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For decades, host cell proteins (HCPs) have been investigated as putative contaminants in downstream processing of biopharmaceutical products of Chinese hamster ovary (CHO) cells. However, little is still known about the composition of the entire protein and vesicle environment in CHO cultivations. Ever evolving mass spectrometry techniques allow more and more insights into cell-cell communication processes and the composition of extracellular matrix, proteases, and further actively segregated compounds such as extracellular vesicles (EVs). EVs themselves are a heterologous group consisting of exosomes, ectosomes, and apoptotic vesicles. To specifically analyze these subsets of the secretome and determine beneficial and detrimental factors for a production process, targeted separation and purification techniques are necessary.In this chapter, we present our optimized workflows for a clear differentiation between directly secreted proteins and the vesicular protein content of different fractions (especially exosomal small EVs) from CHO cell supernatant for proteomic analysis by NanoLC ESI-MS., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum- a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization ( eut ) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes and BMC formation are highly increased in wild-type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM 1 , EutM 2 , and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN , eutL / eutM 1 / eutM 2 , or eutL / eutM 1 / eutM 2 / eutN not only affected BMC formation but also ethanolamine utilization, causing cell growth defects with ethanolamine as a nutrient. BMCs are also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that the BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence., Importance: The oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, thereby promoting the development of colorectal cancer and inducing preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

A comprehensive understanding of microbial community dynamics is fundamental to the advancement of environmental microbiology, human health, and biotechnology. Metaproteomics, defined as the analysis of all proteins present within a microbial community, provides insights into these complex systems. Microbial adaptation and activity depend to an important extent on newly synthesized proteins (nP), however, the distinction between nP and bulk proteins is challenging. The application of BONCAT with click chemistry has demonstrated efficacy in the enrichment of nP in pure cultures for proteomics. However, the transfer of this technique to microbial communities and metaproteomics has proven challenging and thus it has not not been used on microbial communities before. To address this, a new workflow with efficient and specific nP enrichment was developed using a laboratory-scale mixture of labelled Escherichia coli and unlabeled yeast. This workflow was then successfully applied to an anaerobic microbial community with initially low bioorthogonal non-canonical amino acid tagging efficiency. A substrate shift from glucose to ethanol selectively enriched nP with minimal background. The identification of bifunctional alcohol dehydrogenase and a syntrophic interaction between an ethanol-utilizing bacterium and two methanogens (hydrogenotrophic and acetoclastic) demonstrates the potential of metaproteomics targeting nP to trace microbial activity in complex microbial communities., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Plant-derived triterpenoids are in high demand due to their valuable applications in cosmetic, nutraceutical, and pharmaceutical industries. To meet this demand, microbial production of triterpenoids is being developed for large-scale production. However, a prominent limitation of microbial synthesis is the intracellular accumulation, requiring cell disruption during downstream processing. Destroying the whole-cell catalyst drives up production costs and limits productivity and product yield per cell. Here, in situ product extraction of triterpenoids into a second organic phase was researched to address this limitation. An organic solvent screening identified water-immiscible isopropyl myristate as a suitable in situ extractant, enabling extraction of up to 90% of total triterpenoids from engineered Saccharomyces cerevisiae. Combining isopropyl myristate and β-cyclodextrins improved extraction efficiency. In a first configuration, repeated batch fermentation with sequential product extraction and cell recycling resulted in 1.8 times higher production than a reference fermentation without in situ product extraction. In the second configuration, yeast cells were in contact with the second organic phase throughout a fed-batch fermentation to continuously extract triterpenoids. This resulted in 90% product extraction and an extended production phase. Further improvement of triterpenoid production was not achieved due to microbial host limitations uncovered through omics analyses., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

A bacterial strain was isolated from pathogenic lesions of Acer campestre tree leaves from the Teutoburg Forest in North Rhine-Westphalia, Germany, by culture on non-selective agar plates. 16S rRNA sequencing revealed 100% similarity to Sanguibacter keddieii and Sanguibacter inulinus , as well as 99% similarity to Sanguibacter gelidistatuariae and Sanguibacter antarcticus . Here, we used genome-based taxonomy with the Type (Strain) Genome Server (TYGS), which suggests the isolation of a novel prokaryotic strain. According to TYGS-analysis, using whole genome digital DNA-DNA hybridization, only 65.5% similarity to the closest relative S. inulinus was revealed, suggesting a novel species. Growth was observed at both aerobic and anaerobic conditions. Bacterial cells depicted coryneform motile rods, with a length of 1.1-3.3 µm and a constant diameter of 0.5 µm. Cells did not form spores under the tested conditions and stain Gram-positive. Growth occurred between 0.5 and 4% NaCl (optimal: 1%), at pH 5.5-9.5 (optimal: 8.0-9.0). The strain was mesophilic with an optimal growth at 25   °C. Major cellular fatty acids of the novel strain were anteiso-C 15 : 0 and C 16 : 0 .

