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Most Escherichia coli strains associated with neonatal meningitis express the K1 capsule, a sialic acid polysaccharide that is directly related to their pathogenicity. Metabolic oligosaccharide engineering (MOE) has mostly been developed in eukaryotes, but has also been successfully applied to the study of several oligosaccharides or polysaccharides constitutive of the bacterial cell wall. However, bacterial capsules are seldom targeted despite their important role as virulence factors, and the K1 polysialic acid (PSA) antigen that shields bacteria from the immune system still remains untackled. Herein, we report a fluorescence microplate assay that allows the fast and facile detection of K1 capsules with an approach that combines MOE and bioorthogonal chemistry. We exploit the incorporation of synthetic analogues of N-acetylmannosamine or N-acetylneuraminic acid, metabolic precursors of PSA, and copper-catalysed azide-alkyne cycloaddition (CuAAC) as the click chemistry reaction to specifically label the modified K1 antigen with a fluorophore. The method was optimized, validated by capsule purification and fluorescence microscopy, and applied to the detection of whole encapsulated bacteria in a miniaturized assay. We observe that analogues of ManNAc are readily incorporated into the capsule while those of Neu5Ac are less efficiently metabolized, which provides useful information regarding the capsule biosynthetic pathways and the promiscuity of the enzymes involved. Moreover, this microplate assay is transferable to screening approaches and may provide a platform to identify novel capsule-targeted antibiotics that would circumvent resistance issues.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.

International audience; The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.

International audience; A series of ferrocenyl-containing γ-hydroxy-γ-lactam tetramates were prepared in 2-3 steps through ring opening-ring closure (RORC) process of γ-ylidene-tetronate derivatives in the presence of ferrocenyl alkylamines. The compounds were screened in vitro for their antiplasmodial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (W2) clones of P. falciparum, displaying activity in the range of 0.12-100 μM, with generally good resistance index. The most active ferrocene in these series exhibited IC50 equal to 0.09 μM (3D7) and 0.12 μM (W2). The low cytotoxicity of the ferrocenyl-containing γ-hydroxy-γ-lactam tetramates against Human Umbilical Vein Endothelial (HUVEC) cell line demonstrated selective antiparasitic activity. The redox properties of these ferrocene-derived tetramates were studied and physico-biochemical studies evidenced that these derivatives can exert potent antimalarial activities via a mechanism distinct from ferroquine.

This article describes a methodology for detailed mapping of the lignification capacity of plant cell walls that we have called “REPRISAL” for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G*, and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labeling strategy based on the sequential use of three main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence segmentation algorithm is developed that assigns fluorescent image pixels to three distinct cell wall zones corresponding to cell corners, compound middle lamella and secondary cell walls. The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method [RM] 1) and proportions (RM 2) within the different cell wall zones. We first describe the use of this methodology to map developmentally related changes in the lignification capacity of wild-type Arabidopsis (Arabidopsis thaliana) interfascicular fiber cells. We then apply REPRISAL to analyze the Arabidopsis peroxidase (PRX) mutant prx64 and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. In addition, we also demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar (Populus tremula × Populus alba), flax (Linum usitatissimum), and maize (Zea mays). Finally, we show that the methodology can be used to map the incorporation of a fucose reporter into noncellulosic cell wall polymers.

International audience; Benefiting from the recent advances of synchrotron X-ray nanoprobes, we demonstrate three-dimensional (3D) correlative nano-imaging on malaria-infected human red blood cells. By combining X-ray fluorescence to-mography and phase contrast nanotomography on the same cell with sub-100 nm pixel size, we establish a routine workflow from the data acquisition, data processing to tomographic reconstruction. We quantitatively compare the elemental volumes obtained with different reconstruction methods, the total variation minimization giving the most satisfactory results. We reveal elemental correlations in different cell compartments more reliably on reconstructions as opposed to 2D projections. Finally, we determine for the first time the 3D mass fraction maps of multiple elements at sub-cellular level. The estimated total number of Fe atoms and the total mass of the red blood cell show very good agreement with previously reported values.

