are related across a pseudo-centre of inversion. The piperazine rings have chair conformations with each N-bound substituent occupying an equatorial position so that the dihedral angles between the planes of the triazole and benzene ring are 78.20 (19) and 79.10 (19)° for the two independent mol-ecules, indicating that the mol-ecules have an L-shape. In the crystal, a three-dimensional architecture is stabilized by C-H⋯π inter-actions. The crystal studied was an inversion twin with the fractional contribution of the minor component being 0.27 (9).

5-(Adamantan-1-yl)-3-anilinomethyl-2,3-di-hydro-1,3,4-oxa-diazole-2-thione.

In the title compound, C19H23N3OS, the oxa-diazole and benzene rings are inclined at a dihedral angle of 50.30 (11)°, with the major twist between them occurring at the ring-methyl-ene N-C bond [N-N-C-N torsion angle = -101.2 (2)°]. In the crystal, helical supra-molecular chains along [010] are sustained by N-H⋯S hydrogen bonds. These are linked into layers lying parallel to (-101) by methyl-ene-phenyl C-H⋯π inter-actions.

5-(Adamantan-1-yl)-3-[(4-fluoro-anilino)meth-yl]-2,3-di-hydro-1,3,4-oxa-diazole-2-thione.

In the title compound, C19H22FN3OS, the dihedral angle between the inclined oxa-diazole and benzene rings is 52.7 (3)°. In the crystal, helical supra-molecular chains along [100] are sustained by N-H⋯S hydrogen bonds supported by methyl-ene-benzene C-H⋯π inter-actions. The crystal studied was an inversion twin with the fractional contribution of the minor component being 0.33 (14).

Synthesis and antimicrobial activity of N'-heteroarylidene-1-adamantylcarbohydrazides and (±)-2-(1-adamantyl)-4-acetyl-5-[5-(4-substituted phenyl-3-isoxazolyl)]-1,3,4-oxadiazolines.

The reaction of adamantane-1-carbohydrazide (1) with heterocyclic aldehydes, namely 5-(4-chlorophenyl)isoxazole-3-carboxaldehyde (2a), 5-(4-methylphenyl)isoxazole-3-carboxaldehyde (2b), 5-(4-methoxyphenyl)isoxazole-3-carboxaldehyde (2c), 1H-imidazole-2-carboxaldehyde and 2-butyl-4-chloro-1H-imidazole-5-carboxaldehyde, in ethanol, yielded the corresponding N'-heteroarylidene-1-adamantylcarbohydrazides 3a, 3b, 3c, 4 and 5, respectively, in good yields. The 4-acetyl-1,3,4-oxadiazoline analogues 6a­c were prepared in 48-55% yields by heating their corresponding N'-heteroarylidene-1-adamantylcarbohydrazides 3a-c with acetic anhydride for two hours. Compounds 3a-c, 4, 5 and 6a-c were tested for in vitro activities against a panel of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Compounds 4 and 5 displayed potent broad-spectrum antimicrobial activity, while compounds 3a-c showed good activity against the Gram-positive bacteria.

[Indications and outcome of ventilated patients treated in a neurological intensive care unit]. / Indikationen und Outcome beatmeter Patienten einer neurologischen Intensivstation.

OBJECTIVE: This study characterized artificially ventilated patients in a neurological intensive care unit (NICU) between 2006-2008 in a purely neurological clinic and a so-called stand-alone situation. In addition the long-term prognoses as well as the quality of life of surviving patients were investigated. METHODS: All ventilated patients from October 2006 to December 2008 were enrolled in this descriptive, retrospective study. The duration of stay in intensive care was analyzed and the current quality of life was prospectively assessed based on the patient records. Final diagnoses, duration of intensive care unit and ventilation as well as the highest score in SAPS II (simplified acute physiology score) and complications during hospitalization were determined. The patients were divided into groups based on the diagnoses as vascular, inflammatory, neurodegenerative, hereditary, epileptogenic and others. Additionally patients were contacted and asked to respond by completing questionnaires on the Barthel index (BI) and the modified Rankin scale (mRS). RESULTS: During the study period a total of 512 patients were treated in the NICU of whom 201 required artificial respiration. Cerebrovascular diseases were the main reason for therapy in the NICU in 96 out of 201 cases (47.8%), followed by inflammatory diseases in 46 (22.8%) and epileptogenic diseases in 26 patients (13%). The median duration of artificial respiration was 9 days with a mean treatment duration of 16 days (range 1-57 days). Of the patients 31 (15.4%) died in the NICU and an additional 32 patients (18.8%) died within a median of 2 months after discharge. Outcome data were available from 67 out of 170 sent questionnaires and rehabilitation reports of 86 patients, which enabled the outcome of 121 surviving patients to be analyzed (71.2%). Of these 42.2% showed no or mild impairment in everyday life. However, the remaining 38% had severe impairments according to the BI. The evaluation of the mRS showed that 49.6% of the patients still had severe symptoms. CONCLUSIONS: More than one third of the patients treated in the NICU required artificial ventilation with an emphasis on cerebrovascular diseases, which illustrates the overlap between stroke unit and NICU care. Despite a lengthy duration of ventilation and a long stay in the intensive care unit more than one third of surviving patients showed no or only mild impairment. However, an additional third suffered from severe disability up to nursing care dependency. The study data differ little from the few publications in this field despite the stand alone situation of the NICU. The case mix index per day averaged around 0.3 and underlines the economic importance with respect to other forms of neurological treatment.