Image classification with symbolic hints using limited resources.

Typical machine learning classification benchmark problems often ignore the full input data structures present in real-world classification problems. Here we aim to represent additional information as "hints" for classification. We show that under a specific realistic conditional independence assumption, the hint information can be included by late fusion. In two experiments involving image classification with hints taking the form of text metadata, we demonstrate the feasibility and performance of the fusion scheme. We fuse the output of pre-trained image classifiers with the output of pre-trained text models. We show that calibration of the pre-trained models is crucial for the performance of the fused model. We compare the performance of the fusion scheme with a mid-level fusion scheme based on support vector machines and find that these two methods tend to perform quite similarly, albeit the late fusion scheme has only negligible computational costs.

A comprehensive analysis of pneumococcal two-component system regulatory networks.

Two-component systems are key signal-transduction systems that enable bacteria to respond to a wide variety of environmental stimuli. The human pathogen, Streptococcus pneumoniae (pneumococcus) encodes 13 two-component systems and a single orphan response regulator, most of which are significant for pneumococcal pathogenicity. Mapping the regulatory networks governed by these systems is key to understand pneumococcal host adaptation. Here we employ a novel bioinformatic approach to predict the regulons of each two-component system based on publicly available whole-genome sequencing data. By employing pangenome-wide association studies (panGWAS) to predict genotype-genotype associations for each two-component system, we predicted regulon genes of 11 of the pneumococcal two-component systems. Through validation via next-generation RNA-sequencing on response regulator overexpression mutants, several top candidate genes predicted by the panGWAS analysis were confirmed as regulon genes. The present study presents novel details on multiple pneumococcal two-component systems, including an expansion of regulons, identification of candidate response regulator binding motifs, and identification of candidate response regulator-regulated small non-coding RNAs. We also demonstrate a use for panGWAS as a complementary tool in target gene identification via identification of genotype-to-genotype links. Expanding our knowledge on two-component systems in pathogens is crucial to understanding how these bacteria sense and respond to their host environment, which could prove useful in future drug development.

The Y-ome Conundrum: Insights into Uncharacterized Genes and Approaches for Functional Annotation.

The ever-increasing availability of genome sequencing data has revealed a substantial number of uncharacterized genes without known functions across various organisms. The first comprehensive genome sequencing of E. coli K12 revealed that more than 50% of its open reading frames corresponded to transcripts with no known functions. The group of protein-coding genes without a functional description and/or a recognized pathway, beginning with the letter "Y", is classified as the "y-ome". Several efforts have been made to elucidate the functions of these genes and to recognize their role in biological processes. This review provides a brief update on various strategies employed when studying the y-ome, such as high-throughput experimental approaches, comparative omics, metabolic engineering, gene expression analysis, and data integration techniques. Additionally, we highlight recent advancements in functional annotation methods, including the use of machine learning, network analysis, and functional genomics approaches. Novel approaches are required to produce more precise functional annotations across the genome to reduce the number of genes with unknown functions.

Substrate space analysis of the bacterial proton-coupled oligopeptide transporter YdgR by cheminformatics.

Proton-dependent oligopeptide transporters (POTs) are recognized for their substrate promiscuity due to their ability to transport a wide range of substrates. POTs are conserved in all forms of life ranging from bacteria to humans. A dipeptide-fluorophore conjugate, H-(ß-Ala)-Lys(AMCA)-OH, is a well-known substrate of the transporter YdgR that is commonly used as a fluorescent reporter. In order to understand the substrate space of YdgR, we used this dipeptide as a bait reference, when screening an ensemble of compounds (previously tested in PEPT/PTR/NPF space) via a cheminformatic analysis based on the Tanimoto similarity index. Eight compounds (sinalbin, abscisic acid, carnosine, jasmonic acid, N-acetyl-aspartate, N-acetyl-lysine, aspartame, and N-acetyl-aspartylglutamate), covering a wide range on the Tanimoto scale, were tested for YdgR-mediated transport. Carnosine was the only compound observed to be a YdgR substrate based on cell-based transport assays and molecular docking. The other compounds tested were neither inhibitors nor substrates. Thus, we found that neither the Tanimoto similarity index nor ADME (absorption, distribution, metabolism, and excretion) properties appear useful for the identification of substrates (e.g., dipeptides) in YdgR-mediated drug transport.

