Ru(II)-Catalyzed C-H Activation/[4+2] Annulation of Sulfoxonium Ylide with Maleimide: Access to Fused Benzo[e]isoindole-1,3,5-trione.

A ruthenium-catalyzed C-H activation and a concomitant [4+2] annulation of sulfoxonium ylide with maleimide have been demonstrated. This tandem reaction results in the formation of fused benzo[e]isoindole-1,3,5-trione. The method employs mild conditions and is free of metal oxidants. The reaction pathway predominantly involves protodemetalation over ß-hydride elimination due to the lack of syn ß-hydrogens.

Distinct Bin/Amphiphysin/Rvs (BAR) family proteins may assemble on the same tubule to regulate membrane organization in vivo.

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of Drosophila ICA69 (dICA69) (an N-BAR protein) and Drosophila CIP4 (dCIP4) (an F-BAR protein), focusing on their impact on in vivo membrane tubule organization. In contrast to the prevailing models of BAR-domain protein function, we observed colocalization of endogenous dICA69 with dCIP4-induced tubules, indicating their potential recruitment for tubule formation and maintenance. Moreover, actin-regulatory proteins such as Wasp, SCAR, and Arp2/3 were recruited at the site of CIP4-induced tubule formation. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width, possibly by recruiting actin modulators during the generation of tubules. Our data suggests that cooperative activities of distinct BAR-domain family proteins may determine the length and width of the membrane tubule in vivo.

Ru(II)-Catalyzed C-H Alkylation of N-Benzyltriflamide with Maleimides: Synthesis of o-Succinimide Substituted Benzaldehydes.

A ruthenium-catalyzed N-benzyltriflamide assisted C-H alkylation with maleimide followed by hydrolysis of in situ generated imine has been developed for the first time. This synthetic method results in the efficient synthesis of o-succinimide derivatives of benzaldehydes. This reaction involves less expensive and mild reaction conditions and shows excellent site selectivity and good functional group compatibility.

dAsap regulates cellular protrusions via an Arf6-dependent actin regulatory pathway in S2R+ cells.

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape.

Beadex, the Drosophila LIM only protein, is required for the growth of the larval neuromuscular junction.

The appropriate growth of the neurons, accurate organization of their synapses, and successful neurotransmission are indispensable for sensorimotor activities. These processes are highly dynamic and tightly regulated. Extensive genetic, molecular, physiological, and behavioral studies have identified many molecular candidates and investigated their roles in various neuromuscular processes. In this article, we show that Beadex (Bx), the Drosophila LIM only (LMO) protein, is required for motor activities and neuromuscular growth of Drosophila. The larvae bearing Bx7, a null allele of Bx, and the RNAi-mediated neuronal-specific knockdown of Bx show drastically reduced crawling behavior, a diminished synaptic span of the neuromuscular junctions (NMJs) and an increased spontaneous neuronal firing with altered motor patterns in the central pattern generators (CPGs). Microarray studies identified multiple targets of Beadex that are involved in different cellular and molecular pathways, including those associated with the cytoskeleton and mitochondria that could be responsible for the observed neuromuscular defects. With genetic interaction studies, we further show that Highwire (Hiw), a negative regulator of synaptic growth at the NMJs, negatively regulates Bx, as the latter's deficiency was able to rescue the phenotype of the Hiw null mutant, HiwDN. Thus, our data indicate that Beadex functions downstream of Hiw to regulate the larval synaptic growth and physiology.NEW & NOTEWORTHY A novel role for Beadex (Bx) regulates the larval neuromuscular junction (NMJ) structure and function in a tissue-specific manner. Bx is expressed in a subset of Toll-6-expressing neurons and is involved in regulating synaptic span and physiology, possibly through its negative interaction with Highwire (Hiw). The findings of this study provide insights into the molecular mechanisms underlying NMJ development and function and warrant further investigation to understand the role of Bx in these processes fully.

Total synthesis of diplofuranone A and diapolic acid A.