Understanding the genetic and fitness consequences of anthropogenic bottlenecks is crucial for biodiversity conservation. However, studies of bottlenecked populations combining genomic approaches with fitness data are rare. Theory predicts that severe bottlenecks deplete genetic diversity, exacerbate inbreeding depression and decrease population viability. However, actual outcomes are complex and depend on how a species' unique demography affects its genetic load. We used population genetic and veterinary pathology data, demographic modelling, whole-genome resequencing and forward genetic simulations to investigate the genomic and fitness consequences of a near-extinction event in the northern elephant seal. We found no evidence of inbreeding depression within the contemporary population for key fitness components, including body mass, blubber thickness and susceptibility to parasites and disease. However, we detected a genomic signature of a recent extreme bottleneck (effective population size = 6; 95% confidence interval = 5.0-7.5) that will have purged much of the genetic load, potentially leading to the lack of observed inbreeding depression in our study. Our results further suggest that deleterious genetic variation strongly impacted the post-bottleneck population dynamics of the northern elephant seal. Our study provides comprehensive empirical insights into the intricate dynamics underlying species-specific responses to anthropogenic bottlenecks., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Motivation: Using a single linear reference genome poses a limitation to exploring the full genomic diversity of a species. The release of a draft human pangenome underscores the increasing relevance of pangenomics to overcome these limitations. Pangenomes are commonly represented as graphs, which can represent billions of base pairs of sequence. Presently, there is a lack of scalable software able to perform key tasks on pangenomes, such as quantifying universally shared sequence across genomes (the core genome) and measuring the extent of genomic variability as a function of sample size (pangenome growth)., Results: We introduce Panacus (pangenome-abacus), a tool designed to rapidly perform these tasks and visualize the results in interactive plots. Panacus can process GFA files, the accepted standard for pangenome graphs, and is able to analyze a human pangenome graph with 110 million nodes in <1 h., Availability and Implementation: Panacus is implemented in Rust and is published as Open Source software under the MIT license. The source code and documentation are available at https://github.com/marschall-lab/panacus. Panacus can be installed via Bioconda at https://bioconda.github.io/recipes/panacus/README.html., (© The Author(s) 2024. Published by Oxford University Press.)

Sulfolobus acidocaldarius , a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of the saci&#95;2122 promoter (P ara ), providing low basal activity and high pentose-dependent induction. However, the available expression vector does not include a 5'-terminal untranslated region (5'-UTR), a typical element found in bacterial expression vectors that usually enhances protein production in bacteria. To establish S. acidocaldarius as a production strain in biotechnology in the long term, it is intrinsically relevant to optimize its tools and capacities to increase production efficiencies. Here we show that protein production is increased by the integration of S. acidocaldarius 5'-UTRs into P ara expression plasmids. Using the esterase Saci&#95;1116 as a reporter protein, we observed a four-fold increase in soluble and active protein yield upon insertion of the saci&#95;1322 ( alba ) 5'-UTR. Screening of four additional 5'-UTRs from other highly abundant proteins ( thα , slaA , slaB, saci&#95;0330 ) revealed a consistent enhancement in target protein production. Additionally, site-directed mutagenesis of the Shine-Dalgarno (SD) motif within the alba 5'-UTR revealed its significance for protein synthesis. Ultimately, the alba 5'-UTR optimized expression vector improved the expression of various proteins, including six glycosyltransferases and one hydroxyacyl-CoA dehydratase from S. acidocaldarius , and a malto-oligosyltrehalose trehalohydrolase from Saccharolobus solfataricus , demonstrating its applicability. Our results show that the integration of SD-motif containing 5'-UTRs significantly enhanced plasmid-based protein production in S. acidocaldarius . This advancement in recombinant expression not only broadens the utility of S. acidocaldarius as an archaeal expression platform but also marks an important step toward potential biotechnological applications., 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 Kuschmierz, Wagner, Schmerling, Busche, Kalinowski, Bräsen and Siebers.)

