The Mechanism of Inhibition of Mycobacterial (p)ppGpp Synthetases by a Synthetic Analog of Erogorgiaene.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

Synthesis, Antimicrobial and Antibiofilm Activities, and Molecular Docking Investigations of 2-(1H-Indol-3-yl)-1H-benzo[d]imidazole Derivatives.

The treatment of many bacterial and fungal infections remains a problem due to increasing antibiotic resistance and biofilm formation by pathogens. In the present article, a methodology for the chemoselective synthesis of 2-(1H-indol-3-yl)-1H-benzo[d]imidazole derivatives is presented. We report on the antimicrobial activity of synthesized 2-(1H-indol-3-yl)-1H-benzo[d]imidazoles with significant activity against Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300 (MRSA), Mycobacterium smegmatis (mc(2)155/ATCC 700084), and Candida albicans ATCC 10231. High activity against staphylococci was shown by indolylbenzo[d]imidazoles 3ao and 3aq (minimum inhibitory concentration (MIC) < 1 µg/mL) and 3aa and 3ad (MIC 3.9-7.8 µg/mL). A low MIC was demonstrated by 2-(1H-indol-3-yl)-1-methyl-1H-benzo[d]imidazole (3ag) against M. smegmatis and against C. albicans (3.9 µg/mL and 3.9 µg/mL, respectively). 2-(5-Bromo-1H-indol-3-yl)-6,7-dimethyl-1H-benzo[d]imidazole (3aq) showed a low MIC of 3.9 µg/mL against C. albicans. Compounds 3aa, 3ad, 3ao, and 3aq exhibited excellent antibiofilm activity, inhibiting biofilm formation and killing cells in mature biofilms. Molecular docking analysis identified three potential interaction models for the investigated compounds, implicating (p)ppGpp synthetases/hydrolases, FtsZ proteins, or pyruvate kinases in their antibacterial action mechanism.

The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives.

The treatment of many bacterial diseases remains a significant problem due to the increasing antibiotic resistance of their infectious agents. Among others, this is related to Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) and Mycobacterium tuberculosis. In the present article, we report on antibacterial compounds with activity against both S. aureus and MRSA. A straightforward approach to 2-(1H-indol-3-yl)quinazolin-4(3H)-one and their analogues was developed. Their structural and functional relationships were also considered. The antimicrobial activity of the synthesized compounds against Mycobacterium tuberculosis H37Rv, S. aureus ATCC 25923, MRSA ATCC 43300, Candida albicans ATCC 10231, and their role in the inhibition of the biofilm formation of S. aureus were reported. 2-(5-Iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) showed a low minimum inhibitory concentration (MIC) of 0.98 µg/mL against MRSA. The synthesized compounds were assessed via molecular docking for their ability to bind long RSH (RelA/SpoT homolog) proteins using mycobacterial and streptococcal (p)ppGpp synthetase structures as models. The cytotoxic activity of some synthesized compounds was studied. Compounds 3c, f, g, k, r, and 3z displayed significant antiproliferative activities against all the cancer cell lines tested. Indolylquinazolinones 3b, 3e, and 3g showed a preferential suppression of the growth of rapidly dividing A549 cells compared to slower growing fibroblasts of non-tumor etiology.

First genome-scale insights into the virulence of the snow mold causal fungus Microdochium nivale.

Pink snow mold, caused by a phytopathogenic and psychrotolerant fungus, Microdochium nivale, is a severe disease of winter cereals and grasses that predominantly occurs under snow cover or shortly after its melt. Snow mold has significantly progressed during the past decade, often reaching epiphytotic levels in northern countries and resulting in dramatic yield losses. In addition, M. nivale gradually adapts to a warmer climate, spreading to less snowy territories and causing different types of plant diseases throughout the growing period. Despite its great economic importance, M. nivale is poorly investigated; its genome has not been sequenced and its crucial virulence determinants have not been identified or even predicted. In our study, we applied a hybrid assembly based on Oxford Nanopore and Illumina reads to obtain the first genome sequence of M. nivale. 11,973 genes (including 11,789 protein-encoding genes) have been revealed in the genome assembly. To better understand the genetic potential of M. nivale and to obtain a convenient reference for transcriptomic studies on this species, the identified genes were annotated and split into hierarchical three-level functional categories. A file with functionally classified M. nivale genes is presented in our study for general use. M. nivale gene products that best meet the criteria for virulence factors have been identified. The genetic potential to synthesize human-dangerous mycotoxins (fumonisin, ochratoxin B, aflatoxin, and gliotoxin) has been revealed for M. nivale. The transcriptome analysis combined with the assays for extracellular enzymatic activities (conventional virulence factors of many phytopathogens) was carried out to assess the effect of host plant (rye) metabolites on the M. nivale phenotype. In addition to disclosing plant-metabolite-upregulated M. nivale functional gene groups (including those related to host plant protein destruction and amino acid metabolism, xenobiotic detoxication (including phytoalexins benzoxazinoids), cellulose destruction (cellulose monooxygenases), iron transport, etc.), the performed analysis pointed to a crucial role of host plant lipid destruction and fungal lipid metabolism modulation in plant-M. nivale interactions.