Physiological response in E. coli to YdgR overexpression depends on whether the protein has an intact function.

Membrane transport proteins are essential for the transport of a wide variety of molecules across the cell membrane to maintain cellular homeostasis. Generally, these transport proteins can be overexpressed in a suitable host (bacteria, yeast, or mammalian cells), and it is well documented that overexpression of membrane proteins alters the global metabolomic and proteomic profiles of the host cells. In the present study, we investigated the physiological consequences of overexpression of a membrane transport protein YdgR that belongs to the POT/PTR family from E. coli by using the lab strain BL21 (DE3)pLysS in its functional and attenuated mutant YdgR-E33Q. We found significant differences between the omics (metabolomics and proteomics) profiles of the cells expressing functional YdgR as compared to cells expressing attenuated YdgR, e.g., upregulation of several uncharacterized y-proteins and enzymes involved in the metabolism of peptides and amino acids. Furthermore, molecular network analysis suggested a relatively higher presence of proline-containing tripeptides in cells expressing functional YdgR. We envisage that an in-depth investigation of physiological alterations due to protein over-expression may be used for the deorphanization of the y-gene transportome.

Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae.

Introduction: Whole genome sequencing offers great opportunities for linking genotypes to phenotypes aiding in our understanding of human disease and bacterial pathogenicity. However, these analyses often overlook non-coding intergenic regions (IGRs). By disregarding the IGRs, crucial information is lost, as genes have little biological function without expression. Methods/Results: In this study, we present the first complete pangenome of the important human pathogen Streptococcus pneumoniae (pneumococcus), spanning both the genes and IGRs. We show that the pneumococcus species retains a small core genome of IGRs that are present across all isolates. Gene expression is highly dependent on these core IGRs, and often several copies of these core IGRs are found across each genome. Core genes and core IGRs show a clear linkage as 81% of core genes are associated with core IGRs. Additionally, we identify a single IGR within the core genome that is always occupied by one of two highly distinct sequences, scattered across the phylogenetic tree. Discussion: Their distribution indicates that this IGR is transferred between isolates through horizontal regulatory transfer independent of the flanking genes and that each type likely serves different regulatory roles depending on their genetic context.

Redshifted and thermally bistable one-way quantitative hemithioindigo-derived photoswitches enabled by isomer-specific excited state intramolecular proton transfer.

We report bistable indole-containing hemithioindigos (HTIs) with one-way quantitative photoswitching properties. Supported by state-averaged CASPT2/CASSCF calculations, we propose a mechanism for the observed one-way photoswitching that involves an isomer-specific excited state intramolecular proton transfer (ESIPT). Additionally, we developed a thermally bistable oligomer-inspired bipyrrole-containing HTI, which displays large band separation and bidirectional near-quantitative photoisomerization in the near-infrared, bio-optical window.

Global transcriptional responses of pneumococcus to human blood components and cerebrospinal fluid.

Streptococcus pneumoniae (pneumococcus) is a leading cause of severe invasive infectious diseases such as sepsis and meningitis. Understanding how pneumococcus adapts and survive in the human bloodstream environment and cerebrospinal fluid (CSF) is important for development of future treatment strategies. This study investigates the global transcriptional response of pneumococcus to human blood components and CSF acquired from discarded and anonymized patient samples. Extensive transcriptional changes to human blood components were observed during early stages of interaction. Plasma-specific responses were primarily related to metabolic components and include strong downregulation of fatty acid biosynthesis genes, and upregulation of nucleotide biosynthesis genes. No transcriptional responses specific to the active plasma proteins (e.g., complement proteins) were observed during early stages of interaction as demonstrated by a differential expression analysis between plasma and heat-inactivated plasma. The red blood cell (RBC)-specific response was far more complex, and included activation of the competence system, differential expression of several two-component systems, phosphotransferase systems and transition metal transporter genes. Interestingly, most of the changes observed for CSF were also observed for plasma. One of the few CSF-specific responses, not observed for plasma, was a strong downregulation of the iron acquisition system piuBCDA. Intriguingly, this transcriptomic analysis also uncovers significant differential expression of more than 20 small non-coding RNAs, most of them in response to RBCs, including small RNAs from uncharacterized type I toxin-antitoxin systems. In summary, this transcriptomic study identifies key pneumococcal metabolic pathways and regulatory genes involved with adaptation to human blood and CSF. Future studies should uncover the potential involvement of these factors with virulence in-vivo.

