Orthogonal Recurrent Neural Networks and Batch Normalization in Deep Neural Networks

Despite the recent success of various machine learning techniques, there are still numerous obstacles that must be overcome. One obstacle is known as the vanishing/exploding gradient problem. This problem refers to gradients that either become zero or unbounded. This is a well known problem that commonly occurs in Recurrent Neural Networks (RNNs). In this work we describe how this problem can be mitigated, establish three different architectures that are designed to avoid this issue, and derive update schemes for each architecture. Another portion of this work focuses on the often used technique of batch normalization. Although found to be successful in decreasing training times and in preventing overfitting, it is still unknown why this technique works. In this paper we describe batch normalization and provide a potential alternative with the end goal of improving our understanding of how batch normalization works

Hydraulic adjustment to an obstacle in a rotating channel

Author Posting. © Cambridge University Press, 2000. This article is posted here by permission of Cambridge University Press for personal use, not for redistribution. The definitive version was published in Journal of Fluid Mechanics 404 (2000):117-149, doi:10.1017/S0022112099007065.In order to gain insight into the hydraulics of rotating-channel flow, a set of initial-value problems analogous to Long's towing experiments is considered. Specifically, we calculate the adjustment caused by the introduction of a stationary obstacle into a steady, single-layer flow in a rotating channel of infinite length. Using the semigeostrophic approximation and the assumption of uniform potential vorticity, we predict the critical obstacle height above which upstream influence occurs. This height is a function of the initial Froude number, the ratio of the channel width to an appropriately defined Rossby radius of deformation, and a third parameter governing how the initial volume flux in sidewall boundary layers is partitioned. (In all cases, the latter is held to a fixed value specifying zero flow in the right-hand (facing downstream) boundary layer.) The temporal development of the flow according to the full, two-dimensional shallow water equations is calculated numerically, revealing numerous interesting features such as upstream-propagating shocks and separated rarefying intrusions, downstream hydraulic jumps in both depth and stream width, flow separation, and two types of recirculations. The semigeostrophic prediction of the critical obstacle height proves accurate for relatively narrow channels and moderately accurate for wide channels. Significantly, we find that contact with the left-hand wall (facing downstream) is crucial to most of the interesting and important features. For example, no instances are found of hydraulic control of flow that is separated from the left-hand wall at the sill, despite the fact that such states have been predicted by previous semigeostrophic theories. The calculations result in a series of regime diagrams that should be very helpful for investigators who wish to gain insight into rotating, hydraulically driven flow.The authors have been supported by the National Science Foundation through Grants (OCE-9810599 for L.J.P. and K.R.H. and OCE-9711186 for EPC). L.J.P. also received support from the Office of Naval Research under Grant N00014-95-1-0456 and K.R.H. under grant N00014-93-1-0263

Rapidly Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in NSCLC Cell Lines through De-Repression of FGFR2 and FGFR3 Expression

Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5′ flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors

An unusual flavin-dependent halogenase from the metagenome of the marine sponge Theonella swinhoei WA

The authors thank EU BlueGenics (Seventh Framework Programme, Collaborative project “BlueGenics”, Grant no. 311848 RJMG and JP), the SNF (Grant no.205321_165695 to JP), the Helmut Horten Foundation (JP), and ERAIB (Grant no. 031A338A KHVP and RJMG) for funding.Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin dependent halogenase with a broad substrate specificity, and with potential as a useful new biocatalytic tool is described.PostprintPeer reviewe

Synergism of anisotropic and computational NMR methods reveals the likely configuration of phormidolide A.

Characterization of the complex molecular scaffold of the marine polyketide natural product phormidolide A represents a challenge that has persisted for nearly two decades. In light of discordant results arising from recent synthetic and biosynthetic reports, a rigorous study of the configuration of phormidolide A was necessary. This report outlines a synergistic effort employing computational and anisotropic NMR investigation, that provided orthogonal confirmation of the reassigned side chain, as well as supporting a further correction of the C7 stereocenter

A community resource for paired genomic and metabolomic data mining

Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.Peer reviewe

MIBiG 3.0 : a community-driven effort to annotate experimentally validated biosynthetic gene clusters

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Genome- and Ecology-Based Strategies for the Discovery of Bioactive Polyketides and Peptides

