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Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia-derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Peptidergic intercellular communication controls a wide range of physiological and behavioral responses and occurs throughout eukaryotes. TheChlamydomonas reinhardtiigenome encodes what appear to be numerous prepropeptides and enzymes homologous to those used to convert metazoan prepropeptides into bioactive peptide products. SinceC. reinhardtii, a green alga, lack the dense core vesicles in which metazoan peptides are processed and stored, we explored the hypothesis that propeptide processing and secretion occur through the regulated release of ciliary ectosomes. A synthetic peptide (GATI-amide) that could be generated from a 91-kDa peptide precursor (proGATI) serves as a chemotactic modulator, attractingminusgametes while repellingplusgametes. Here we dissect the processing pathway that leads to formation of an amidated peptidergic sexual signal specifically on the ciliary ectosomes ofplusgametes. Unlike metazoan propeptides, modeling studies identified stable domains in proGATI. Mass spectrometric analysis of a potential prohormone convertase and the amidated proGATI-derived products found in cilia and mating ectosomes link endoproteolytic cleavage to ectosome entry. Extensive posttranslational modification of proGATI confers stability to its amidated product. Analysis of this pathway affords insight into the evolution of peptidergic signaling; this will facilitate studies of the secretory functions of metazoan cilia.

The present paper elicits a marvelous synthesis of a series of sulfonamide containing 1,4-disubstituted 1,2,3-triazoles through click reaction of terminal alkynes with aromatic azides. The synthesi...

Extracellular vesicles (EVs) provide a mechanism for intercellular communication that transports complex signals in membrane delimited structures between cells, tissues and organisms. Cells secrete EVs of various subtypes defined by the pathway leading to release and by the pathological condition of the cell. Cilia are evolutionarily conserved organelles that can act as sensory structures surveilling the extracellular environment. Here we discuss the secretory functions of cilia and their biological implications. Studies in multiple species - from the nematode Caenorhabditis elegans and the chlorophyte alga Chlamydomonas reinhardtii to mammals - have revealed that cilia shed bioactive EVs (ciliary EVs or ectosomes) by outward budding of the ciliary membrane. The content of ciliary EVs is distinct from that of other vesicles released by cells. Peptides regulate numerous aspects of metazoan physiology and development through evolutionarily conserved mechanisms. Intriguingly, cilia-derived vesicles have recently been found to mediate peptidergic signaling. C. reinhardtii releases the peptide α-amidating enzyme (PAM), bioactive amidated products and components of the peptidergic signaling machinery in ciliary EVs in a developmentally regulated manner. Considering the origin of cilia in early eukaryotes, it is likely that release of peptidergic signals in ciliary EVs represents an alternative and ancient mode of regulated secretion that cells can utilize in the absence of dedicated secretory granules.

Peptidergic intercellular communication occurs throughout the eukaryotes, and regulates a wide range of physiological and behavioral responses. Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Our previous study revealed the presence of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), in cilia and its key role in ciliogenesis. Furthermore, PAM and its amidated products are released in ciliary ectosomes from the green alga Chlamydomonas reinhardtii. One amidated product (GATI-amide) serves as a chemotactic modulator for C. reinhardtii gametes, attracting minus gametes while repelling plus gametes. Here we dissect the complex processing pathway that leads to formation of this amidated peptidergic sexual signal specifically on the ectosomes of plus gametes. We also identify a potential prohormone convertase that undergoes domain rearrangement during ectosomal secretion as a substrate for PAM. Analysis of this pathway affords insight into how single-celled organisms lacking dense core vesicles engage in regulated secretion, and provides a paradigm for understanding how amidated peptides that transmit sexual and other signals through cilia are generated.