This article describes a new methodology for detailed mapping of the lignification capacity of plant cell walls that we have called “REPRISAL” for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G* and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labelling strategy based on the sequential use of 3 main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence (AI) segmentation algorithm is developed that assigns fluorescent image pixels to 3 distinct cell wall zones corresponding to cell corners (CC), compound middle lamella (CML) and secondary cell walls (SCW). The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method 1) and proportions (ratiometric method 2) within the different cell wall zones. In order to demonstrate the potential of REPRISAL for investigating lignin formation we firstly describe its use to map developmentally-related changes in the lignification capacity of WT Arabidopsis interfascicular fiber cells. We then show how it can be used to reveal subtle phenotypical differences in lignification by analyzing the Arabidopsis prx64 peroxidase mutant and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. Finally, we demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar, flax and maize.

Correction for ‘EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification’ by Clémence Simon et al., Chem. Commun., 2021, DOI: 10.1039/D0CC05218C.

International audience; The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site-specific incorporation of unnatural amino acids in proteins, and post-synthetic labelling of nucleic acids. While a majority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

International audience; Although their impact in biology and cellular communication are now well established, in situ visualization of glycans has long remained elusive due to the lack of suitable and general imaging techniques. Twenty years ago, the bioorthogonal chemical reporter strategy based on chemoselective and biocompatible reactions has emerged to become the state-of-the-art labeling technology for studying glycans. In this chapter, after providing an update on recent developments in bioorthogonal chemistry, we review how this strategy can be implemented to the visualization and biochemical analysis of different glycans in the bacterial cell envelope.

International audience; In bioimaging, bioorthogonal chemistry is most often used to visualize chemical reporters by fluorescence in their native environment. Herein, we show that TEMPO-based probes can be ligated to monolignol reporters by Diels–Alder chemistry in plant cell walls, paving the way for the study of lignification by EPR spectroscopy and imaging.

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin contents in ugt72e1, ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71, which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer., SCOPUS: ar.j, info:eu-repo/semantics/published

Non‐tuberculous mycobacterium (NTM) infections, such as those caused by Mycobacterium abscessus, are increasing globally. Due to their intrinsic drug resistance, M. abscessus pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against M. abscessus and Mycobacterium tuberculosis, the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against M. abscessus. Structure‐activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD‐88 as a new promising active analogue against M. abscessus. Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half‐life of 3.2 hours., Molecular modelling: Molecular representation of the interaction of PIPD1 and M. abscessus MmpL3 predictive model. On one side, PIPD1 is hydrogen‐bound to the carboxylic acid group of D618 through its amino moiety. On the other side, its hydroxyl group is H‐bound to the hydroxyl of Y219 and a potent interaction with the carboxylic acid group of D258 can occur. A stabilization occurred through π‐π interactions between the toluene moiety of PIPD1 and phenyl rings of F262 and F622.

International audience; The conjugation of organometallic complexes to known bioactive organic frameworks is a proven strategy revered for devising new drug molecules with novel modes of action. This approach holds great promise for the generation of potent drug leads in the quest for therapeutic chemotypes with the potential to overcome the development of clinical resistance. Herein, we present the in vitro antiplasmodial and antiproliferative investigation of ferrocenyl α‐aminocresol conjugates assembled by amalgamation of the organometallic ferrocene unit and an α‐aminocresol scaffold possessing antimalarial activity. The compounds pursued in the study exhibited higher toxicity towards the chemosensitive (3D7) and ‐resistant (Dd2) strains of the Plasmodium falciparum parasite than to the human HCC70 triple‐negative breast cancer cell line. Indication of cross‐resistance was absent for the compounds evaluated against the multi‐resistant Dd2 strain. Structure‐activity analysis revealed that the phenolic hydroxy group and rotatable σ bond between the α‐carbon and NH group of the α‐amino‐o‐cresol skeleton are crucial for the biological activity of the compounds. Spectrophotometric techniques and in silico docking simulations performed on selected derivatives suggest that the compounds show a dual mode of action involving hemozoin inhibition and DNA interaction via minor‐groove binding. Lastly, compound 9 a, identified as a possible lead, exhibited preferential binding for the plasmodial DNA isolated from 3D7 P. falciparum trophozoites over the mammalian calf thymus DNA, thereby substantiating the enhanced antiplasmodial activity of the compounds. The presented research demonstrates the strategy of incorporating organometallic complexes into known biologically active organic scaffolds as a viable avenue to fashion novel multimodal compounds with potential to counter the development drug resistance.

