Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella.

KEY MESSAGE: Characterization of Physcomitrella 3'UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3'UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3'UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3'UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3'UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

Mosses.

Often overlooked, these small but otherwise brilliant plants began covering Earth's land masses more than 450 million years ago. They saw the dinosaurs come and go, and they saw us humans coming. Mosses, liverworts and hornworts comprise the bryophytes, the second largest monophyletic clade of land plants (embryophytes), after the vascular plants (tracheophytes). Like all embryophytes, mosses exhibit a haplodiplontic life cycle. This alternation of generations (originally termed Generationswechsel in German) between the haploid gametophyte and the diploid sporophyte implies that every plant genome encodes two distinct ontogenies, in contrast to animal genomes. Contrary to tracheophytes, the haploid gametophyte is the dominant generation in mosses. Haploidy of the major tissues facilitates gene-function annotation via reverse genetics. Nevertheless, the diploid sporophyte of mosses, the spore capsule, is a visible structure unlike the gametophyte of flowering plants, which is largely reduced and embedded in the sporophyte. Visibility of both generations on one plant facilitates the analysis of the alternation of generations, and in a broader sense evo-devo studies. Whereas the conservation of moss morphology over hundreds of millions of years suggests stasis, molecular data reveal fast evolving moss genomes, leaving an enigma for evolutionary biologists. Finally, the extraordinary resilience of mosses may provide lessons for current man-made climate change. In this Primer, we will highlight some of the peculiarities of mosses from historical observations to current genomic data, with an emphasis on their development, reproduction, evolution, biotic interactions, and potential for biotechnology. Mosses from three genera - the living fossil Takakia, the ecosystems engineer Sphagnum, and the model moss Physcomitrella - exemplify the scientific insights and the applications mosses have to offer.

Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Long-Distance Electrical and Calcium Signals Evoked by Hydrogen Peroxide in Physcomitrella.

Electrical and calcium signals in plants are some of the basic carriers of information that are transmitted over a long distance. Together with reactive oxygen species (ROS) waves, electrical and calcium signals can participate in cell-to-cell signaling, conveying information about different stimuli, e.g. abiotic stress, pathogen infection or mechanical injury. There is no information on the ability of ROS to evoke systemic electrical or calcium signals in the model moss Physcomitrella nor on the relationships between these responses. Here, we show that the external application of hydrogen peroxide (H2O2) evokes electrical signals in the form of long-distance changes in the membrane potential, which transmit through the plant instantly after stimulation. The responses were calcium-dependent since their generation was inhibited by lanthanum, a calcium channel inhibitor (2 mM), and EDTA, a calcium chelator (0.5 mM). The electrical signals were partially dependent on glutamate receptor (GLR) ion channels since knocking-out the GLR genes only slightly reduced the amplitude of the responses. The basal part of the gametophyte, which is rich in protonema cells, was the most sensitive to H2O2. The measurements carried out on the protonema expressing fluorescent calcium biosensor GCaMP3 proved that calcium signals propagated slowly (>5 µm/s) and showed a decrement. We also demonstrate upregulation of a stress-related gene that appears in a distant section of the moss 8 min after the H2O2 treatment. The results help understand the importance of both types of signals in the transmission of information about the appearance of ROS in the plant cell apoplast.

Structural modelling of human complement FHR1 and two of its synthetic derivatives provides insight into their in-vivo functions.

Human complement is the first line of defence against invading pathogens and is involved in tissue homeostasis. Complement-targeted therapies to treat several diseases caused by a dysregulated complement are highly desirable. Despite huge efforts invested in their development, only very few are currently available, and a deeper understanding of the numerous interactions and complement regulation mechanisms is indispensable. Two important complement regulators are human Factor H (FH) and Factor H-related protein 1 (FHR1). MFHR1 and MFHR13, two promising therapeutic candidates based on these regulators, combine the dimerization and C5-regulatory domains of FHR1 with the central C3-regulatory and cell surface-recognition domains of FH. Here, we used AlphaFold2 to model the structure of these two synthetic regulators. Moreover, we used AlphaFold-Multimer (AFM) to study possible interactions of C3 fragments and membrane attack complex (MAC) components C5, C7 and C9 in complex with FHR1, MFHR1, MFHR13 as well as the best-known MAC regulators vitronectin (Vn), clusterin and CD59, whose experimental structures remain undetermined. AFM successfully predicted the binding interfaces of FHR1 and the synthetic regulators with C3 fragments and suggested binding to C3. The models revealed structural differences in binding to these ligands through different interfaces. Additionally, AFM predictions of Vn, clusterin or CD59 with C7 or C9 agreed with previously published experimental results. Because the role of FHR1 as MAC regulator has been controversial, we analysed possible interactions with C5, C7 and C9. AFM predicted interactions of FHR1 with proteins of the terminal complement complex (TCC) as indicated by experimental observations, and located the interfaces in FHR11-2 and FHR14-5. According to AFM prediction, FHR1 might partially block the C3b binding site in C5, inhibiting C5 activation, and block C5b-7 complex formation and C9 polymerization, with similar mechanisms of action as clusterin and vitronectin. Here, we generate hypotheses and give the basis for the design of rational approaches to understand the molecular mechanism of MAC inhibition, which will facilitate the development of further complement therapeutics.

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention.

The auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens), three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Furthermore, the pinC mutants have a higher portion of differentially expressed genes related to spermatogenesis, increased fertility and an increased abortion rate of premeiotic sporophytes. Here, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters.

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge-network organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis.

Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.

Ralf Reski.

Interview with Ralf Reski, who works on fundamental and applied issues using moss plants.

Spindle motility skews division site determination during asymmetric cell division in Physcomitrella.

