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Importance: Dysbiosis has been robustly demonstrated in Parkinson disease (PD), and fecal microbiota transplantation (FMT) has shown promising effects in preclinical PD models., Objective: To assess the safety and symptomatic efficacy of colonic single-dose anaerobically prepared FMT., Design, Setting, and Participants: This was a double-blind, placebo-controlled, randomized clinical trial conducted between November 2020 and June 2023 with a follow-up period of 12 months at 4 hospitals in Finland. Patients with PD aged 35 to 75 years in Hoehn & Yahr stage 1-3 with a mild to moderate symptom burden and dysbiosis of fecal microbiota were included. Of 229 patients screened, 48 were randomized and 47 received the intervention. One patient discontinued due to worsening of PD symptoms. Two further patients were excluded before analysis and 45 were included in the intention-to-treat analysis., Intervention: Participants were randomized in a 2:1 ratio to receive FMT or placebo via colonoscopy., Main Outcomes and Measures: The primary end point was the change of Movement Disorder Society Unified Parkinson's Disease Rating Scale parts I-III (part III off medication) at 6 months. Safety was assessed by recording adverse events (AEs)., Results: The median (IQR) age was 65 (52.5-70.0) years in the placebo group and 66 (59.25-69.75) years in the FMT group; 9 (60.0%) and 16 (53.3%) patients were male in the placebo group and the FMT group, respectively. The primary outcome did not differ between the groups (0.97 points, 95% CI, -5.10 to 7.03, P = .75). Gastrointestinal AEs were more frequent in the FMT group (16 [53%] vs 1 [7%]; P = .003). Secondary outcomes and post hoc analyses showed stronger increase of dopaminergic medication and improvement of certain motor and nonmotor outcomes in the placebo group. Microbiota changes were more pronounced after FMT but differed by donor. Nevertheless, dysbiosis status was reversed more frequently in the placebo group., Conclusions and Relevance: FMT was safe but did not offer clinically meaningful improvements. Further studies-for example, through modified FMT approaches or bowel cleansing-are warranted regarding the specific impact of donor microbiota composition and dysbiosis conversion on motor and nonmotor outcomes as well as medication needs in PD., Trial Registration: ClinicalTrials.gov Identifier: NCT04854291.

ATP-BINDING CASSETTE SUBFAMILY E MEMBER (ABCE) proteins are one of the most conserved proteins across eukaryotes and archaea. Yeast and most animals possess a single ABCE gene encoding the critical translational factor ABCE1. In several plant species, including Arabidopsis thaliana and Oryza sativa , two or more ABCE gene copies have been identified, however information related to plant ABCE gene family is still missing. In this study we retrieved ABCE gene sequences of 76 plant species from public genome databases and comprehensively analyzed them with the reference to A. thaliana ABCE2 gene ( AtABCE2 ). Using bioinformatic approach we assessed the conservation and phylogeny of plant ABCEs. In addition, we performed haplotype analysis of AtABCE2 and its paralogue AtABCE1 using genomic sequences of 1,135 A. thaliana ecotypes. Plant ABCE proteins showed overall high sequence conservation, sharing at least 78% of amino acid sequence identity with AtABCE2. We found that over half of the selected species have two to eight ABCE genes, suggesting that in plants ABCE genes can be classified as a low-copy gene family, rather than a single-copy gene family. The phylogenetic trees of ABCE protein sequences and the corresponding coding sequences demonstrated that Brassicaceae and Poaceae families have independently undergone lineage-specific split of the ancestral ABCE gene. Other plant species have gained ABCE gene copies through more recent duplication events. We also noticed that ploidy level but not ancient whole genome duplications experienced by a species impacts ABCE gene family size. Deeper analysis of AtABCE2 and AtABCE1 from 1,135 A. thaliana ecotypes revealed four and 35 non-synonymous SNPs, respectively. The lower natural variation in AtABCE2 compared to AtABCE1 is in consistence with its crucial role for plant viability. Overall, while the sequence of the ABCE protein family is highly conserved in the plant kingdom, many plants have evolved to have more than one copy of this essential translational factor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jakobson, Mõttus, Suurväli, Sõmera, Tarassova, Nigul, Smolander and Sarmiento.)