Reported herein is an in vivo triple labelling strategy to monitor the formation of plant cell walls. Based on a combination of copper-catalysed alkyne–azide cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), and Diels–Alder reaction with inverse electronic demand (DARinv), this methodology can be applied to various plant species of interest in research. It allowed detection of the differential incorporation of alkynyl-, azido-, and methylcyclopropenyl-tagged reporters of the three main monolignols into de novo biosynthesized lignin in different tissues, cell types, or cell wall layers. In addition, this triple labelling was implemented with different classes of chemical reporters, using two monolignol reporters in conjunction with alkynylfucose to simultaneously monitor the biosynthesis of lignin and non-cellulosic polysaccharides. This allowed observation of their deposition occurring contemporaneously in the same cell wall.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix. ispartof: HUMAN MOLECULAR GENETICS vol:27 issue:17 pages:3029-3045 ispartof: location:England status: published

A new series of cyrhetrenyl and ferrocenyl sulfonamides were designed, synthesized and characterized. The reactions of organometallic amines (P2 and P3) with the corresponding arylsulfonyl chlorides produced cyrhetrenyl (2a-c) and ferrocenyl (3a-c) sulfonamide derivatives. All the compounds were characterized by conventional spectroscopic techniques (FT-IR, 1H and 13C NMR and mass spectrometry). The proposed molecular structures of 2a and 3a were confirmed by single-crystal X-ray crystallography. Theoretical calculations at the DFT level were also carried out as an approximation to rationalize the influences of the electronic natures of the organometallic fragments into the sulfonamide skeleton. All the complexes were screened in vitro against Mycobacterium tuberculosis and exhibited only modest activities in the micromolar range.

A series of new organometallic tosyl hydrazones of the general formula [{(η5-C5H4)-C(R) = NNHSO2C6H4CH3}]M(CO)3 (M = Re, Mn; R = H, CH3) were prepared by the reaction between formyl or acetyl organometallic precursors with p-toluenesulfonyl hydrazide. All compounds were characterized by conventional spectroscopic techniques (infrared, 1H and 13C NMR, mass spectrometry and elemental analysis). In addition, the molecular structures of the cymantrenyl hydrazones 2a and 2b have been determined by single-crystal X-ray diffraction. In the solid state, both compounds showed an E-configuration around the C N moiety. Evaluation of antitubercular activity, measured in vitro against Mycobacterium tuberculosis mc26230 strain, indicate that all organometallic tosyl hydrazones are considerably less active than the reference drug isoniazid. The antiproliferative activity against non-small cell lung cancer cells (H1299 cells) was also evaluated. Even though the IC50 measured for hydrazones 1–3 resulted 2–3-fold higher than cisplatin, biological results demonstrate that the electronic effects of the substituents on the hydrazone carbon are influencing factor in the anticancer activity.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis. Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane–associated mycolic acid flippase in M. tuberculosis. Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria. 294;46

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis. Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc(2)6230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane–associated mycolic acid flippase in M. tuberculosis. Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.

Copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) has been utilized for preparing a series of 1H-1,2,3-triazoles with the purpose of probing structure-activity relationships among a uracil-ferrocene-triazole conjugate family. The anti-tubercular evaluation studies revealed an improvement in activity with the introduction of a ferrocene nucleus among N-alkylazido uracil precursors, with a preference for a bromo-substituent along with moderate chain lengths of n=2-6. The reported protocol is a successful approach for integrating uracil-ferrocene-chalcone functionalities tethered via 1H-1,2,3-triazole rings with apparent physicochemical stability. This article is protected by copyright. All rights reserved.

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.