Host-glycan metabolism is regulated by a species-conserved two-component system in Streptococcus pneumoniae.

Pathogens of the Streptococcus genus inhabit many different environmental niches during the course of an infection in a human host and the bacteria must adjust their metabolism according to available nutrients. Despite their lack of the citric-acid cycle, some streptococci proliferate in niches devoid of a readily available carbohydrate source. Instead they rely on carbohydrate scavenging for energy acquisition, which are obtained from the host. Here we discover a two-component system (TCS07) of Streptococcus pneumoniae that responds to glycoconjugated structures on proteins present on the host cells. Using next-generation RNA sequencing we find that the uncharacterized TCS07 regulon encodes proteins important for host-glycan processing and transporters of the released glycans, as well as intracellular carbohydrate catabolizing enzymes. We find that a functional TCS07 allele is required for growth on the glycoconjugated model protein fetuin. Consistently, we see a TCS07-dependent activation of the glycan degradation pathway. Thus, we pinpoint the molecular constituents responsible for sensing host derived glycans and link this to the induction of the proteins necessary for glycan degradation. Furthermore, we connect the TCS07 regulon to virulence in a mouse model, thereby establishing that host-derived glycan-metabolism is important for infection in vivo. Finally, a comparative phylogenomic analysis of strains from the Streptococcus genus reveal that TCS07 and most of its regulon is specifically conserved in species that utilize host-glycans for growth.

sRNA-mediated control in bacteria: An increasing diversity of regulatory mechanisms.

Small regulatory RNAs (sRNAs) act as post-transcriptional regulators controlling bacterial adaptation to environmental changes. Our current understanding of the mechanisms underlying sRNA-mediated control is mainly based on studies in Escherichia coli and Salmonella. Ever since the discovery of sRNAs decades ago, these Gram-negative species have served as excellent model organisms in the field of sRNA biology. More recently, the role of sRNAs in gene regulation has become the center of attention in a broader range of species, including Gram-positive model organisms. Here, we highlight some of the most apparent similarities and differences between Gram-negative and Gram-positive bacteria with respect to the mechanisms underlying sRNA-mediated control. Although key aspects of sRNA regulation appear to be highly conserved, novel themes are arising from studies in Gram-positive species, such as a clear abundance of sRNAs acting through multiple C-rich motifs, and an apparent lack of RNA-binding proteins with chaperone activity.

Intraintestinal and Parenteral Administration of an Insulin Analogue Leads to Comparable Activation of Signaling Downstream of the Insulin Receptor in the Small Intestine.

BACKGROUND: Oral delivery of insulin was recently demonstrated to have therapeutic relevance in patients with diabetes. Insulin receptors are expressed in the gastrointestinal tract and can be activated by insulin in the bloodstream, but it is not known if the large amount of insulin in the intestinal lumen required for sufficient oral delivery will induce a different effect. The aim of this study was to compare the acute effect in the intestine of insulin administered in the intestinal lumen with that of insulin administered by a parenteral route. METHOD: Intraintestinal (ii) injection in the mid-jejunum of anaesthetized rats with insulin analogue 106 (I106), formulated with the absorption-enhancer sodium caprate, was used as an animal model of oral insulin administration. As control treatment, rats were treated with I106 by iv infusion according to algorithms which precisely mimicked the pharmacokinetic and pharmacodynamic properties of ii administered I106. Several fold more I106 was administered by ii injection than by iv infusion. Phosphorylated Akt (Ser473) was used as indicator of insulin-stimulated acute effects in the intestine. RESULTS: Treatment with I106 resulted in activation of Akt in the intestine, with no significant difference between the effects of ii or iv administration. CONCLUSION: The results from this rat model of orally administered insulin indicate that the unabsorbed insulin in the intestinal lumen after oral administration will not result in an enhanced acute effect in the intestine.

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors.

Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.

Selective Acetylene Hydrogenation over Single-Atom Alloy Nanoparticles by Kinetic Monte Carlo.