Introduction: 33-70% of lung cancer (LC) patients develop recurrence after radical treatment. Previous studies have shown the importance of clinical-pathological characteristics for the risk of recurrence. The role of molecular mechanisms remains unclear. The aim was analyzing genomic features in LC patients with local (LR) versus distant recurrence (DR) to predict the risk and type of recurrence., Materials and Methods: Patients previously curatively treated with LC recurrences from 2015 to 2017 were retrospectively enrolled. Histological specimens collected at the time of LC diagnosis were sent for whole exome sequencing (WES). Genomic data was analyzed for single nucleotide polymorphisms (SNPs) and insertion-deletion mutations (INDELs)., Results: 191 patients were included. 33% of patients had LR and 67% DR, with median recurrence-free survival (RFS) 15.4 versus 11.2 months (p = 0.20) and overall survival (OS) after recurrence 12.9 versus 8.5 months (p = 0.007), respectively. Of various laboratory parameters studied, lymphocytes were significantly decreased at recurrence (p < 0.0001) in the DR group. In genetic analysis, significantly enriched INDEL mutations were found in 38 and 98 genes and SNP mutations in 63 and 179 genes in DR and LR groups, respectively. DMXL2 and ABCC9 gene mutations caused by INDELs appeared exclusively in the DR group. Enrichment analysis detected genes, like KNTC1, CLASP1, CLASP2 and CENPE, responsible of microtubule disturbance in the DR group. Furthermore, genes related to cytosolic Ca2+ such as STIM1, ITPR3 and RYR3, were significantly enriched in DR group whereas in LR group enrichment of pathways related to endoplasmic/sarcoplasmic reticulum Ca2+ was observed., Conclusion: Our findings indicate distinct genomic signatures in the LR and DR cohorts, with microtubule disturbance and calcium regulation playing a crucial role in invasiveness in DR of LC. Understanding molecular mechanisms of LC recurrence may lead to the discovery of novel drug targets that could potentially stop spread of cancer cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Water-in-oil droplets allow performing massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, analyzing such vast arrays of droplets usually requires advanced expertise and sophisticated workflow tools, which limits accessibility for a wider user base in the fields of chemistry and biology. Thus, there is a need for more user-friendly tools for droplet analysis. In this article, we deliver a set of analytical pipelines for user-friendly analysis of typical scenarios in droplet experiments. We built pipelines that combine various open-source image-analysis software with a custom-developed data processing tool called "EasyFlow". Our pipelines are applicable to the typical experimental scenarios that users encounter when working with droplets: i) mono- and polydisperse droplets, ii) brightfield and fluorescent images, iii) droplet and object detection, iv) signal profile of droplets and objects (e.g., fluorescence)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system and the leading cause of progressive neurological disability in young adults. It decreases the patient's lifespan by about 10 years and affects women more than men. No medication entirely restricts or reverses neurological degradation. However, early diagnosis and treatment increase the possibility of a better outcome. To identify new MS biomarkers, we tested the expression of six potential markers (P2X4, P2X7, CXCR4, RGS1, RGS16 and VAV1) using qPCR in peripheral blood mononuclear cells (PBMC) of MS patients treated with interferon β (IFNβ), with glatiramer acetate (GA) or untreated. We showed that P2X7 and VAV1 are significantly induced in MS patients. In contrast, the expression of P2X4, CXCR4, RGS1 and RGS16 was not significantly modified by MS in PBMC. P2X7 and VAV1 are essentially induced in female patients, suggesting these markers are connected to sex-specific mechanisms. Strikingly, VAV1 expression is higher in healthy women than healthy men and IFNβ treatment of MS reduced VAV1 expression in female MS patients while it up-regulated VAV1 in male MS patients. Our data point to the differential, sex-dependent value of MS markers and treatment effects. Although rgs16 expression in PBMC was not a valid MS marker in patients, the strong upregulation of P2X4 and P2X7 induced in the spinal cord of WT mice by EAE was abrogated in rgs16KO mice suggesting that rgs16 is required for P2X4 and P2X7 induction by neurological diseases., (© 2023 The Scandinavian Foundation for Immunology.)

Reduction in responsiveness to gonadotropins or hyporesponsiveness may lead to the failure of in vitro fertilization (IVF), due to a low number of retrieved oocytes. The ovarian sensitivity index (OSI) is used to reflect the ovarian responsiveness to gonadotropin stimulation before IVF. Although introduced to clinical practice already years ago, its usefulness to predict clinical outcomes requires further research. Nevertheless, pathophysiological mechanisms of ovarian hyporesponse, along with advanced maternal age and in younger women, have not been fully elucidated. Follicles consist of multiple cell types responsible for a repertoire of biological processes including responding to pituitary gonadotropins necessary for follicle growth and oocyte maturation as well as ovulation. Encouraging evidence suggests that hyporesponse could be influenced by many contributing factors, therefore, investigating the variability of ovarian follicular cell types and their gene expression in hyporesponders is highly informative for increasing their prognosis for IVF live birth. Due to advancements in single-cell analysis technologies, the role of somatic cell populations in the development of infertility of ovarian etiology can be clarified. Here, somatic cells were collected from the fluid of preovulatory ovarian follicles of patients undergoing IVF, and RNA-seq was performed to study the associations between OSI and gene expression. We identified 12 molecular pathways differentially regulated between hypo- and normoresponder patient groups (FDR<0.05) from which extracellular matrix organization, post-translational protein phosphorylation, and regulation of Insulin-like Growth Factor (IGF) transport and uptake by IGF Binding Proteins were regulated age-independently. We then generated single-cell RNA-seq data from matching follicles revealing 14 distinct cell clusters. Using cell cluster-specific deconvolution from the bulk RNA-seq data of 18 IVF patients we integrated the datasets as a novel approach and discovered that the abundance of three cell clusters significantly varied between hypo- and normoresponder groups suggesting their role in contributing to the deviations from normal ovarian response to gonadotropin stimulation. Our work uncovers new information regarding the differences in the follicular gene expression between hypo- and normoresponders. In addition, the current study fills the gap in understanding the inter-patient variability of cell types in human preovulatory follicles, as revealed by single-cell analysis of follicular fluid cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roos, Rooda, Keif, Liivrand, Smolander, Salumets and Velthut-Meikas.)