N-Heterocyclic compounds like 1,2,3-triazoles serve as a key scaffolds among organic compounds having diverse applications in the field of drug discovery, bioconjugation, material science, liquid crystals, pharmaceutical chemistry and solid phase organic synthesis. Various drugs containing 1,2,3-triazole ring which are commonly available in market includes Rufinamide, Cefatrizine, Tazobactam etc., Stability to acidic/basic hydrolysis along with significant dipole moment support triazole moiety for appreciable participation in hydrogen bonding and dipole-dipole interactions with biological targets. Huisgen 1,3-dipolar azide-alkyne cycloaddition culminate into a mixture of 1,4 and 1,5- disubstituted 1,2,3-triazoles. In 2001, Sharpless and Meldal came across with a copper(I) catalyzed regioselective synthesis of 1,4-disubstituted 1,2,3-triazoles by cycloaddition between azides and terminal alkynes. This azide-alkyne cycloaddition has been labelled as a one of the important key click reaction. Click synthesis describes chemical reactions that are simple to perform, gives high selectivity, wide in scope, fast reaction rate and high yields. Click reactions are not single specific reaction, but serve as a pathway for construction of simple to complex molecules from a variety of starting materials. In the last few decades, 1,2,3-triazoles attracted attention of researchers all over the world because of their broad spectrum of biological activities. Keeping in view the biological importance of 1,2,3-triazole, in this review we focus on the various synthetic routes for the syntheisis of 1,4-disubstituted 1,2,3-triazoles. This review involves various synthetic protocols which involves copper and non-copper catalysts, different solvents as well as substrates. It will boost synthetic chemists to explore new pathway for the development of newer biologically active 1,2,3-triazoles.

A series of 21 amide linked 1,4-disubstituted-1,2,3-triazoles were achieved via one-pot synthesis through Cu(I) catalyzed click reaction between terminal alkynes and 2-azido-N-substituted acetamides. Newly formed triazoles were characterized by various spectroscopic techniques (FT-IR, 1H NMR, 13C NMR spectroscopy, and HRMS) and investigated for in vitro antitubercular evaluation against bacteria, i.e., Mycobacterium tuberculosis and antimicrobial evaluation against Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. Some of the synthesized triazole derivatives were found to exhibit moderate inhibitory activity against the tested antitubercular strain, whereas one compound displayed a significant inhibitory activity against most of the tested microbial strains.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adultPamMyh6-cKO/cKOatria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures ofPam0-Cre-cKO/cKOatrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM inPamMyh6-cKO/cKOatrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels inPamMyh6-cKO/cKOmyocytes; the inability of these drugs to inhibit basal proANP secretion byPamMyh6-cKO/cKOmyocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in thecis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway.SignificanceTransmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure.

A series of 21 2-(4-(hydroxyalkyl)-1H-1,2,3-triazol-1-yl)-N-substituted propanamides (1,4-disubstituted 1,2,3-triazoles having amide linkage and hydroxyl group) have been synthesized from click reaction between terminal alkyne and 2-azido-N-substituted propanamide (generated in situ from reaction of 2-bromo-N-substituted propanamide and sodium azide) and characterized by FTIR, 1H NMR, 13C NMR spectroscopy, and HRMS. All the newly synthesized triazoles were tested in vitro for antimicrobial activity against four bacterial cultures – Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Staphylococcus aureus – and two fungal cultures – Candida albicans and Aspergillus niger. The synthesized 1,4-disubstituted 1,2,3-triazoles displayed moderate to good antimicrobial potential against the tested strains.

Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that both receive information from the environment and secrete bioactive extracellular vesicles (ectosomes). However, the nature of these secreted signals and their biological functions remain poorly understood. Here, we report the developmentally regulated release of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), and the presence of peptidergic signaling machinery (including propeptide precursors, subtilisin-like prohormone convertases, amidated products, and receptors) in ciliary ectosomes from the green alga Chlamydomonas. One identified amidated PAM product serves as a chemoattractant for mating-type minus gametes but repels plus gametes. Thus, cilia provide a previously unappreciated route for the secretion of amidated signaling peptides. Our study in Chlamydomonas and the presence of PAM in mammalian cilia suggest that ciliary ectosome-mediated peptidergic signaling dates to the early eukaryotes and plays key roles in metazoan physiology., Peptide-mediated intercellular communication is an essential process, and those peptides are usually released from secretory granules. This study demonstrates that peptidergic signals are also secreted through vesicular ectosomes that bud from the ciliary membrane.