Single-atom alloys, which are prepared by embedding isolated metal sites in host metals, are promising systems for improved catalyst selectivity. For technical applications, catalysts based on nanoparticles are preferred thanks to a large surface area. Herein, we investigate hydrogenation of acetylene to ethylene using kinetic Monte Carlo simulations based on density functional theory and compare the performance of Pd/Cu nanoparticles with Pd(111) and Pd/Cu(111). We find that embedding Pd in Cu systems strongly enhances the selectivity and that the reaction mechanism is fundamentally different for nanoparticles and extended surfaces. The reaction mechanism on nanoparticles is complex and involves elementary steps that proceed preferentially over different sites. Edge and corner sites on nanoparticles are predicted to lower the selectivity, and we infer that a rational design strategy in selective acetylene hydrogenation is to maximize the number of (111) sites in relation to edge sites for Pd/Cu nanoparticles.

Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins.

Rearrangement of the 1q12 pericentromeric heterochromatin and subsequent amplification of the 1q arm is commonly associated with cancer development and progression and may result from epigenetic deregulation. In many premalignant and malignant cells, loss of 1q12 satellite DNA methylation causes the deposition of polycomb factors and formation of large polycomb aggregates referred to as polycomb bodies. Here, we show that SSX proteins can destabilize 1q12 pericentromeric heterochromatin in melanoma cells when it is present in the context of polycomb bodies. We found that SSX proteins deplete polycomb bodies and promote the unfolding and derepression of 1q12 heterochromatin during replication. This further leads to segregation abnormalities during anaphase and generation of micronuclei. The structural rearrangement of 1q12 pericentromeric heterochromatin triggered by SSX2 is associated with loss of polycomb factors, but is not mediated by diminished polycomb repression. Instead, our studies suggest a direct effect of SSX proteins facilitated though a DNA/chromatin binding, zinc finger-like domain and a KRAB-like domain that may recruit chromatin modifiers or activate satellite transcription. Our results demonstrate a novel mechanism for generation of 1q12-associated genomic instability in cancer cells.

Design and Synthesis of 2,3- trans-Proline Analogues as Ligands for Ionotropic Glutamate Receptors and Excitatory Amino Acid Transporters.

Development of pharmacological tools for the ionotropic glutamate receptors (iGluRs) is imperative for the study and understanding of the role and function of these receptors in the central nervous system. We report the synthesis of 18 analogues of (2 S,3 R)-2-carboxy-3-pyrrolidine acetic acid (3a), which explores the effect of introducing a substituent on the ε-carbon (3c-q). A new synthetic method was developed for the efficient synthesis of racemic 3a and applied to give expedited access to 13 racemic analogues of 3a. Pharmacological characterization was carried out at native iGluRs, cloned homomeric kainate receptors (GluK1-3), NMDA receptors (GluN1/GluN2A-D), and excitatory amino acid transporters (EAAT1-3). From the structure-activity relationship studies, several new ligands emerged, exemplified by triazole 3p-d1, GluK3-preferring (GluK1/GluK3 Ki ratio of 15), and the structurally closely related tetrazole 3q-s3-4 that displayed 4.4-100-fold preference as an antagonist for the GluN1/GluN2A receptor ( Ki = 0.61 µM) over GluN1/GluN2B-D ( Ki = 2.7-62 µM).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

MonteCoffee: A programmable kinetic Monte Carlo framework.

Kinetic Monte Carlo (kMC) is an essential tool in heterogeneous catalysis enabling the understanding of dominant reaction mechanisms and kinetic bottlenecks. Here we present MonteCoffee, which is a general-purpose object-oriented and programmable kMC application written in python. We outline the implementation and provide examples on how to perform simulations of reactions on surfaces and nanoparticles and how to simulate sorption isotherms in zeolites. By permitting flexible and fast code development, MonteCoffee is a valuable alternative to previous kMC implementations.

Influence of atomic site-specific strain on catalytic activity of supported nanoparticles.