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene's numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses., Results: The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models., Conclusions: The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Background: Hsa-miR-548ba expressed in ovarian granulosa cells targets PTEN and LIFR, which are essential for ovarian follicle activation and growth. The expression pattern of hsa-miR-548ba correlates with its host gene follicle-stimulating hormone receptor (FSHR), and FSH has a positive influence on hsa-miR-548ba expression. However, hsa-miR-548ba is a member of a large hsa-mir-548 family with potentially overlapping targets. The current study aims to investigate the co-expression of hsa-mir-548 family members in FSHR-positive reproductive tissues and to explore the potential co-regulation of pathways., Results: For the above-described analysis, small RNA sequencing data from public data repositories were used. Sequencing results revealed that hsa-miR-548ba was expressed at the highest level in the ovarian granulosa cells and uterine myometrial samples together with another twelve and one hsa-miR-548 family members, respectively. Pathway enrichment analysis of microRNA targets in the ovarian samples revealed the hsa-miR-548ba and hsa-miR-548b-5p co-regulation of RAB geranylgeranylation in mural granulosa cells. Moreover, other hsa-mir-548 family members co-regulate pathways essential for ovarian functions (PIP3 activates AKT signalling and signalling by ERBB4). In addition to hsa-miR-548ba, hsa-miR-548o-3p is expressed in the myometrium, which separately targets the peroxisome proliferator-activated receptor alpha (PPARA) pathway., Conclusion: This study reveals that hsa-mir-548 family members are expressed in variable combinations in the reproductive tract, where they potentially fulfil different regulatory roles. The results provide a reference for further studies of the hsa-mir-548 family role in the reproductive tract., (© 2021. The Author(s).)

Droplet microfluidics has revealed innovative strategies in biology and chemistry. This advancement has delivered novel quantification methods, such as droplet digital polymerase chain reaction (ddPCR) and an antibiotic heteroresistance analysis tool. For droplet analysis, researchers often use image-based detection techniques. Unfortunately, the analysis of images may require specific tools or programming skills to produce the expected results. In order to address the issue, we explore the potential use of standalone freely available software to perform image-based droplet detection. We select the four most popular software and classify them into rule-based and machine learning-based types after assessing the software's modules. We test and evaluate the software's (i) ability to detect droplets, (ii) accuracy and precision, and (iii) overall components and supporting material. In our experimental setting, we find that the rule-based type of software is better suited for image-based droplet detection. The rule-based type of software also has a simpler workflow or pipeline, especially aimed for non-experienced users. In our case, CellProfiler (CP) offers the most user-friendly experience for both single image and batch processing analyses., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients ( p = 0.04) containing eight differentially expressed microRNAs ( p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes; however, -the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, a time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that strokeinduced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke, genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is an increase in saccharides, sugar phosphates, ceramides and free fatty acids and a decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery of AAV1-MANF to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Tree architecture has evolved to support a top-heavy above-ground biomass, but this integral feature poses a weight-induced challenge to trunk stability. Maintaining an upright stem is expected to require vertical proprioception through feedback between sensing stem weight and responding with radial growth. Despite its apparent importance, the principle by which plant stems respond to vertical loading forces remains largely unknown. Here, by manipulating the stem weight of downy birch (Betula pubescens) trees, we show that cambial development is modulated systemically along the stem. We carried out a genetic study on the underlying regulation by combining an accelerated birch flowering program with a recessive mutation at the ELIMÄKI locus (EKI), which causes a mechanically defective response to weight stimulus resulting in stem collapse after just 3 months. We observed delayed wood morphogenesis in eki compared with WT, along with a more mechanically elastic cambial zone and radial compression of xylem cell size, indicating that rapid tissue differentiation is critical for cambial growth under mechanical stress. Furthermore, the touch-induced mechanosensory pathway was transcriptionally misregulated in eki, indicating that the ELIMÄKI locus is required to integrate the weight-growth feedback regulation. By studying this birch mutant, we were able to dissect vertical proprioception from the gravitropic response associated with reaction wood formation. Our study provides evidence for both local and systemic responses to mechanical stimuli during secondary plant development., Competing Interests: Declaration of Interests The authors declare no competing interest, (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.