The mammalian mono-&alpha, 2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish &alpha, 2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of &alpha, 2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

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Three new series of quinoline, quinolone, and benzimidazole derivatives were synthesized and evaluated in vitro against Trypanosoma brucei gambiense . In the quinoline series, the metallo antimalarial drug candidate (ferroquine, FQ) and its ruthenium analogue (ruthenoquine, RQ, compound 13 ) showed the highest in vitro activities with IC 50 values around 0.1 μM. Unfortunately, both compounds failed to cure Trypanosoma brucei brucei infected mice in vivo. The other heterocyclic compounds were active in vitro with IC 50 values varying from 0.8 to 34 μM. One of the most interesting results was a fluoroquinolone derivative (compound 2 ) that was able to offer a survival time of 8 days after a treatment at the single dose of 100 μmol/kg by intraperitoneal route. Although no clear-cut structure–activity relationships emerged, further pharmacomodulations are worth to be developed in this series.

A new series of palladium (II) and platinum (II) complexes containing ferrocenyl and cyrhetrenyl thiosemicarbazone ligands were synthesized and characterized. The two-step reaction of the organometallic thiosemicarbazones with i) K2MCl4 and ii) PPh3 and their subsequent recrystallization from CH2Cl2/hexane yielded the binuclear complexes [M {MLn(η5-C5H4)C(H)=NN=C(S)-NHR}-(Cl)(PPh3)] (M′=Pd, Pt; MLn=Re(CO)3, FeCp; R=H, CH3). The structures of the products were inferred from elemental analyses and IR, 1H and 31P NMR spectroscopies. The molecular structures of 2b and 3d were confirmed by single crystal X-ray analysis. All complexes were screened in vitro against Mycobacterium tuberculosis and exhibited only moderate activity in the low micromolar range., R.A. and A.H.K. acknowledge FONDECYT-Chile (Project 11130443 and 1110669) and D.I. Pontificia Universidad Católica de Valparaíso.

Despite the tremendous progress in medicine, cancer remains one of the most serious global health problems awaiting new effective therapies. Here we present ferroquine (FQ), the next generation antimalarial drug, as a promising candidate for repositioning as cancer therapeutics. We report that FQ potently inhibits autophagy, perturbs lysosomal function and impairs prostate tumor growth in vivo. We demonstrate that FQ negatively regulates Akt kinase and hypoxia-inducible factor-1α (HIF-1α) and is particularly effective in starved and hypoxic conditions frequently observed in advanced solid cancers. FQ enhances the anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant to existing anticancer therapy. Alike its parent compound chloroquine (CQ), FQ accumulates within and deacidifies lysosomes. Further, FQ induces lysosomal membrane permeabilization, mitochondrial depolarization and caspase-independent cancer cell death. Overall, our work identifies ferroquine as a promising new drug with a potent anticancer activity.

The development of metabolic oligosaccharide engineering (MOE) over the past two decades enabled the bioimaging studies of glycosylation processes in physio-pathological contexts. Herein, we successfully applied the chemical reporter strategy to image the fate of sialylated glycoconjugates in healthy and sialin-deficient patient fibroblasts. This chemical glycomics enrichment is a powerful tool for tracking sialylated glycoconjugates and probing lysosomal recycling capacities. Thus, such strategies appear fundamental for the characterization of lysosomal storage diseases.

A better in vivo understanding of lignin formation within plant cell walls will contribute to improving the valorization of plant-derived biomass. Although bioorthogonal chemistry provides a promising platform to study the lignification process, methodologies that simultaneously detect multiple chemical reporters in living organisms are still scarce. Here, we have developed an original bioorthogonal labeling imaging sequential strategy (BLISS) to visualize and analyze the incorporation of both p-hydroxyphenyl (H) and guaiacyl (G) units into lignin in vivo with a combination of strain-promoted and copper-catalyzed azide-alkyne cycloadditions. On our path to BLISS, we designed a new azide-tagged monolignol reporter for H units in metabolic lignin engineering and used it in conjunction with an alkyne-tagged G unit surrogate to study lignification dynamics in flax. Here, we show that BLISS provides precise spatial information on the zones of active lignification and reveals polarization in single-cell lignification dynamics.