The first and concise syntheses of the anticancer agent diplofuranone A and the fatty acid-derived metabolite diapolic acid A have been demonstrated using easily accessible and commercially available starting materials. The key feature of these syntheses is the efficient diversification of highly stereo- and chemoselectively constructed (E,E)-1,6-dioxo-2,4-dienes using ruthenium catalytic conditions, which enabled straightforward access to diversely substituted bioactive molecules.

A palladium catalyzed asymmetric desymmetrization approach to enantioenriched 1,3-disubstituted isoindolines.

Herein, we report the first palladium/MPAA catalyzed enantioselective C-H activation/[4 + 1] annulation of diarylmethyltriflamide and olefins to construct chiral cis-1,3-disubstituted isoindoline derivatives. The use of a readily accessible mono-N-protected amino acid as a chiral ligand improves the efficiency and enantioselectivity of the catalytic transformation. The developed method provides access to both enantiomers of a product using either d or l-phenylalanine derivative as a chiral ligand facilitating the synthesis of both optically active 1,3-disubstituted isoindoline derivatives.

AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila.

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. Here, we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. We propose a model in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.

Diversification of (E,E)-1,6-Dioxo-2,4-Dienes for the Synthesis of (+)-Aspicillin, Isolaurepan, and ß-Parinaric Acid.

A divergent formal synthesis of polyhydroxylated macrocyclic lactone (+)-aspicillin and polyene bioactive natural product ß-parinaric acid and the total synthesis of non-terpenoid metabolite isolaurepan have been achieved using a ruthenium-catalyzed stereo- and chemoselective oxidative coupling reaction of easily accessible vinyl ketones and acrylates. The crucial transformation involves the efficient synthesis and functionalization of stereodefined (E,E)-1,6-dioxo-2,4-dienes using simple reaction protocols, which enabled straightforward access to a diverse range of bioactive natural products.

Role of Bin-Amphiphysin-Rvs (BAR) domain proteins in mediating neuronal signaling and disease.

Several proteins contain signaling domains that can regulate the cell membrane dynamics as well as the underlying cytoskeleton. Among these, Bin-Amphiphysin-Rvs (BAR) domain-containing proteins, with their membrane deforming properties, have emerged as the key players in regulating neuronal morphology and inducing neuronal signaling that can modulate synaptic architecture. While the biochemical and structural basis of membrane deformation by the BAR-domain proteins has been extensively studied, the in vivo contexts in which these proteins function remain to be elucidated. Despite the discovery of BAR-domain proteins over 25 years ago, most of the studies have primarily focused on understanding the structural and biochemical properties and cell biological processes regulated by these proteins. Understanding the functional requirements of these proteins at the level of multicellular organisms and the way these proteins regulate biological processes remains a topic of intensive study. In this review, we discuss the functional roles of BAR-domain proteins in the context of membrane dynamics and cellular signaling. We highlight recent developments describing the functional role of these proteins in neuronal morphogenesis, synaptic function, and disease.

Synthesis of Polyene Bioactive Natural Products: FR252921 and Vitamin A.

A formal synthesis of FR252921, a potent macrocyclic immunosuppressive agent, and a six-step synthesis of vitamin A have been demonstrated. The application of a ruthenium-catalyzed step-economic and environmentally benign strategy for the highly stereo- and chemoselective construction of valuable polyene motifs of FR252921 and vitamin A highlights the syntheses. The key features for the synthesis FR252921 include preparation of the triene moiety followed by two consecutive peptide couplings of the three fragments.

Ruthenium-catalyzed stereo- and chemoselective oxidative coupling of vinyl ketones: efficient access to (E,E)-1,6-dioxo-2,4-dienes.

A Ru-catalyzed direct oxidative coupling reaction of vinyl ketones was developed. It offers a straightforward and atom-economical protocol for the synthesis of functionalized (E,E)-1,6-dioxo-2,4-diene derivatives in moderate to good yields with excellent stereo- and chemoselectivities. In addition, the synthetic utility of this method was further demonstrated by its application to the synthesis of bioactive natural products such as (7E,9E)-henicosa-7,9-diene-6,11-dione (sex pheromone), ostopanic acid (plant anticancer agent), JA (anti-tumor agent) and the southern part (C1-C13) of the antibiotic macrolactin-T.