P2X purinergic receptors are extracellular ATP-gated ion channel receptors present on the cell plasma membrane. P2X receptors have been found in Metazoa, fungi, amoebas, and in plants. In mammals, P2X7 is expressed by a large number of cell types and is involved in inflammation and immunity. Remarkably, P2X7 does not desensitize as other P2X do, a feature linked to a "C-cysteine anchor" intra-cytoplasmic motif encoded by exon 11. Another specific feature of P2X7 is its C-terminal cytoplasmic ballast domain (exon 13) which contains a zinc (Zn) coordinating cysteine motif and a GDP-binding region. To determine the origin of P2X7, we analyzed and compared sequences and protein motifs of the C-terminal intra-cytoplasmic region across all main groups of Metazoa. We identified proteins with typical ballast domains, sharing a remarkably conserved Zn-coordinating cysteine motif. Apart from vertebrates, these ballast domains were not associated with a typical P2X architecture. These results strongly suggest that P2X7 resulted from the fusion of a P2X gene, highly similar to P2X4, with an exon encoding a ballast domain. Our work brings new evidence on the origin of the P2X7 purinergic receptor and identifies the Zn-coordinating cysteine domain as the fundamental feature of the ancient ballast fold., (Copyright © 2020 Rump, Smolander, Rüütel Boudinot, Kanellopoulos and Boudinot.)

The novel Acidipropionibacterium genus encompasses species of industrial importance but also those associated with food spoilage. In particular, Acidipropionibacterium acidipropionici , Acidipropionibacterium thoenii , and Acidipropionibacterium jensenii play an important role in food fermentation, as biopreservatives, or as potential probiotics. Notably, A. jensenii and A. thoenii can cause brown spot defects in Swiss-type cheeses, which have been tied to the rhamnolipid pigment granadaene. In the pathogenic bacterium Streptococcus agalactiae, production of granadaene depends on the presence of a cyl gene cluster, an important virulence factor linked with haemolytic activity. Here, we show that the production of granadaene in pigmented Acidipropionibacterium, including A. jensenii, A. thoenii, and Acidipropionibacterium virtanenii , is tied to haemolytic activity and the presence of a cyl -like gene cluster. Furthermore, we propose a PCR-based test, which allows pinpointing acidipropionibacteria with the cyl -like gene cluster. Finally, we present the first two whole genome sequence analyses of the A. jensenii strains as well as testing phenotypic characteristics important for industrial applications. In conclusion, the present study sheds light on potential risks associated with the presence of pigmented Acidipropionibacterium strains in food fermentation. In addition, the results presented here provide ground for development of a quick and simple diagnostic test instrumental in avoiding potential negative effects of Acidipropionibacterium strains with haemolytic activity on food quality.

Unknown sequences, or gaps, are present in many published genomes across public databases. Gap filling is an important finishing step in de novo genome assembly, especially in large genomes. The gap filling problem is nontrivial and while there are many computational tools partially solving the problem, several have shortcomings as to the reliability and correctness of the output, i.e. the gap filled draft genome. SSPACE-LongRead is a scaffolding tool that utilizes long reads from multiple third-generation sequencing platforms in finding links between contigs and combining them. The long reads potentially contain sequence information to fill the gaps created in the scaffolding, but SSPACE-LongRead currently lacks this functionality. We present an automated pipeline called gapFinisher to process SSPACE-LongRead output to fill gaps after the scaffolding. gapFinisher is based on the controlled use of a previously published gap filling tool FGAP and works on all standard Linux/UNIX command lines. We compare the performance of gapFinisher against two other published gap filling tools PBJelly and GMcloser. We conclude that gapFinisher can fill gaps in draft genomes quickly and reliably. In addition, the serial design of gapFinisher makes it scale well from prokaryote genomes to larger genomes with no increase in the computational footprint., Competing Interests: Patrik Koskinen, one of the co-authors of our manuscript, currently is an employee of Valuemotive Ltd. Finland, a private company. He has also been an employee of another private company Blueprint Genetics Ltd. during 2016-2018. He was employed by the institution represented by the rest of the authors (Institute of Biotechnology, University of Helsinki) at the time when most of the conceptual work on this study was carried out. The two private companies mentioned are in no relation to the study under submission. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche 1,2 . Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α) 3 , and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.

Background: The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata., Results: The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins., Conclusions: The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.

Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l p.S78G knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

An increasing number of mammalian species have been shown to have a history of hybridization and introgression based on genetic analyses. Only relatively few fossils, however, preserve genetic material, and morphology must be used to identify the species and determine whether morphologically intermediate fossils could represent hybrids. Because dental and cranial fossils are typically the key body parts studied in mammalian palaeontology, here we bracket the potential for phenotypically extreme hybridizations by examining uniquely preserved cranio-dental material of a captive hybrid between grey and ringed seals. We analysed how distinct these species are genetically and morphologically, how easy it is to identify the hybrids using morphology and whether comparable hybridizations happen in the wild. We show that the genetic distance between these species is more than twice the modern human-Neanderthal distance, but still within that of morphologically similar species pairs known to hybridize. By contrast, morphological and developmental analyses show grey and ringed seals to be highly disparate, and that the hybrid is a predictable intermediate. Genetic analyses of the parent populations reveal introgression in the wild, suggesting that grey-ringed seal hybridization is not limited to captivity. Taken together, we postulate that there is considerable potential for mammalian hybridization between phenotypically disparate taxa., Competing Interests: We have no competing interests.

A Gram-stain-positive, catalase-positive and pleomorphic rod organism was isolated from malted barley in Finland, classified initially by partial 16S rRNA gene sequencing and originally deposited in the VTT Culture Collection as a strain of Propionibacterium acidipropionici (currently Acidipropionibacterium acidipropionici). The subsequent comparison of the whole 16S rRNA gene with other representatives of the genus Acidipropionibacterium revealed that the strain belongs to a novel species, most closely related to Acidipropionibacterium microaerophilum and Acidipropionibacterium acidipropionici, with similarity values of 98.46 and 98.31 %, respectively. The whole genome sequencing using PacBio RS II platform allowed further comparison of the genome with all of the other DNA sequences available for the type strains of the Acidipropionibacterium species. Those comparisons revealed the highest similarity of strain JS278 T to A. acidipropionici, which was confirmed by the average nucleotide identity analysis. The genome of strain JS278 T is intermediate in size compared to the A. acidipropionici and Acidipropionibacterium jensenii at 3 432 872 bp, the G+C content is 68.4 mol%. The strain fermented a wide range of carbon sources, and produced propionic acid as the major fermentation product. Besides its poor ability to grow at 37 °C and positive catalase reaction, the observed phenotype was almost indistinguishable from those of A. acidipropionici and A. jensenii. Based on our findings, we conclude that the organism represents a novel member of the genus Acidipropionibacterium, for which we propose the name Acidipropionibacteriumvirtanenii sp. nov. The type strain is JS278 T (=VTT E-113202 T =DSM 106790 T ).

In Swiss-type cheeses, characteristic nut-like and sweet flavor develops during the cheese ripening due to the metabolic activities of cheese microbiota. Temperature changes during warm and cold room ripening, and duration of ripening can significantly change the gene expression of the cheese microbiota, which can affect the flavor formation. In this study, a metagenomic and metatranscriptomic analysis of Swiss-type Maasdam cheese was performed on samples obtained during ripening in the warm and cold rooms. We reconstructed four different bacterial genomes (Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus helveticus, and Propionibacterium freudenreichii subsp. shermanii strain JS) from the Maasdam cheese to near completeness. Based on the DNA and RNA mean coverage, Lc. lactis strongly dominated (~80-90%) within the cheese microbial community. Genome annotation showed the potential for the presence of several flavor forming pathways in these species, such as production of methanethiol, free fatty acids, acetoin, diacetyl, acetate, ethanol, and propionate. Using the metatranscriptomic data, we showed that, with the exception of Lc. lactis, the central metabolism of the microbiota was downregulated during cold room ripening suggesting that fewer flavor compounds such as acetoin and propionate were produced. In contrast, Lc. lactis genes related to the central metabolism, including the vitamin biosynthesis and homolactic fermentation, were upregulated during cold room ripening., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Stroke is the most common cause of adult disability in developed countries, largely because spontaneous recovery is often incomplete, and no pharmacological means to hasten the recovery exist. It was recently shown that mesencephalic astrocyte-derived neurotrophic factor (MANF) induces alternative or M2 activation of immune cells after retinal damage in both fruit fly and mouse and mediates retinal repair. Therefore, we set out to study whether poststroke MANF administration would enhance brain tissue repair and affect behavioral recovery of rats after cerebral ischemic injury. We used the distal middle cerebral artery occlusion (dMCAo) model of ischemia-reperfusion injury and administered MANF either as a recombinant protein or via adeno-associated viral (AAV) vector. We discovered that, when MANF was administered to the peri-infarct region 2 or 3 days after stroke, it promoted functional recovery of the animals without affecting the lesion volume. Further, AAV7-MANF treatment transiently increased the number of phagocytic macrophages in the subcortical peri-infarct regions. In addition, the analysis of knockout mice revealed the neuroprotective effects of endogenous MANF against ischemic injury, although endogenous MANF had no effect on immune cell-related gene expression. The beneficial effect of MANF treatment on the reversal of stroke-induced behavioral deficits implies that MANF-based therapies could be used for the repair of brain tissue after stroke.