To explore anticancer and antioxidant agents with improved potency, we synthesized a series of amide linked 1,4-disubstituted 1,2,3-triazoles through click chemistry approach. The structure of synthesized triazoles were characterized by- FTIR, 1H NMR, 13C NMR spectroscopy and HRMS. All the synthesized compounds were screened for their anticancer activity against four different cell lines- PC3 (prostate cancer), A549 (lung cancer), MIAPACA (liver cancer), Fr2 (Breast epithelial), reflecting compounds 7e and 7f to possess good activity. The antioxidant activity was evaluated by using stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and compound 7d showed promising activity having IC50 value 1.61 µg/ml. Molecular docking studies of compounds 7e and 7f was carried out in active site of human epidermal growth factor receptor 2 revealed high binding affinities and within toxicity limits. The experimental results were in good agreement with docking studies. In-silico ADME studies of synthesized compounds also have good dispositional profile and are patient compliant, may be potential future candidates for anticancer treatment.

A library of twenty five 1,2,3-triazoles bridged with amine-amide functionalities have been synthesized from reaction of N-substituted(prop-2-yn-1-yl)amines (2a-2e), 2-bromo-N-arylacetamides (4a-4e) and sodium azide through copper(I)-catalyzed alkyne-azide cycloaddition. The synthesized compounds were characterized by using FTIR, 1H NMR, 13C NMR, and HRMS techniques. The structures of synthesized 5a (CCDC 1569245) and 5h (CCDC 1569249) were also confirmed by X-ray crystallography. Antifungal evaluation of newly synthesized triazoles was carried out against - Candida albicans and Aspergillus niger. Biological screening of synthesized 1,2,3-triazoles revealed moderate to good antifungal activity against tested strains.

Mutations in the progressive ankylosis protein (NP_473368, human ANKH) cause craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and widened metaphyses in long bones. The pathogenesis of CMD remains largely unknown, and treatment for CMD is limited to surgical intervention. We have reported that knock-in mice (AnkKI/KI) carrying a F377del mutation in ANK (NM_020332, mouse ANK) replicate many features of CMD. Interestingly, ablation of the Ank gene in AnkKO/KO mice also leads to several CMD-like phenotypes. Mutations causing CMD led to decreased steady-state levels of ANK/ANKH protein due to rapid degradation. While wild type (wt) ANK was mostly associated with plasma membranes, endoplasmic reticulum (ER), Golgi apparatus and lysosomes, CMD-linked mutant ANK was aberrantly localized in cytoplasm. Inhibitors of proteasomal degradation significantly restored levels of overexpressed mutant ANK, whereas endogenous CMD-mutant ANK/ANKH levels were more strongly increased by inhibitors of lysosomal degradation. However, these inhibitors do not correct the mislocalization of mutant ANK. Co-expressing wt and CMD-mutant ANK in cells showed that CMD-mutant ANK does not negatively affect wt ANK expression and localization, and vice versa. In conclusion, our finding that CMD mutant ANK/ANKH protein is short-lived and mislocalized in cells may be part of the CMD pathogenesis.

