The Computational Structure of Spike Trains

Neurons perform computations, and convey the results of those computations through the statistical structure of their output spike trains. Here we present a practical method, grounded in the information-theoretic analysis of prediction, for inferring a minimal representation of that structure and for characterizing its complexity. Starting from spike trains, our approach finds their causal state models (CSMs), the minimal hidden Markov models or stochastic automata capable of generating statistically identical time series. We then use these CSMs to objectively quantify both the generalizable structure and the idiosyncratic randomness of the spike train. Specifically, we show that the expected algorithmic information content (the information needed to describe the spike train exactly) can be split into three parts describing (1) the time-invariant structure (complexity) of the minimal spike-generating process, which describes the spike train statistically; (2) the randomness (internal entropy rate) of the minimal spike-generating process; and (3) a residual pure noise term not described by the minimal spike-generating process. We use CSMs to approximate each of these quantities. The CSMs are inferred nonparametrically from the data, making only mild regularity assumptions, via the causal state splitting reconstruction algorithm. The methods presented here complement more traditional spike train analyses by describing not only spiking probability and spike train entropy, but also the complexity of a spike train's structure. We demonstrate our approach using both simulated spike trains and experimental data recorded in rat barrel cortex during vibrissa stimulation.Comment: Somewhat different format from journal version but same conten

Striving for sustainable biosynthesis:discovery, diversification, and production of antimicrobial drugs in<i> Escherichia coli</i>

New antimicrobials need to be discovered to fight the advance of multidrug-resistant pathogens. A promising approach is the screening for antimicrobial agents naturally produced by living organisms. As an alternative to studying the native producer, it is possible to use genetically tractable microbes as heterologous hosts to aid the discovery process, facilitate product diversification through genetic engineering, and ultimately enable environmentally friendly production. In this mini-review, we summarize the literature from 2017 to 2022 on the application of Escherichia coli and E. coli-based platforms as versatile and powerful systems for the discovery, characterization, and sustainable production of antimicrobials. We highlight recent developments in high-throughput screening methods and genetic engineering approaches that build on the strengths of E. coli as an expression host and that led to the production of antimicrobial compounds. In the last section, we briefly discuss new techniques that have not been applied to discover or engineer antimicrobials yet, but that may be useful for this application in the future

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts

The bioprospecting of secondary metabolites from endophytic fungi received great attention in the 1990s and 2000s, when the controversy around taxol production from Taxus spp. endophytes was at its height. Since then, hundreds of reports have described the isolation and characterization of putative secondary metabolites from endophytic fungi. However, only very few studies also report the genetic basis for these phenotypic observations. With low sequencing cost and fast sample turnaround, genetics- and genomics-based approaches have risen to become comprehensive approaches to study natural products from a wide-range of organisms, especially to elucidate underlying biosynthetic pathways. However, in the field of fungal endophyte biology, elucidation of biosynthetic pathways is still a major challenge. As a relatively poorly investigated group of microorganisms, even in the light of recent efforts to sequence more fungal genomes, such as the 1000 Fungal Genomes Project at the Joint Genome Institute (JGI), the basis for bioprospecting of enzymes and pathways from endophytic fungi is still rather slim. In this review we want to discuss the current approaches and tools used to associate phenotype and genotype to elucidate biosynthetic pathways of secondary metabolites in endophytic fungi through the lens of bioprospecting. This review will point out the reported successes and shortcomings, and discuss future directions in sampling, and genetics and genomics of endophytic fungi. Identifying responsible biosynthetic genes for the numerous secondary metabolites isolated from endophytic fungi opens the opportunity to explore the genetic potential of producer strains to discover novel secondary metabolites and enhance secondary metabolite production by metabolic engineering resulting in novel and more affordable medicines and food additives

Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli

O-Methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ∼30 enzymes against five substrates in 3 days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus far uncharacterized O-methyltransferases for future use as biocatalysts

Artemisinin-Type Drugs in Tumor Cell Death:Mechanisms, Combination Treatment with Biologics and Nanoparticle Delivery