Biosynthetic precursors of NAD + can replenish a decreased cellular NAD + pool and, supposedly via sirtuin (SIRT) deacetylases, improve mitochondrial function. We found decreased hepatic NAD + concentration and downregulated biosynthesis in Bcs1l p.S78G knock-in mice with respiratory chain complex III deficiency and mitochondrial hepatopathy. Aiming at ameliorating disease progression via NAD + repletion and improved mitochondrial function, we fed these mice nicotinamide riboside (NR), a NAD + precursor. A targeted metabolomics verified successful administration and suggested enhanced NAD + biosynthesis in the treated mice, although hepatic NAD + concentration was unchanged at the end point. In contrast to our expectations, NR did not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1l p.S78G mice. We linked this lack of therapeutic effect to NAD + -independent activation of SIRT-1 and -3 via AMPK and cAMP signaling related to the starvation-like metabolic state of Bcs1l p.S78G mice. In summary, we describe an unusual metabolic state with NAD + depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative metabolism. Our study highlights that the knowledge of the underlying complex metabolic alterations is critical when designing therapies for mitochondrial dysfunction.-Purhonen, J., Rajendran, J., Tegelberg, S., Smolander, O.-P., Pirinen, E., Kallijärvi, J., Fellman, V. NAD + repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation.

Background: Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species., Results: We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm., Conclusion: The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Reticulons (RTNs) are a large family of membrane associated proteins with various functions. NOGO-A/RTN4A has a well-known function in limiting neurite outgrowth and restricting the plasticity of the mammalian central nervous system. On the other hand, Reticulon 4 proteins were shown to be involved in forming and maintaining endoplasmic reticulum (ER) tubules. Using comparative transcriptome analysis and qPCR, we show here that NOGO-B/RTN4B and NOGO-A/RTN4A are simultaneously expressed in cultured epithelial, fibroblast and neuronal cells. Electron tomography combined with immunolabelling reveal that both isoforms localize preferably to curved membranes on ER tubules and sheet edges. Morphological analysis of cells with manipulated levels of NOGO-B/RTN4B revealed that it is required for maintenance of normal ER shape; over-expression changes the sheet/tubule balance strongly towards tubules and causes the deformation of the cell shape while depletion of the protein induces formation of large peripheral ER sheets.

Background: The white-rot Agaricomycetes species Phlebia radiata is an efficient wood-decaying fungus degrading all wood components, including cellulose, hemicellulose, and lignin. We cultivated P. radiata in solid state cultures on spruce wood, and extended the experiment to 6 weeks to gain more knowledge on the time-scale dynamics of protein expression upon growth and wood decay. Total proteome and transcriptome of P. radiata were analyzed by peptide LC-MS/MS and RNA sequencing at specific time points to study the enzymatic machinery on the fungus' natural growth substrate., Results: According to proteomics analyses, several CAZy oxidoreductase class-II peroxidases with glyoxal and alcohol oxidases were the most abundant proteins produced on wood together with enzymes important for cellulose utilization, such as GH7 and GH6 cellobiohydrolases. Transcriptome additionally displayed expression of multiple AA9 lytic polysaccharide monooxygenases indicative of oxidative cleavage of wood carbohydrate polymers. Large differences were observed for individual protein quantities at specific time points, with a tendency of enhanced production of specific peroxidases on the first 2 weeks of growth on wood. Among the 10 class-II peroxidases, new MnP1-long, characterized MnP2-long and LiP3 were produced in high protein abundances, while LiP2 and LiP1 were upregulated at highest level as transcripts on wood together with the oxidases and one acetyl xylan esterase, implying their necessity as primary enzymes to function against coniferous wood lignin to gain carbohydrate accessibility and fungal growth. Majority of the CAZy encoding transcripts upregulated on spruce wood represented activities against plant cell wall and were identified in the proteome, comprising main activities of white-rot decay., Conclusions: Our data indicate significant changes in carbohydrate-active enzyme expression during the six-week surveillance of P. radiata growing on wood. Response to wood substrate is seen already during the first weeks. The immediate oxidative enzyme action on lignin and wood cell walls is supported by detected lignin substructure sidechain cleavages, release of phenolic units, and visual changes in xylem cell wall ultrastructure. This study contributes to increasing knowledge on fungal genetics and lignocellulose bioconversion pathways, allowing us to head for systems biology, development of biofuel production, and industrial applications on plant biomass utilizing wood-decay fungi.