This study aimed to reveal interactions of the stress response sigma subunits (factors) σ D and σ H of RNA polymerase and promoters in Gram-positive bacterium Corynebacterium glutamicum by combining wet-lab obtained data and in silico modeling. Computer modeling-guided point mutagenesis of C. glutamicum σ H subunit led to the creation of a panel of σ H variants. Their ability to initiate transcription from naturally occurring hybrid σ D /σ H -dependent promoter Pcg0441 and two control canonical promoters (σ D -dependent PrsdA and σ H -dependent PuvrD3) was measured and interpreted using molecular dynamics simulations of homology models of all complexes. The results led us to design the artificial hybrid promoter PD 35 H 10 combining the -10 element of the PuvrD3 promoter and the -35 element of the PrsdA promoter. This artificial hybrid promoter PD 35-rsdA H 10-uvrD3 showed almost optimal properties needed for the bio-orthogonal transcription (not interfering with the native biological processes)., Competing Interests: Declarations. Conflict of interests: The authors declare that this work was carried out in the absence of any personal, professional, or financial relationships that could potentially be construed as a conflict of interest. Data Availability: No datasets were generated or analysed during the current study., (© 2024. The Author(s).)

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup-, while only one Hup-  Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a, and 3b according to Søndergaard, Pedersen, and Greening (HydDB: a web tool for hydrogenase classification and analysis. Sci Rep 2016;6:34212. https://doi.org/10.1038/srep34212.); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h, and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1 h and group 3b are not., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Background: Evolutionary divergence and genetic variation are often linked to differences in microbial community structure and diversity. While environmental factors and diet heavily influence gut microbial communities, host species contributions are harder to quantify. Closely related species living in sympatry provide a unique opportunity to investigate species differences without the confounding effects of habitat and dietary variation. We therefore compared and contrasted the gut microbiota of three sympatric plover species: the widespread Kittlitz's and white-fronted plovers (Anarhynchus pecuarius and A. marginatus) and the endemic and vulnerable Madagascar plover (A. thoracicus)., Results: We found no significant differences in the beta diversity (composition) of the gut microbiota of the three species. However, A. thoracicus exhibited higher intraspecific compositional similarity (i.e. lower pairwise distances) than the other two species; this pattern was especially pronounced among juveniles. By contrast, microbial alpha diversity varied significantly among the species, being highest in A. pecuarius, intermediate in A. marginatus and lowest in A. thoracicus. This pattern was again stronger among juveniles. Geographical distance did not significantly affect the composition of the gut microbiota, but genetic relatedness did., Conclusion: While patterns of microbial diversity varied across species, the lack of compositional differences suggests that habitat and diet likely exert a strong influence on the gut microbiota of plovers. This may be enhanced by their precocial, ground-dwelling nature, which could facilitate the horizontal transmission of microbes from the environment. We hypothesise that gut microbiota diversity in plovers primarily reflects the ecological pool of microbiota, which is subsequently modified by host-specific factors including genetics. The reduced microbial and genetic diversity of the endemic A. thoracicus may hinder its ability to adapt to environmental changes, highlighting the need for increased conservation efforts for this vulnerable species., Competing Interests: Declarations. Ethics approval and consent to participate: Sampling of the three species of Madagascar plovers was approved by the Direction des Aires Protégées, des Ressources Naturellement Renouvelables et des Ecosystèmes from Madagascar (permits n $$^{\circ }$$ ∘ 386/21 and 054/21/MEDD/SG/DGGE/DAPRNE/SCBE.Re). Consent for publication: Not applicable. Completing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Photosynthetic organisms often interact with heterotrophic microbes. We here report the complete genome sequences of the bacterial strains Asticcacaulis sp. MM231 and Brevundimonas subvibrioides MM232. Both bacteria were co-isolated from a single green colony originating from an aquatic sample taken from a pond at the Bielefeld University campus., Competing Interests: The authors declare no conflict of interest.

We cultivated bacteria contained in a sandy soil sample, isolated DNA from a single bacterial colony, and assembled from genomic reads the full genome sequence of Chitinophaga and Microbacterium strains, termed MM2321 and MM2322. Besides the genome sequences, the phylogenetic classifications of both strains are reported., Competing Interests: The authors declare no conflict of interest.