Heterogeneous catalysis is an enabling technology that utilises transition metal nanoparticles (NPs) supported on oxides to promote chemical reactions. Structural mismatch at the NP-support interface generates lattice strain that could affect catalytic properties. However, detailed knowledge about strain in supported NPs remains elusive. We experimentally measure the strain at interfaces, surfaces and defects in Pt NPs supported on alumina and ceria with atomic resolution using high-precision scanning transmission electron microscopy. The largest strains are observed at the interfaces and are predominantly compressive. Atomic models of Pt NPs with experimentally measured strain distributions are used for first-principles kinetic Monte Carlo simulations of the CO oxidation reaction. The presence of only a fraction of strained surface atoms is found to affect the turnover frequency. These results provide a quantitative understanding of the relationship between strain and catalytic function and demonstrate that strain engineering can potentially be used for catalyst design.

Activation of insulin receptors and IGF-1 receptors in COLO-205 colon cancer xenografts by insulin and insulin analogue X10 does not enhance growth under normo- or hypoglycaemic conditions.

AIMS/HYPOTHESIS: Recent studies with normal rats and mouse allograft models have reported that insulin and insulin analogues do not activate the IGF-1 receptor in vivo, and that this characteristic therefore cannot be responsible for the increased incidence of mammary tumours observed for the insulin analogue X10 in chronic toxicity studies with Sprague Dawley rats. This is in clear contrast to reports of insulin and insulin analogues in vitro. Clarification of this is important for understanding the mechanisms behind possible growth-promoting effects of insulin analogues, and will have implications for the development of novel insulin analogues. METHODS: We established a xenograft model in BALB/c nude mice with the human colon cancer cell line COLO-205, which expresses human insulin and IGF-1 receptors, and explored the acute and chronic effects of treatment with supra-pharmacological doses of human insulin, insulin analogue X10 and human IGF-1. With a novel antibody, acute IGF-1 receptor activation was also examined in various tissues from normal rats treated with human insulin, insulin analogue X10 or human IGF-1. Finally, the effects of pharmacologically relevant doses of human insulin and insulin analogue X10 on receptor activation and growth of COLO-205 xenograft were explored in BALB/c nude mice with alloxan-induced hyperglycaemia. RESULTS: In normal rats and in BALB/c nude mice bearing a COLO-205 cell xenograft, treatment with supra-pharmacological doses of human insulin, insulin analogue X10 or human IGF-1 resulted in activation of insulin receptors as well as IGF-1 receptors. Treatment of diabetic nude mice with pharmacologically relevant doses of human insulin or insulin analogue X10, which decreased blood glucose from hyperglycaemic levels to the normoglycaemic range, did not increase IGF-1 receptor activation. Furthermore, repeated treatment with supra-pharmacological as well as pharmacological doses of human insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts. CONCLUSIONS/INTERPRETATION: This study demonstrates that activation of IGF-1 receptors in cancer cells by insulin and insulin analogues cannot be considered as a purely in vitro phenomenon. It does occur in vivo in animal models, although only after treatment with supra-pharmacological doses. Furthermore, treatment with insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts under normo- or hypoglycaemic conditions. Further studies are needed before a conclusion can be reached on whether IGF-1 receptor activation by insulin analogues correlates with increased growth in vivo.

sRNA-dependent control of curli biosynthesis in Escherichia coli: McaS directs endonucleolytic cleavage of csgD mRNA.

Production of curli, extracellular protein structures important for Escherichia coli biofilm formation, is governed by a highly complex regulatory mechanism that integrates multiple environmental signals through the involvement of numerous proteins and small non-coding RNAs (sRNAs). No less than seven sRNAs (McaS, RprA, GcvB, RydC, RybB, OmrA and OmrB) are known to repress the expression of the curli activator CsgD. Many of the sRNAs repress CsgD production by binding to the csgD mRNA at sites far upstream of the ribosomal binding site. The precise mechanism behind sRNA-mediated regulation of CsgD synthesis is largely unknown. In this study, we identify a conserved A/U-rich region in the csgD mRNA 5' untranslated region, which is cleaved upon binding of the small RNAs, McaS, RprA or GcvB, to sites located more than 30 nucleotides downstream. Mutational analysis shows that the A/U-rich region as well as an adjacent stem-loop structure are required for McaS-stimulated degradation, also serving as a binding platform for the RNA chaperone Hfq. Prevention of McaS-activated cleavage completely relieves repression, suggesting that endoribonucleolytic cleavage of csgD mRNA is the primary regulatory effect exerted by McaS. Moreover, we find that McaS-mediated degradation of the csgD 5' untranslated region requires RNase E.