Despite the crucial roles of phytohormones in plant development, comparison of the exact distribution profiles of different hormones within plant meristems has thus far remained scarce. Vascular cambium, a wide lateral meristem with an extensive developmental zonation, provides an optimal system for hormonal and genetic profiling. By taking advantage of this spatial resolution, we show here that two major phytohormones, cytokinin and auxin, display different yet partially overlapping distribution profiles across the cambium. In contrast to auxin, which has its highest concentration in the actively dividing cambial cells, cytokinins peak in the developing phloem tissue of a Populus trichocarpa stem. Gene expression patterns of cytokinin biosynthetic and signaling genes coincided with this hormonal gradient. To explore the functional significance of cytokinin signaling for cambial development, we engineered transgenic Populus tremula × tremuloides trees with an elevated cytokinin biosynthesis level. Confirming that cytokinins function as major regulators of cambial activity, these trees displayed stimulated cambial cell division activity resulting in dramatically increased (up to 80% in dry weight) production of the lignocellulosic trunk biomass. To connect the increased growth to hormonal status, we analyzed the hormone distribution and genome-wide gene expression profiles in unprecedentedly high resolution across the cambial zone. Interestingly, in addition to showing an elevated cambial cytokinin content and signaling level, the cambial auxin concentration and auxin-responsive gene expression were also increased in the transgenic trees. Our results indicate that cytokinin signaling specifies meristematic activity through a graded distribution that influences the amplitude of the cambial auxin gradient., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Propionibacterium freudenreichii subsp. freudenreichii DSM 20271(T) is the type strain of species Propionibacterium freudenreichii that has a long history of safe use in the production dairy products and B12 vitamin. P. freudenreichii is the type species of the genus Propionibacterium which contains Gram-positive, non-motile and non-sporeforming bacteria with a high G + C content. We describe the genome of P. freudenreichii subsp. freudenreichii DSM 20271(T) consisting of a 2,649,166 bp chromosome containing 2320 protein-coding genes and 50 RNA-only encoding genes.

Lactococcus piscium is a psychrotrophic lactic acid bacterium and is known to be one of the predominant species within spoilage microbial communities in cold-stored packaged foods, particularly in meat products. Its presence in such products has been associated with the formation of buttery and sour off-odors. Nevertheless, the spoilage potential of L. piscium varies dramatically depending on the strain and growth conditions. Additional knowledge about the genome is required to explain such variation, understand its phylogeny, and study gene functions. Here, we present the complete and annotated genomic sequence of L. piscium MKFS47, combined with a time course analysis of the glucose catabolism-based transcriptome. In addition, a comparative analysis of gene contents was done for L. piscium MKFS47 and 29 other lactococci, revealing three distinct clades within the genus. The genome of L. piscium MKFS47 consists of one chromosome, carrying 2,289 genes, and two plasmids. A wide range of carbohydrates was predicted to be fermented, and growth on glycerol was observed. Both carbohydrate and glycerol catabolic pathways were significantly upregulated in the course of time as a result of glucose exhaustion. At the same time, differential expression of the pyruvate utilization pathways, implicated in the formation of spoilage substances, switched the metabolism toward a heterofermentative mode. In agreement with data from previous inoculation studies, L. piscium MKFS47 was identified as an efficient producer of buttery-odor compounds under aerobic conditions. Finally, genes and pathways that may contribute to increased survival in meat environments were considered., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The standard digestion-ligation cloning method enables synthesis of large amounts of complementary DNA (cDNA) from a model organism facilitating study of the transcriptome. Here, we used cDNA amplification of the dimorphic yeast Taphrina betulina as an example of how a library construction protocol can significantly increase sequencing throughput. Two modification steps were introduced to the Evrogen standard Mint-2 protocol to improve its suitability for next-generation sequencing projects. We performed two partial Illumina MiSeq sequencing runs with the modified protocol: one with and one without biotin-purified primers. The results demonstrated that biotinylated libraries increased both accuracy and throughput of the modified protocol. Moreover, our sequencing results indicate that a sequence-specific miscall may affect the output of Illumina's MiSeq platform., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.

Background: Inferring operon maps is crucial to understanding the regulatory networks of prokaryotic genomes. Recently, RNA-seq based transcriptome studies revealed that in many bacterial species the operon structure vary with the change of environmental conditions. Therefore, new computational solutions that use both static and dynamic data are necessary to create condition specific operon predictions., Results: In this work, we propose a novel classification method that integrates RNA-seq based transcriptome profiles with genomic sequence features to accurately identify the operons that are expressed under a measured condition. The classifiers are trained on a small set of confirmed operons and then used to classify the remaining gene pairs of the organism studied. Finally, by linking consecutive gene pairs classified as operons, our computational approach produces condition-dependent operon maps. We evaluated our approach on various RNA-seq expression profiles of the bacteria Haemophilus somni, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica. Our results demonstrate that, using features depending on both transcriptome dynamics and genome sequence characteristics, we can identify operon pairs with high accuracy. Moreover, the combination of DNA sequence and expression data results in more accurate predictions than each one alone., Conclusion: We present a computational strategy for the comprehensive analysis of condition-dependent operon maps in prokaryotes. Our method can be used to generate condition specific operon maps of many bacterial organisms for which high-resolution transcriptome data is available.