Biobanking as a Tool for Genomic Research: From Allele Frequencies to Cross-Ancestry Association Studies.

In recent years, great advances have been made in the field of collection, storage, and analysis of biological samples. Large collections of samples, biobanks, have been established in many countries. Biobanks typically collect large amounts of biological samples and associated clinical information; the largest collections include over a million samples. In this review, we summarize the main directions in which biobanks aid medical genetics and genomic research, from providing reference allele frequency information to allowing large-scale cross-ancestry meta-analyses. The largest biobanks greatly vary in the size of the collection, and the amount of available phenotype and genotype data. Nevertheless, all of them are extensively used in genomics, providing a rich resource for genome-wide association analysis, genetic epidemiology, and statistical research into the structure, function, and evolution of the human genome. Recently, multiple research efforts were based on trans-biobank data integration, which increases sample size and allows for the identification of robust genetic associations. We provide prominent examples of such data integration and discuss important caveats which have to be taken into account in trans-biobank research.

Aggregation of Genome-Wide Association Data from FinnGen and UK Biobank Replicates Multiple Risk Loci for Pregnancy Complications.

Complications endangering mother or fetus affect around one in seven pregnant women. Investigation of the genetic susceptibility to such diseases is of high importance for better understanding of the disease biology as well as for prediction of individual risk. In this study, we collected and analyzed GWAS summary statistics from the FinnGen cohort and UK Biobank for 24 pregnancy complications. In FinnGen, we identified 11 loci associated with pregnancy hypertension, excessive vomiting, and gestational diabetes. When UK Biobank and FinnGen data were combined, we discovered six loci reaching genome-wide significance in the meta-analysis. These include rs35954793 in FGF5 (p=6.1×10-9), rs10882398 in PLCE1 (p=8.9×10-9), and rs167479 in RGL3 (p=5.2×10-9) for pregnancy hypertension, rs10830963 in MTNR1B (p=4.5×10-41) and rs36090025 in TCF7L2 (p=3.4×10-15) for gestational diabetes, and rs2963457 in the EBF1 locus (p=6.5×10-9) for preterm birth. In addition to the identified genome-wide associations, we also replicated 14 out of 40 previously reported GWAS markers for pregnancy complications, including four more preeclampsia-related variants. Finally, annotation of the GWAS results identified a causal relationship between gene expression in the cervix and gestational hypertension, as well as both known and previously uncharacterized genetic correlations between pregnancy complications and other traits. These results suggest new prospects for research into the etiology and pathogenesis of pregnancy complications, as well as early risk prediction for these disorders.

Plasma miRNA Profile in High Risk of Preterm Birth during Early and Mid-Pregnancy.

In recent years evidence has been accumulated showing that miRNAs can act as potential biomarkers or targets for therapy of preterm birth, one of the most important problems in modern obstetrics. We have performed a prospective study of the miRNA profile in the plasma during the first and second trimesters in pregnant women with high risk of preterm birth (n = 13 cases and n = 11 controls). For the study group plasma blood samples at 9-13 weeks before diagnosis and at 22-24 weeks after start of therapy were selected. Using high-throughput sequencing technology we detected differences in the levels of 15 miRNAs (3 upregulated-hsa-miR-122-5p, hsa-miR-34a-5p, hsa-miR-34c-5p; 12 downregulated-hsa-miR-487b-3p, hsa-miR-493-3p, hsa-miR-432-5p, hsa-miR-323b-3p, hsa-miR-369-3p, hsa-miR-134-5p, hsa-miR-431-5p, hsa-miR-485-5p, hsa-miR-382-5p, hsa-miR-369-5p, hsa-miR-485-3p, hsa-miR-127-3p) (log2(FC) ≥ 1.5; FDR ≤ 0.05) during the first trimester compared with the control (non-high-risk of preterm birth pregnant women). All downregulated miRNAs in the first trimester from the placenta-specific C14MC cluster. During the second trimester no differentially expressed miRNAs were found. Our results suggest that the miRNA profile in plasma during early pregnancy may predict a high risk of preterm birth, which is important in preventing gestational problems as early as possible.