To study the electronic influence of the organometallic moieties in thiosemicarbazones, two short libraries of cyrhetrenyl thiosemicarbazone and ferrocenyl thiosemicarbazone hybrids were synthesized and tested for their antichagasic and antitubercular activities. The unreported cyrhetrenyl thiosemicarbazone derivatives of the form [(η5-C5H4)–C(R1) = NNHC(S)NHR2]Re(CO)3 (R1 = H, CH3; R2 = H, CH3, CH2CH3, C6H5) were prepared from cyrhetrenylcarbaldehyde (1a) or acetylcyrhetrene (1b) and the corresponding thiosemicarbazide. The 1H and 13C NMR spectra indicate that these compounds have the anti-(E) conformation in solution, and the X-ray crystal structure of formylcyrhetrene 4-methyl-thiosemicarbazone (2b) confirms that this complex also adopts the anti-(E) form in the solid state. The new cyrhetrenyl thiosemicarbazones and their ferrocene analogues were screened in vitro against Trypanosoma cruzi and Mycobacterium tuberculosis. The anti-T. cruzi evaluation showed that the ferrocenyl derivatives were more efficient trypanocidal agents compared to their cyrhetrenyl counterparts. The incorporation of any organometallic fragment into thiosemicarbazone scaffold showed moderate antituberculosis activity against mc27000 strain.

A series of twenty piperazine tethered 7-chloroquinoline-isatin hybrids have been synthesized via either direct nucleophilic substitution or Cu(Ι)Cl mediated Mannich reaction. These new conjugates were evaluated for their antimalarial and anti-tubercular efficacy against a chloroquine resistant strain of Plasmodium falciparum and Mycobacterium tuberculosis, respectively, while the cytotoxic profiles were evaluated against 3T6 cell line, a permanent mouse embryonic fibroblast cell line. The most potent of the test compound with IC50 of 0.22 μM against W2 strain of P. falciparum and 31.62 μM against the embryonic fibroblast cell line (cytotoxicity) displayed a high selective index of 143.73. This article is protected by copyright. All rights reserved.

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongstthe phenotypic changes exhibited by tumor cells, a widerange of glycosylationshas been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspectsof glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches onthree human colon cell lines (HT29, HCT116 and CCD841CoN), we herein report that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affectsoverall N-and O-glycosylation patterns. Interestingly, silencingof OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation.On the other hand, OGT silencing perturbs biosynthesis ofglycosphingolipids resulting ina decreaseingangliosidesandan increase in globosides.Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells. 19;21-22

A new series of cyrhetrenyl and ferrocenyl 1,3,4-thiadiazoles (TDZs) were designed, synthesized and characterized. The condensation–cyclization reaction of organometallic-cyanide with the corresponding thiosemicarbazide produces the ferrocenyl-TDZs (3a–c) and cyrhetrenyl analogues (4ac). The structures of all products were inferred from their elemental analysis, 1H and 13C NMR spectra and MS. The molecular structures of 3b and 4b were confirmed using X-ray crystallography. The electronic effects of cyrhetrenyl (4a–c) and ferrocenyl (3a–c) bound directly to the thiadiazole ring have been correlated with the chemical shift of the exocyclic amine proton and 13C shift of the carbon at the 5-position. The X-ray crystal structure of 3b and 4b indicates that the rhenium compound 4b possesses a more extensive conjugated π-system. The activity of the new organometallic-TDZs was evaluated in vitro against Mycobacterium tuberculosis. The incorporation of any organometallic fragment into the thiadiazole skeleton showed a comparable but lower antitubercular activity than isoniazid.

A Cu-mediated azide–alkyne cycloaddition protocol has been employed for the synthesis of 16 different triazoles to probe the antitubercular structure–activity relationships within the isatin–ferrocene–triazole conjugate family. The antitubercular evaluation studies revealed a marked improvement in activity with the introduction of ferrocene nucleus among precursors N-alkylazido isatins with a prefernce for halogen (F, Cl) substituent at C-5 position of isatin as well as propyl chain length as a spacer. The induction of a chalcone nucleus resulted in the enhanced antimycobacterial efficacy irrespective of the subtituent and alkyl chain length as evidenced by the isatin–ferrocenylchalcone hybrids. The described protocol is the first successful attempt of the amalgamation of ferrocene–isatin nuclei tethered via a triazole linker.