Weakly Coordinating, Hydroxyl Directed Ruthenium Catalyzed C-H Alkylation of Ubiquitous Benzyl Alcohols with Maleimides.

Benzyl alcohols have been employed as effective coupling partners in Ru-catalyzed C-H functionalization reactions, and their annulation with maleimides then offers efficient synthesis of useful ortho substituted succinimide aromatic aldehydes and ketones. Detailed mechanistic studies have been demonstrated by performing preliminary reactions, deuterium studies, and competitive experiments.

Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei.

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.

Fragile X premutation rCGG repeats impair synaptic growth and synaptic transmission at Drosophila larval neuromuscular junction.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell-developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. Here, we used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. We show that presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by riboCGG (rCGG) repeats mediates presynaptically during larval neuromuscular junction development. We also suggest that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats.

Spermatozoa produced during winter are superior in terms of phenotypic characteristics and oviduct explants binding ability in the water buffalo (Bubalus bubalis).

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.

Drosophila Mon1 and Rab7 interact to regulate glutamate receptor levels at the neuromuscular junction.

Regulation of post-synaptic receptors plays an important role in determining synaptic strength and plasticity. The Drosophila larval neuromuscular junction (nmj) has been used extensively as a model to understand some of these processes. In this context, we are interested in the role of Drosophila Monensin sensitivity protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. DMon1 is an evolutionarily conserved protein which, in complex with calcium caffeine zinc sensitivity1 (CCZ1), regulates the conversion of early endosomes to late endosomes through recruitment of Rab7. C-terminal deletion mutants of Dmon1 (Dmon1Δ181) exhibit lethality. The escapers have a short life span and exhibit severe motor defects. At the nmj, these mutants show defects in synaptic morphology and a strong increase in GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear. In this study, we have characterized an EMS mutant referred to as pog1 and demonstrate it to be an allele of Dmon1. Further, we have examined the role of rab7 in regulating GluRIIA. We show that similar to Dmon1, knock-down of rab7 using RNAi in neurons, but not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 and rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1 Δ181 mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscore the importance of the endo-lysosomal pathway in this process.

Drosophila ELYS regulates Dorsal dynamics during development.

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes into functional nuclear pores toward the end of mitosis. Using cellular, molecular, and genetic tools, including fluorescence and Electron microscopy, quantitative PCR, and RNAi-mediated depletion, we report here that the ELYS ortholog (dElys) plays critical roles during Drosophila development. dElys localized to the nuclear rim in interphase cells, but during mitosis it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that dElys has conserved structural features, but contains a noncanonical AT-hook-like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by dElys during development.

A cell-permeant small molecule for the super-resolution imaging of the endoplasmic reticulum in live cells.

The endoplasmic reticulum (ER) constitutes about half of the total membrane of a eukaryotic cell, and defects in the ER have been shown to be linked with a variety of diseases. To investigate these underlying mechanisms in detail, the specific labelling of the ER for high-resolution long-term live-imaging can serve as an important tool. Here, we report the identification of a stimulated emission depletion (STED) microscopy-compatible BODIPY derivative (NH2-BODIPY) to selectively image the ER. In contrast to the conventional ER-Tracker™ dye, NH2-BODIPY selectively labels the ER at a much lower concentration with no detectable cytotoxicity. Conventional imaging agents are often unstable under the intense light field used for STED microscopy, but NH2-BODIPY is stable due to its robust structure and therefore it has the potential to be widely exploited for ER imaging.

Drosophila Choline transporter non-canonically regulates pupal eclosion and NMJ integrity through a neuronal subset of mushroom body.

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/ß core (α/ßc) and ϒ neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/ßc and ϒ neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.