Traditionally, natural products have been isolated by bioactivity-guided fractionation. This strategy, however, led to the frequent re-isolation of known metabolites. An alternative strategy for the discovery of novel specialized metabolites is genome mining. Genome mining is an in-silico natural product discovery strategy which uses genetic information to identify natural product biosynthetic gene clusters (BGCs). The identified BGCs are annotated and the information about the BGC constitution is used to predict the structure of the corresponding secondary metabolite. Based on these predictions the corresponding natural product can be isolated in a targeted fashion. Trans-acyltransferase polyketide synthases (trans-AT PKSs) are giant modular multi-domain enzymes that are responsible for the biosynthesis of complex polyketides. These bacterial assembly-lines, ubiquitously distributed across the bacterial branch of life, were shown to produce bioactive polyketides of pharmaceutical and agricultural value. Phylogenetic analysis of ketosynthase (KS) domains from characterized PKSs, responsible for polyketide chain elongation, revealed a correlation between KS domains that phylogenetically cluster together and the substrates they recognize. Based on the correlation between phylogenetic origin and substrate specificity, referred to as the trans-AT PKS correlation rule, we developed the trans-AT PKS-derived polyketide predictor (TransATor) web application for the precise predictions of trans-AT PKS-derived polyketide structures. The TransATor web-application can be used to conduct in-silico dereplication studies. The TransATor-based in-silico annotation of a BGC to a known metabolite prevents the re-isolation of known metabolites, as exemplified by the isolation of tartrolon D from Gynuella sunshinyii. Moreover, TransATor predictions were employed to prioritize isolation efforts and, most importantly, to guide the purification and structure elucidation workflow. This has led to the isolation of a novel polyketide scaffold from Brevibacillus sp. Leaf182 and a new phormidolide-like polyketide, leptolyngbialide, from Leptolyngbia sp. PCC 7375. The prediction of KS substrate specificities furthermore resulted in the revision of the biosynthetic model for oocydin from Serratia plymuthica, the proposal of a model for sesbanimide biosynthesis in Azorhizobium doebereinerae and the reinvestigation and revision of the absolute stereochemistry of phormidolide isolated from a Leptolyngbia sp. ISBN3-Nov-94-8. A previous study on misakinolide-like polyketides suggested that the corresponding PKS family has evolved through the exchange, deletion or insertion of conserved sequential arrangements of PKS modules (PKS motifs). Driven by the hypothesis that trans-AT PKSs evolved in a mosaic-like manner, through combinatorial recombination of conserved motifs from different pathways, we analyzed all 1724 trans-AT PKSs deposited in GenBank for the presence of motifs shared between different PKSs. In total, 327 conserved sequential arrangements of PKS modules were identified. Based on these conserved motifs, also reflected in the polyketide structure, we were able to isolate new polyketides with conserved substructures. These included the novel glutarimide-containing polyketide secimide from the plant pathogen Pseudomonas syringae pv. syringae and the new phormidolide, gynuellalide, from G. sunshinyii. Moreover, the identification of a conserved PKS gene fragment encoding part of the PKS responsible for the biosynthesis of the cytotoxic spliceostatin in the plant pathogen Xanthomonas campestris pv. cannabis resulted in the identification of the pathogenicity factor of the bacterium. In addition, PKSs that share conserved motifs with other orphan PKSs but do not show any conserved motifs with known polyketides are good candidates for the discovery of structural novelty in the corresponding polyketides. The analysis of conserved PKS motifs shared between different PKSs furthermore paves the way towards evolutionary inspired engineering of non-natural hybrid PKSs to produce novel polyketides. 8 A complementary strategy to genome mining is the activation of silent BGCs. Here, we studied the potential of the actinobacterial signaling metabolite hormaomycin to activate silent BGCs in the producing organism and the model strain Streptomyces coelicolor. The effect of hormaomycin on the metabolome of the producing organism, Streptomyces griseoflavus W-384 was analyzed, and compared to the metabolome of hormaomycin production deficient- and hormaomycin overproducing mutants. Two novel metabolites, xanthoductin and xanthotryptin were only produced in the hormaomycin overproducing mutant. Moreover, the production of pharmaceutically important natural products was either strongly up- or downregulated in the presence of hormaomycin. In order to study its effect on other Streptomyces, we subjected S. coelicolor, either challenged with the hormaomcyin overproducer or purified hormaomycin to matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry and nanospray desorption ionization mass spectrometry (nanoDESI) coupled to molecular network analysis. These analyses revealed that hormaomycin induces the production of siderophores, antibiotics, morphogens and pigments belonging to different classes of natural products. In addition, hormaomycin was able to trigger the production of unknown metabolites. These studies show that hormaomycin can be used as a tool to activate silent BGCs and to increase the production of pharmaceutically relevant specialized metabolites. Intrigued by the chemotype triggered by hormaomycin, we were interested in identifying its molecular target. Using a mutasynthesis strategy, hormaomycin analogs with either alkyne or azide handles were generated. These analogs were coupled to different tags and the obtained probes used for pull-down assays to identify the target of hormaomcyin. These assays revealed the the F-ATP synthase as the putative target molecule. Quantification of intracellular ATP levels in the presence or absence of hormaomycin and the phenotypic comparison of the effect of hormaomycin with the decoupling agent carbonylcyanide m-chlorophenylhydrazone supported our initial hypothesis that hormaomycin exerts its effect by interacting with the F-ATP synthase. Moreover, we conducted a high-throughput bipartite interaction screen of all strains of the At-LSPHERE strain collection (50,000 interactions) of bacterial leaf isolates to identify talented antibiotic producers. Linking these data with genome mining studies (AntiSMASH and BigScape analysis), resulted in the identification of antibiotic producers harboring BGCs that encode for putative biosynthetic and structural novelty. The most promising inhibiting strain, Brevibacillus sp. Leaf182 that contains a dozen BGCs was selected for bioactivity-guided fractionation to identify the antibiotic(s) responsible for the observed inhibition of one third of all strains of the strain collection. Isolation of the metabolites responsible for the majority of the observed antibiotic activity yielded tyrocidin analogs and marthiapeptide A. To our surprise, marthiapeptide A is putatively produced via the non-ribosomal route of peptide biosynthesis, which is remarkable given that two structurally close homologs were previously reported to be ribosomally-synthesized and post-translationally modified peptides. The biosynthesis of the same class of polythiazole/polyoxazole-containing 7-8 membered cyclopeptides from two biorthogonal pathways displays an extreme case of convergent evolution. Moreover, the isolation of a novel trans-AT PKS scaffold from the same organism highlights the huge untapped biosynthetic potential of bacterial isolates from the plant phyllosphere