Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present Feature-Based Molecular Networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. The FBMN method brings quantitative analyses, isomeric resolution, including from ion-mobility spectrometry, into molecular networks.

1Molecular networking has become a key method used to visualize and annotate the chemical space in 2 non-targeted mass spectrometry-based experiments. However, distinguishing isomeric compounds and

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex-depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates. INTRODUCTIONThe nuclear pore complex (NPC) is a large macromolecular structure that fuses and perforates the two nuclear membranes. The 120-million-dalton nuclear pore provides the major route for the active transport of molecules between the nucleus and cytoplasm (for recent review, see Davis, 1995;Goldberg and Allen, 1995;Panté and Aebi, 1995;Simos and Hurt, 1995). From electron microscopy, the pore complex is seen to have a modular organization, consisting of an octasymmetrical framework of eight spokes sandwiched between cytoplasmic and nuclear rings. The spokes embrace a central channel or "transporter" that is thought to carry out the gated aspects of nucleocyto- ¶ Corresponding author: University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.© 1997 by The American Society for Cell Biology 2017 plasmic transport (Unwin and Milligan, 1982;Akey and Radermacher, 1993). Other microscopically visible features include eight cytoplasmic filaments, which extend from the cytoplasmic ring of the pore, and a nuclear basket, which extends from the nuclear ring of the pore into the nucleoplasm. Electron microscopy reveals that the eightf...

Interphase cytosol extracts prepared from Xenopus laevis eggs are active in RNA polymerase III (Pol III) transcription. Addition of recombinant B1 cyclin to these extracts activates mitotic protein kinases that repress transcription. Affinity-purified p34cdc2-cyclin B kinase (mitosis-promoting factor) is sufficient to effect this repression in a simplified Pol III transcription system. This mitotic repression involves the direct phosphorylation of a component of the Pol III transcription initiation factor TFIIIB, which consists of the TATA box-binding protein (TBP) and associated Pol III-specific factors. The transcriptional activity of the TFIIIB-TBP fraction can be modulated in vitro by phosphorylation with mitotic kinases and by dephosphorylation with immobilized alkaline phosphatase.

The need for new drugs for the treatment of various diseases is enormous. From the previous century until the present, numerous peptide and peptide-derived natural products have been isolated from bacteria and fungi. Hence, microorganisms play a pivotal role as sources for novel drugs with an emphasis on anti-infective agents. Various disciplines from biology, chemistry, and medicine are involved in early stages of the search for peptide natural products including taxonomy, microbiology, bioanalytics, bioinformatics, and medicinal chemistry. Under biochemical aspects, small peptide drugs are basically either ribosomally synthesized and post-translationally modified (RiPPs) or synthesized by multimodular nonribosomal peptide synthetases (NRPSs). Within the context of current developments on bioactive peptide natural products, this Account predominantly highlights recent discoveries, approaches, and research from our laboratory on RiPPs and NRPSs from bacteria and fungi. In our search for peptides showing bioactivities of interest, different approaches were applied: classical screening, in silico prediction, in vitro reconstitution, site-directed mutagenesis, chemoenzymatics, heterologous expression, and total synthesis including structure-activity relationship (SAR) studies in the research on the labyrinthopeptins, albicidin, and the cyclodepsipeptides (CDPs). The ribosomally synthesized labyrinthopeptins, class III lanthipeptides, which have been discovered in a classical screening campaign, display highly attractive antiallodynic (against neuropathic pain caused by dysfunction of the nervous system) and antiviral activities. Therefore, the biosynthetic assembly was investigated by extensive enzymatic studies of the modifying enzymes, and site-directed mutagenesis was performed for the generation of analogs. By genome mining, other class III lanthipeptides have been uncovered, while synthetic access proved to be an unmet challenge for the labyrinthopeptins. In contrast, for the gyrase inhibitor albicidin, the establishment of a chemical synthesis followed by medicinal chemistry studies was the only viable option to gain access to derivatives. Albicidin, which has been discovered investigating plant host-pathogen interactions, has a strong activity against Gram-negative bacteria, for example, Escherichia coli and Pseudomonas aeruginosa, and a future synthetic derivative may become a lead structure for development of an anti-Gram-negative drug. The compound class of the cyclodepsipeptides contributes already two marketed drugs, enniatin (fusafungine) and emodepside. Cyclodepsipeptides show general antibacterial and antifungal effects, whereas specific insecticidal and anthelmintic activities provide lead structures for drug development. Hence, exploiting the chances of reprogramming NRPSs, the generation of chimeric or otherwise designed synthetases could render a new untapped structural space and thus novel bioactivities. While current developments in the fields of genomics, bioinformatics, and molecular biology f...

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.

Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high-and low-molecularweight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria, and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of Slayer biogenesis, and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.

We introduce VAMPIRE, 1 a lightweight pretraining framework for effective text classification when data and computing resources are limited. We pretrain a unigram document model as a variational autoencoder on in-domain, unlabeled data and use its internal states as features in a downstream classifier. Empirically, we show the relative strength of VAMPIRE against computationally expensive contextual embeddings and other popular semi-supervised baselines under low resource settings. We also find that fine-tuning to indomain data is crucial to achieving decent performance from contextual embeddings when working with limited supervision. We accompany this paper with code to pretrain and use VAMPIRE embeddings in downstream tasks.