Cadaverine biosynthesis contributes to decreased Escherichia coli susceptibility to antibiotics.

Some bacterial stress responses are involved in survival under antibiotic treatment and contribute to less susceptible microbial forms selection. Here, we tested the role of cadaverine, one of the biogenic polyamines considered as universal adaptogens, in the processes. The expression of ldcC and cadA genes, encoding cadaverine-producing lysine decarboxylase, increased in Escherichia coli cells exposed to ß-lactams and fluoroquinolones but not aminoglycosides. The transcriptional regulators RpoS and SoxS controlled the expression of ldcC and cadA, respectively, in response to antibiotics. Exogenous cadaverine had little effect on E. coli antibiotic susceptibility, whereas non-antibiotic-induced endogenous cadaverine contributed to its tolerance to ß-lactams, fluoroquinolones, and aminoglycosides. Antibiotic-induced cadaverine synthesis promoted bacterial survival under fluoroquinolone exposure, as well as could contribute to low-resistant bacterial forms development. Selection under the fluoroquinolone levofloxacin exposure toward bacteria with an increased ability to synthesize cadaverine and negative correlation between LdcC activity and fluoroquinolone susceptibility in the selected forms were demonstrated. The same correlation in a special group of low-level resistant clinical E. coli isolates was revealed. So, cadaverine biosynthesis appeared to be a significant player in decreased E. coli antibiotic susceptibility development.

Identification of Key Metabolic Pathways and Biomarkers Underlying Flowering Time of Guar (Cyamopsis tetragonoloba (L.) Taub.) via Integrated Transcriptome-Metabolome Analysis.

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an annual legume crop native to India and Pakistan. Seeds of the plant serve as a source of galactomannan polysaccharide (guar gum) used in the food industry as a stabilizer (E412) and as a gelling agent in oil and gas fracturing fluids. There were several attempts to introduce this crop to countries of more northern latitudes. However, guar is a plant of a short photoperiod, therefore, its introduction, for example, to Russia is complicated by a long day length during the growing season. Breeding of new guar varieties insensitive to photoperiod slowed down due to the lack of information on functional molecular markers, which, in turn, requires information on guar genome. Modern breeding strategies, e.g., genomic predictions, benefit from integration of multi-omics approaches such as transcriptome, proteome and metabolome assays. Here we present an attempt to use transcriptome-metabolome integration to understand the genetic determination of flowering time variation among guar plants that differ in their photoperiod sensitivity. This study was performed on nine early- and six delayed-flowering guar varieties with the goal to find a connection between 63 metabolites and 1,067 differentially expressed transcripts using Shiny GAM approach. For the key biomarker of flowering in guar myo-inositol we also evaluated the KEGG biochemical pathway maps available for Arabidopsis thaliana. We found that the phosphatidylinositol signaling pathway is initiated in guar plants that are ready for flowering through the activation of the phospholipase C (PLC) gene, resulting in an exponential increase in the amount of myo-inositol in its free form observed on GC-MS chromatograms. The signaling pathway is performed by suppression of myo-inositol phosphate kinases (phosphorylation) and alternative overexpression of phosphatases (dephosphorylation). Our study suggests that metabolome and transcriptome information taken together, provide valuable information about biomarkers that can be used as a tool for marker-assisted breeding, metabolomics and functional genomics of this important legume crop.

Transcriptomic Analysis of Radish (Raphanus sativus L.) Spontaneous Tumor.

Spontaneous tumors can develop in different organs of various plant species without any pathogen infection and, as a rule, appear in plants with a certain genotype: Mutants, interspecific hybrids, etc. In particular, among the inbred lines of radish (Raphanus sativus L.), lines that form spontaneous tumors on the taproot during the flowering period were obtained many years ago. In this work, we analyzed the differential gene expression in the spontaneous tumors of radish versus the lateral roots using the RNA-seq method. Data were obtained indicating the increased expression of genes associated with cell division and growth (especially genes that regulate G2-M transition and cytokinesis) in the spontaneous tumor. Among genes downregulated in the tumor tissue, genes participating in the response to stress and wounding, mainly involved in the biosynthesis of jasmonic acid and glucosinolates, were enriched. Our data will help elucidate the mechanisms of spontaneous tumor development in higher plants.

A synthetic diterpene analogue inhibits mycobacterial persistence and biofilm formation by targeting (p)ppGpp synthetases.