Artemisinin, the most famous anti-malaria drug initially extracted from Artemisia annua L., also exhibits anti-tumor properties in vivo and in vitro. To improve its solubility and bioavailability, multiple derivatives have been synthesized. However, to reveal the anti-tumor mechanism and improve the efficacy of these artemisinin-type drugs, studies have been conducted in recent years. In this review, we first provide an overview of the effect of artemisinin-type drugs on the regulated cell death pathways, which may uncover novel therapeutic approaches. Then, to overcome the shortcomings of artemisinin-type drugs, we summarize the recent advances in two different therapeutic approaches, namely the combination therapy with biologics influencing regulated cell death, and the use of nanocarriers as drug delivery systems. For the former approach, we discuss the superiority of combination treatments compared to monotherapy in tumor cells based on their effects on regulated cell death. For the latter approach, we give a systematic overview of nanocarrier design principles used to deliver artemisinin-type drugs, including inorganic-based nanoparticles, liposomes, micelles, polymer-based nanoparticles, carbon-based nanoparticles, nanostructured lipid carriers and niosomes. Both approaches have yielded promising findings in vitro and in vivo, providing a strong scientific basis for further study and upcoming clinical trials

Genomic and metabolomic analysis of the endophytic fungus <i>Fusarium</i> sp. VM-40 isolated from the medicinal plant <i> Vinca minor</i>

The genus Fusarium is well-known to comprise many pathogenic fungi that affect cereal crops worldwide, causing severe damage to agriculture and the economy. In this study, an endophytic fungus designated Fusarium sp. VM-40 was isolated from a healthy specimen of the traditional European medicinal plant Vinca minor. Our morphological characterization and phylogenetic analysis reveal that Fusarium sp. VM-40 is closely related to Fusarium paeoniae, belonging to the F. tricinctum species complex (FTSC), the genomic architecture and secondary metabolite profile of which have not been investigated. Thus, we sequenced the whole genome of Fusarium sp. VM-40 with the new Oxford Nanopore R10.4 flowcells. The assembled genome is 40 Mb in size with a GC content of 47.72%, 15 contigs (≥50,000 bp; N 50~4.3 Mb), and 13,546 protein-coding genes, 691 of which are carbohydrate-active enzyme (CAZyme)-encoding genes. We furthermore predicted a total of 56 biosynthetic gene clusters (BGCs) with antiSMASH, 25 of which showed similarity with known BGCs. In addition, we explored the potential of this fungus to produce secondary metabolites through untargeted metabolomics. Our analyses reveal that this fungus produces structurally diverse secondary metabolites of potential pharmacological relevance (alkaloids, peptides, amides, terpenoids, and quinones). We also employed an epigenetic manipulation method to activate cryptic BGCs, which led to an increased abundance of several known compounds and the identification of several putative new compounds. Taken together, this study provides systematic research on the whole genome sequence, biosynthetic potential, and metabolome of the endophytic fungus Fusarium sp. VM-40. </p

Heterologous Naringenin Production in the Filamentous Fungus Penicillium rubens

Naringenin is a natural product with several reported bioactivities and is the key intermediate for the entire class of plant flavonoids. The translation of flavonoids into modern medicine as pure compounds is often hampered by their low abundance in nature and their difficult chemical synthesis. Here, we investigated the possibility to use the filamentous fungus Penicillium rubens as a host for flavonoid production. P. rubens is a well-characterized, highly engineered, traditional "workhorse" for the production of β-lactam antibiotics. We integrated two plant genes encoding enzymes in the naringenin biosynthesis pathway into the genome of the secondary metabolite-deficient P. rubens 4xKO strain. After optimization of the fermentation conditions, we obtained an excellent molar yield of naringenin from fed p-coumaric acid (88%) with a titer of 0.88 mM. Along with product accumulation over 36 h, however, we also observed rapid degradation of naringenin. Based on high-resolution mass spectrometry analysis, we propose a naringenin degradation pathway in P. rubens 4xKO, which is distinct from other flavonoid-converting pathways reported in fungi. Our work demonstrates that P. rubens is a promising host for recombinant flavonoid production, and it represents an interesting starting point for further investigation into the utilization of plant biomass by filamentous fungi. </p