Summary We identified osteoclast defects in craniometaphyseal dysplasia (CMD) using an easy-to-use protocol for differentiating osteoclasts from human induced pluripotent stem cells (hiPSCs). CMD is a rare genetic bone disorder, characterized by life-long progressive thickening of craniofacial bones and abnormal shape of long bones. hiPSCs from CMD patients with an in-frame deletion of Phe377 or Ser375 in ANKH are more refractory to in vitro osteoclast differentiation than control hiPSCs. To exclude differentiation effects due to genetic variability, we generated isogenic hiPSCs, which have identical genetic background except for the ANKH mutation. Isogenic hiPSCs with ANKH mutations formed fewer osteoclasts, resorbed less bone, expressed lower levels of osteoclast marker genes, and showed decreased protein levels of ANKH and vacuolar proton pump v-ATP6v0d2. This proof-of-concept study demonstrates that efficient and reproducible differentiation of isogenic hiPSCs into osteoclasts is possible and a promising tool for investigating mechanisms of CMD or other osteoclast-related disorders., Highlights • An easy-to-use protocol for differentiating osteoclasts (OCs) from hiPSCs • The use of isogenic hiPSCs to study OC defects in craniometaphyseal dysplasia (CMD) • Isogenic hiPSCs with CMD mutations in ANKH have defective osteoclastogenesis • hiPSC-OCs from CMD patients show decreased levels of ANKH and vacuolar proton pump, In this article, Chen and colleagues generated isogenic hiPSCs with or without ANKH mutations identified in patients with craniometaphyseal dysplasia (CMD). By differentiating hiPSC into osteoclasts, they demonstrate that mutations in ANKH lead to decreased osteoclast formation, marker gene expression, and bone resorption. Furthermore, hiPSC osteoclasts from CMD patients have decreased protein levels of ANKH and the vacuolar proton pump ATP6v0d2.

The SIN3 family of co-repressors is a family of highly conserved eukaryotic repressor proteins that regulates diverse functions in yeasts and animals but remains largely uncharacterized functionally even in plants like Arabidopsis. The sole SIN3 homologue in banana, MaSIN3, was identified as a 1408 amino acids, nuclear localized protein conserved to other SIN3s in the PAH, HID and HCR domains. Interestingly, MaSIN3 over-expression in Arabidopsis mimics a state of reduced ABA responses throughout plant development affecting growth processes such as germination, root growth, stomatal closure and water loss, flowering and senescence. The reduction in ABA responses is not due to reduced ABA levels but due to suppression of expression of several transcription factors mediating ABA responses. Transcript levels of negative regulators of germination (ABI3, ABI5, PIL5, RGL2 and RGL3) are reduced post-imbibition while those responsible for GA biosynthesis are up-regulated in transgenic MaSIN3 over-expressers. ABA-associated transcription factors are also down-regulated in response to ABA treatment. The HDAC inhibitors, SAHA and sodium butyrate, in combination with ABA differentially suppress germination in control and transgenic lines suggesting the recruitment by MaSIN3 of HDACs involved in suppression of ABA responses in different processes. The studies provide an insight into the ability of MaSIN3 to specifically affect a subset of developmental processes governed largely by ABA.

Twenty ester-linked 1,4-disubstituted 1,2,3-triazoles having a furyl/thienyl moiety have been synthesized from heteroaryl prop-2-yn-1-yl carboxylate and aromatic azides via a Cu(I) catalyzed 1,3-dipolar cycloaddition. All the synthesized compounds were characterized by FTIR, [Formula: see text]H NMR, [Formula: see text]C NMR spectroscopy and HRMS. Synthesized triazoles were tested in vitro for antimicrobial evaluation against Gram-negative bacteria-Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae; Gram-positive bacteria-Staphylococcus aureus and two fungal strains-Candida albicans and Aspergillus niger, reflecting moderate to good activity. The structure of compound 6f was also confirmed by X-ray crystallography (CCDC 1469326).

A convenient synthesis of some new 1,4-disubstituted 1,2,3-bistriazoles (3a–3f, 4a–4f, 6a–6b, 7a–7b) is reported via copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of various terminal alkynes with 1,4-bis(azidomethyl)benzene and 1,6-diazidohexane. The synthesized compounds were characterized by spectral techniques including infrared, 1H NMR, 13C NMR, and high-resolution mass spectrometry and tested in vitro for antimicrobial potential against Bacillus subtilis and Staphylococcus aureus (Gram-positive bacteria), Pseudomonas aeruginosa and Escherichia coli (Gram-negative bacteria), and Candida albicans and Aspergillus niger (fungi). Among the synthesized 1,4-disubstituted 1,2,3-bistriazoles, compounds 6a, 6b, and 7b displayed excellent antimicrobial potential against most of the tested strains.