Background: Gene expression in Escherichia coli is regulated by several mechanisms. We measured in single cells the expression level of a single copy gene coding for green fluorescent protein (GFP), integrated into the genome and driven by a tetracycline inducible promoter, for varying induction strengths. Also, we measured the transcriptional activity of a tetracycline inducible promoter controlling the transcription of a RNA with 96 binding sites for MS2-GFP., Results: The distribution of GFP levels in single cells is found to change significantly as induction reaches high levels, causing the Fano factor of the cells' protein levels to increase with mean level, beyond what would be expected from a Poisson-like process of RNA transcription. In agreement, the Fano factor of the cells' number of RNA molecules target for MS2-GFP follows a similar trend. The results provide evidence that the dynamics of the promoter complex formation, namely, the variability in its duration from one transcription event to the next, explains the change in the distribution of expression levels in the cell population with induction strength., Conclusions: The results suggest that the open complex formation of the tetracycline inducible promoter, in the regime of strong induction, affects significantly the dynamics of RNA production due to the variability of its duration from one event to the next.

Background: A gene network's capacity to process information, so as to bind past events to future actions, depends on its structure and logic. From previous and new microarray measurements in Saccharomyces cerevisiae following gene deletions and overexpressions, we identify a core gene regulatory network (GRN) of functional interactions between 328 genes and the transfer functions of each gene. Inferred connections are verified by gene enrichment., Results: We find that this core network has a generalized clustering coefficient that is much higher than chance. The inferred Boolean transfer functions have a mean p-bias of 0.41, and thus similar amounts of activation and repression interactions. However, the distribution of p-biases differs significantly from what is expected by chance that, along with the high mean connectivity, is found to cause the core GRN of S. cerevisiae's to have an overall sensitivity similar to critical Boolean networks. In agreement, we find that the amount of information propagated between nodes in finite time series is much higher in the inferred core GRN of S. cerevisiae than what is expected by chance., Conclusions: We suggest that S. cerevisiae is likely to have evolved a core GRN with enhanced information propagation among its genes.

We present a delayed stochastic model of transcription at the single nucleotide level. The model accounts for the promoter open complex formation and includes alternative pathways to elongation, namely pausing, arrest, misincorporation and editing, pyrophosphorolysis, and premature termination. We confront the dynamics of this detailed model with a single-step multi-delayed stochastic model and with measurements of expression of a repressed gene at the single molecule level. At low expression rates both models match the experiments but, at higher rates the two models differ significantly, with consequences to cell-to-cell phenotypic variability. The alternative pathway reactions, due to, for example, causing polymerases to collide more often on the template, are the cause for the difference in dynamical behaviors. Next, we confront the model with measurements of the transcriptional dynamics at the single RNA level of an induced gene and show that RNA production, besides its bursting dynamics, also exhibits pulses (2 or more RNAs produced in intervals smaller than the smallest interval between initiations). The distribution of occurrences and amplitudes of pulses match the experimental measurements. This pulsing and the noise at the elongation stage are shown to play a role in the dynamics of a genetic switch.

Background: There is an increasing interest to model biochemical and cell biological networks, as well as to the computational analysis of these models. The development of analysis methodologies and related software is rapid in the field. However, the number of available models is still relatively small and the model sizes remain limited. The lack of kinetic information is usually the limiting factor for the construction of detailed simulation models., Results: We present a computational toolbox for generating random biochemical network models which mimic real biochemical networks. The toolbox is called Random Models for Biochemical Networks. The toolbox works in the Matlab environment, and it makes it possible to generate various network structures, stoichiometries, kinetic laws for reactions, and parameters therein. The generation can be based on statistical rules and distributions, and more detailed information of real biochemical networks can be used in situations where it is known. The toolbox can be easily extended. The resulting network models can be exported in the format of Systems Biology Markup Language., Conclusion: While more information is accumulating on biochemical networks, random networks can be used as an intermediate step towards their better understanding. Random networks make it possible to study the effects of various network characteristics to the overall behavior of the network. Moreover, the construction of artificial network models provides the ground truth data needed in the validation of various computational methods in the fields of parameter estimation and data analysis.

Motivation: Simulation of dynamic biochemical systems is receiving considerable attention due to increasing availability of experimental data of complex cellular functions. Numerous simulation tools have been developed for numerical simulation of the behavior of a system described in mathematical form. However, there exist only a few evaluation studies of these tools. Knowledge of the properties and capabilities of the simulation tools would help bioscientists in building models based on experimental data., Results: We examine selected simulation tools that are intended for the simulation of biochemical systems. We choose four of them for more detailed study and perform time series simulations using a specific pathway describing the concentration of the active form of protein kinase C. We conclude that the simulation results are convergent between the chosen simulation tools. However, the tools differ in their usability, support for data transfer to other programs and support for automatic parameter estimation. From the experimentalists' point of view, all these are properties that need to be emphasized in the future.