Bacterial persistence coupled with biofilm formation is directly associated with failure of antibiotic treatment of tuberculosis. We have now identified 4-(4,7-DiMethyl-1,2,3,4-tetrahydroNaphthalene-1-yl)Pentanoic acid (DMNP), a synthetic diterpene analogue, as a lead compound that was capable of suppressing persistence and eradicating biofilms in Mycobacterium smegmatis. By using two reciprocal experimental approaches - ΔrelMsm and ΔrelZ gene knockout mutations versus relMsm and relZ overexpression technique - we showed that both RelMsm and RelZ (p)ppGpp synthetases are plausible candidates for serving as targets for DMNP. In vitro, DMNP inhibited (p)ppGpp-synthesizing activity of purified RelMsm in a concentration-dependent manner. These findings, supplemented by molecular docking simulation, suggest that DMNP targets the structural sites shared by RelMsm, RelZ, and presumably by a few others as yet unidentified (p)ppGpp producers, thereby inhibiting persister cell formation and eradicating biofilms. Therefore, DMNP may serve as a promising lead for development of antimycobacterial drugs.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles.

Phloem fibers are important elements of plant architecture and the target product of many fiber crops. A key stage in fiber development is intrusive elongation, the mechanisms of which are largely unknown. Integrated analysis of miRNA and mRNA expression profiles in intrusivelygrowing fibers obtained by laser microdissection from flax (Linum usitatissimum L.) stem revealed all 124 known flax miRNA from 23 gene families and the potential targets of differentially expressed miRNAs. A comparison of the expression between phloem fibers at different developmental stages, and parenchyma and xylem tissues demonstrated that members of miR159, miR166, miR167, miR319, miR396 families were down-regulated in intrusively growing fibers. Some putative target genes of these miRNA families, such as those putatively encoding growth-regulating factors, an argonaute family protein, and a homeobox-leucine zipper family protein were up-regulated in elongating fibers. miR160, miR169, miR390, and miR394 showed increased expression. Changes in the expression levels of miRNAs and their target genes did not match expectations for the majority of predicted target genes. Taken together, poorly understood intrusive fiber elongation, the key process of phloem fiber development, was characterized from a miRNA-target point of view, giving new insights into its regulation.

Stationary-phase genes upregulated by polyamines are responsible for the formation of Escherichia coli persister cells tolerant to netilmicin.

Persisters are rare phenotypic variants of regular bacterial cells that survive lethal antibiotics or stresses owing to slowing down of their metabolism. Recently, we have shown that polyamine putrescine can upregulate persister cell formation in Escherichia coli via the stimulation of rpoS expression, encoding a master regulator of general stress response. We hypothesized that rmf and yqjD, the stationary-phase genes responsible for ribosome inactivation, might be good candidates for the similar role owing to their involvement in translational arrest and the ability to be affected by polyamines. Using reporter gene fusions or single and multiple knockout mutations in rpoS, rmf and yqjD genes, we show in this work that (i) E. coli polyamines spermidine and cadaverine can upregulate persistence, like putrescine; (ii) polyamine effects on persister cell formation are mediated through stimulation of expression of rpoS, rmf and yqjD genes; (iii) these genes are involved in persister cell formation sequentially in a dynamic fashion as cells enter the stationary phase. The data obtained in this work can be used to develop novel tools relying on a suppression of polyamine metabolism in bacteria to combat persister cells as an important cause of infections refractory to antibiotics.

Putrescine controls the formation of Escherichia coli persister cells tolerant to aminoglycoside netilmicin.

Persisters are suggested to be the products of a phenotypic variability that are quasi-dormant forms of regular bacterial cells highly tolerant to antibiotics. Our previous investigations revealed that a decrease in antibiotic tolerance of Escherichia coli cells could be reached through the inhibition of key enzymes of polyamine synthesis (putrescine, spermidine). We therefore assumed that polyamines could be involved in persister cell formation. Data obtained in our experiments with the polyamine-deficient E. coli strain demonstrate that the formation of persisters tolerant to netilmicin is highly upregulated by putrescine in a concentration-dependent manner when cells enter the stationary phase. This period is also accompanied by dissociation of initially homogenous subpopulation of persister cells to some fractions differing in their levels of tolerance to netilmicin. With three independent experimental approaches, we demonstrate that putrescine-dependent upregulation of persister cell formation is mediated by stimulation of rpoS expression. Complementary activity of putrescine and RpoS results in ~ 1000-fold positive effect on persister cell formation.

ATP/ADP alteration as a sign of the oxidative stress development in Escherichia coli cells under antibiotic treatment.