Asymmetric cell division (ACD) underlies the development of multicellular organisms. In animal ACD, the cell division site is determined by active spindle-positioning mechanisms. In contrast, it is considered that the division site in plants is determined prior to mitosis by the microtubule-actin belt known as the preprophase band (PPB) and that the localization of the mitotic spindle is typically static and does not govern the division plane. However, in some plant species, ACD occurs in the absence of PPB. Here, we isolate a hypomorphic mutant of the conserved microtubule-associated protein TPX2 in the moss Physcomitrium patens (Physcomitrella) and observe spindle motility during PPB-independent cell division. This defect compromises the position of the division site and produces inverted daughter cell sizes in the first ACD of gametophore (leafy shoot) development. The phenotype is rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we identify an active spindle-positioning mechanism that, reminiscent of acentrosomal ACD in animals, involves microtubules and actin filaments, and sets the division site in plants.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

Recombinant Spider Silk: Promises and Bottlenecks.

Spider silk threads have exceptional mechanical properties such as toughness, elasticity and low density, which reach maximum values compared to other fibre materials. They are superior even compared to Kevlar and steel. These extraordinary properties stem from long length and specific protein structures. Spider silk proteins can consist of more than 20,000 amino acids. Polypeptide stretches account for more than 90% of the whole protein, and these domains can be repeated more than a hundred times. Each repeat unit has a specific function resulting in the final properties of the silk. These properties make them attractive for innovative material development for medical or technical products as well as cosmetics. However, with livestock breeding of spiders it is not possible to reach high volumes of silk due to the cannibalistic behaviour of these animals. In order to obtain spider silk proteins (spidroins) on a large scale, recombinant production is attempted in various expression systems such as plants, bacteria, yeasts, insects, silkworms, mammalian cells and animals. For viable large-scale production, cost-effective and efficient production systems are needed. This review describes the different types of spider silk, their proteins and structures and discusses the production of these difficult-to-express proteins in different host organisms with an emphasis on plant systems.

Process Engineering of Biopharmaceutical Production in Moss Bioreactors via Model-Based Description and Evaluation of Phytohormone Impact.

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first 2 hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

Unexpected Arabinosylation after Humanization of Plant Protein N-Glycosylation.

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with ß1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human ß1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric ß1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human ß1,4-galactosyltransferase. Stable FTGT expression led to the desired ß1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of ß1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

A synthetic protein as efficient multitarget regulator against complement over-activation.

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g., COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may be alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH, including SCR 13. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as therapeutic for complement-associated diseases.

O-methylated N-glycans Distinguish Mosses from Vascular Plants.

In the animal kingdom, a stunning variety of N-glycan structures have emerged with phylogenetic specificities of various kinds. In the plant kingdom, however, N-glycosylation appears to be strictly conservative and uniform. From mosses to all kinds of gymno- and angiosperms, land plants mainly express structures with the common pentasaccharide core substituted with xylose, core α1,3-fucose, maybe terminal GlcNAc residues and Lewis A determinants. In contrast, green algae biosynthesise unique and unusual N-glycan structures with uncommon monosaccharides, a plethora of different structures and various kinds of O-methylation. Mosses, a group of plants that are separated by at least 400 million years of evolution from vascular plants, have hitherto been seen as harbouring an N-glycosylation machinery identical to that of vascular plants. To challenge this view, we analysed the N-glycomes of several moss species using MALDI-TOF/TOF, PGC-MS/MS and GC-MS. While all species contained the plant-typical heptasaccharide with no, one or two terminal GlcNAc residues (MMXF, MGnXF and GnGnXF, respectively), many species exhibited MS signals with 14.02 Da increments as characteristic for O-methylation. Throughout all analysed moss N-glycans, the level of methylation differed strongly even within the same family. In some species, methylated glycans dominated, while others had no methylation at all. GC-MS revealed the main glycan from Funaria hygrometrica to contain 2,6-O-methylated terminal mannose. Some mosses additionally presented very large, likewise methylated complex-type N-glycans. This first finding of the methylation of N-glycans in land plants mirrors the presumable phylogenetic relation of mosses to green algae, where the O-methylation of mannose and many other monosaccharides is a common trait.

Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella.

KEY MESSAGE: In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA-DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

Viral suppressor of RNA silencing in vascular plants also interferes with the development of the bryophyte Physcomitrella patens.

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR-encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB-11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant-virus interactions.

Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms.

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrium patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding Physcomitrella genes and their 87,533 annotated transcripts in a web application, physCO, for automatized optimization. A thus optimized cDNA results in about twelve times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects occur also in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.

Automated and semi-automated enhancement, segmentation and tracing of cytoskeletal networks in microscopic images: A review.

Cytoskeletal filaments are structures of utmost importance to biological cells and organisms due to their versatility and the significant functions they perform. These biopolymers are most often organised into network-like scaffolds with a complex morphology. Understanding the geometrical and topological organisation of these networks provides key insights into their functional roles. However, this non-trivial task requires a combination of high-resolution microscopy and sophisticated image processing/analysis software. The correct analysis of the network structure and connectivity needs precise segmentation of microscopic images. While segmentation of filament-like objects is a well-studied concept in biomedical imaging, where tracing of neurons and blood vessels is routine, there are comparatively fewer studies focusing on the segmentation of cytoskeletal filaments and networks from microscopic images. The developments in the fields of microscopy, computer vision and deep learning, however, began to facilitate the task, as reflected by an increase in the recent literature on the topic. Here, we aim to provide a short summary of the research on the (semi-)automated enhancement, segmentation and tracing methods that are particularly designed and developed for microscopic images of cytoskeletal networks. In addition to providing an overview of the conventional methods, we cover the recently introduced, deep-learning-assisted methods alongside the advantages they offer over classical methods.