The extensively discussed idea of oxidative stress development under antibiotic treatment was confirmed using an antioxidant gene expression (soxRS-, oxyR-regulon) approach, including microaerobic cultivation conditions. The killing action of antibiotics and their ability to cause peroxide oxidative stress in Escherichia coli cells was comparable to a similar hydrogen peroxide capacity; therefore, the involvement of intracellular hydrogen peroxide production in the killing action of antibiotics seems plausible under conditions studied. The temporary increase of ATP/ADP (which returned to untreated levels in 10 min) and the intensification of respiration preceded the development of oxidative stress. The sharp rise in ATP/ADP was due to the accumulation of ATP with a slight increase in the ADP content. We proposed that ATP accumulation was not a result of increased respiration but was due to the inhibition of energy-consuming processes. The association of reactive oxygen species formation under antibiotic treatment with the inhibition of direct electron flow pathway along the respiratory chain, and a possible role of a sharp rise in ATP/ADP in this process is hypothesized.

Polyamines reduce oxidative stress in Escherichia coli cells exposed to bactericidal antibiotics.

Bactericidal antibiotics (fluoroquinolones, aminoglycosides and cephalosporins) at their sublethal concentrations were able to produce hydroxyl radicals, hydrogen peroxide and superoxide anions (ROS) in Escherichia coli cells, which resulted in damage to proteins and DNA. The cells responded to oxidative stress by a 2-3-fold increase in cell polyamines (putrescine, spermidine) produced as a consequence of upregulation of ornithine decarboxylase (ODC). Relief of oxidative stress by cessation of culture aeration or addition of antioxidants substantially diminished or even completely abolished polyamine accumulation observed in response to antibiotics. Alternatively, inhibition of polyamine synthesis resulted in enhancement of oxidative stress in antibiotic-processed cells. When added to antibiotic-inhibited culture, polyamines reduced intracellular ROS production and thereby prevented damage to proteins and DNA. These effects eventually resulted in a substantial increase in cell viability, growth recovery and antibiotic resistance that were more strongly expressed in polyamine-deficient mutants.

Mimicking the biological activity of diazobenzo[b]fluorene natural products with electronically tuned diazofluorene analogs.

Under appropriate electronic modulation, simple diazofluorene analogs recapitulate the DNA cleavage activity of kinamycin D under thiol-based reducing conditions. Achieving DNA cleavage under these reducing conditions is key to anticancer activity, as the most active compound, 1-methoxydiazofluorene, inhibits the proliferation of HeLa cells.

RNA-mediated synthesis of palladium nanoparticles on Au surfaces.

RNA catalysts for the shape-controlled synthesis of Pd particles from the precursor complex trisdibenzylideneacetone dipalladium ([Pd2(DBA)3] were recently discovered in our laboratory (J. Am. Chem. Soc. 2005, 127, 17814-17818). In the work described here, RNA codes for hexagonal Pd platelets and Pd cubes were covalently immobilized on gold surfaces and evaluated for their activity toward particle synthesis. When coupled to gold via oligoethylene glycol linkers, both RNA sequences were able to catalyze the formation of Pd particles with the same shape control previously observed in solution. For low surface coverages, the average distance between RNA molecules on the surface was estimated at ca. 300 nm, yet large (e.g., dimensions of hundreds of nanometers) Pd hexagons and cubes still formed. This surprising result suggests that a single RNA molecule may be sufficient for nucleating and controlling the shapes of these particles. Finally, the use of surface-bound RNA as a tool for directing the orthogonal synthesis of materials on surfaces was demonstrated. Patterning the RNA code for Pd hexagons next to the code for Pd cubes, followed by incubation in a solution containing [Pd2(DBA)3], resulted in the spontaneous formation of spatially distinct spots of hexagonal and cubic particles.

Assembly and characterization of biomolecule-gold nanoparticle conjugates and their use in intracellular imaging.

In this chapter, we outline protocols for assembling and characterizing peptide-gold nanoparticle conjugates. We describe two strategies for attaching peptides to gold nanoparticles. One involves the covalent coupling of cysteine-terminated peptides directly to a particle surface via a sulfur-gold bond. Alternatively, peptides are coupled to bovine serum albumin (BSA) via a bifunctional molecular crosslinker. We also describe a number of characterization methods for determining the number of crosslinkers per BSA, peptides per BSA, and peptides adsorbed per particle. Finally, we show that the enormous visible light extinction properties of gold nano particles make them excellent imaging agents for tracking the trajectories of peptides inside living cells.