The Antarctic fur seal (Arctocephalus gazella) is an important top predator and indicator of the health of the Southern Ocean ecosystem. Although abundant, this species narrowly escaped extinction due to historical sealing and is currently declining as a consequence of climate change. Genomic tools are essential for understanding these anthropogenic impacts and for predicting long-term viability. However, the current reference genome ("arcGaz3") shows considerable room for improvement in terms of both completeness and contiguity. We therefore combined PacBio sequencing, haplotype-aware HiRise assembly, and scaffolding based on Hi-C information to generate a refined assembly of the Antarctic fur seal reference genome ("arcGaz4&#95;h1"). The new assembly is 2.53 Gb long, has a scaffold N50 of 55.6 Mb and includes 18 chromosome-sized scaffolds, which correspond to the 18 chromosomes expected in otariids. Genome completeness is greatly improved, with 23,408 annotated genes and a Benchmarking Universal Single-Copy Orthologs score raised from 84.7% to 95.2%. We furthermore included the new genome in a reference-free alignment of the genomes of 11 pinniped species to characterize evolutionary conservation across the Pinnipedia using genome-wide Genomic Evolutionary Rate Profiling. We then implemented Gene Ontology enrichment analyses to identify biological processes associated with those genes showing the highest levels of either conservation or differentiation between the 2 major pinniped families, the Otariidae and Phocidae. We show that processes linked to neuronal development, the circulatory system, and osmoregulation are overrepresented both in conserved as well as in differentiated regions of the genome., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Frankia cluster-2 strains are diazotrophs that engage in root nodule symbiosis with actinorhizal plants of the Cucurbitales and the Rosales. Previous studies have shown that an assimilated nitrogen source, presumably arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), while a different metabolite is exported in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported to the host by cluster-2 strains, and which metabolite would be exported in Ceanothus, we analysed gene expression levels, metabolite profiles, and enzyme activities in nodules. We conclude that the export of assimilated nitrogen in symbiosis seems to be a common feature for Frankia cluster-2 strains, but which source is host-dependent. The export of assimilated ammonium to the host suggests that 2-oxoglutarate is drawn from the TCA cycle at a high rate. This specialised metabolism obviates the need for the reductive branch of the TCA cycle. We found several genes encoding enzymes of the central carbon and nitrogen metabolism were lacking in Frankia cluster-2 genomes: the glyoxylate shunt and succinate semialdehyde dehydrogenase. This led to a linearization of the TCA cycle, and we hypothesize this could explain the low saprotrophic potential of Frankia cluster-2., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Cellular levels of the essential micronutrient manganese (Mn) need to be carefully balanced within narrow borders. In cyanobacteria, a sufficient Mn supply is critical for ensuring the function of the oxygen-evolving complex as the central part of the photosynthetic machinery. However, Mn accumulation is fatal for the cells. The reason for the observed cytotoxicity is unclear. To understand the causality behind Mn toxicity in cyanobacteria, we investigated the impact of excess Mn on physiology and global gene expression in the model organism Synechocystis sp. PCC 6803. We compared the response of the WT and the knock-out mutant in the Mn e x porter (Mnx), ∆ mnx , which is disabled in the export of surplus Mn and thus functions as a model for toxic Mn overaccumulation. While growth and pigment accumulation in ∆ mnx were severely impaired 24 h after the addition of tenfold Mn, the WT was not affected and thus mounted an adequate transcriptional response. RNA-seq data analysis revealed that the Mn stress transcriptomes partly resembled an iron limitation transcriptome. However, the expression of iron limitation signature genes isiABDC was not affected by the Mn treatment, indicating that Mn excess is not accompanied by iron limitation in Synechocystis . We suggest that the ferric uptake regulator, Fur, gets partially mismetallated under Mn excess conditions and thus interferes with an iron-dependent transcriptional response. To encounter mismetallation and other Mn-dependent problems on a protein level, the cells invest in transcripts of ribosomes, proteases and chaperones. In the case of the ∆ mnx mutant, the consequences of the disability to export excess Mn from the cytosol manifest in additionally impaired energy metabolism and oxidative stress transcriptomes with a fatal outcome. This study emphasizes the central importance of Mn homeostasis and the transporter Mnx's role in restoring and holding it.

Nanoscale zero valent iron (nZVI) is used to remediate aquifers polluted by organochlorines or heavy metals and was also suggested to eliminate harmful algal blooms. nZVI can therefore affect microorganisms in the vicinity of the application area, including microalgae. However, studies on early transcriptomic effects of microalgae after exposure to nZVI are rare. Here, we described the early physiological and transcriptomic response of the freshwater ecological indicator green microalga, Raphidocelis subcapitata ATCC 22662, to 100 mg/L of reactive nZVI and non-reactive nano-magnetite (nFe 3 O 4 ). The combined effect of shading and the release of total iron from nZVI posed a short-term inhibition effect leading to 15 % of deformed cells and cytosol leakage, while cells viability increased after 24 h. nZVI triggered a more pronounced transcriptomic response with (7380 differentially expressed genes [DEGs]) compared to nFe 3 O 4 (4601 DEGs) after 1 h. nZVI, but not nFe 3 O 4 increased the expression of genes function in DNA repair and replication, while deactivated carbohydrate-energy metabolisms, mitochondria signaling, and transmembrane ion transport. This study highlights an early fate assessment of algal cells under nZVI and nFe 3 O 4 exposure using next-generation risk assessment methods and will serve as valuable information for safe and sustainable application of nZVI in water remediation., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Process intensification has become an important strategy to lower production costs and increase manufacturing capacities for biopharmaceutical products. In particular for the production of viral vectors like lentiviruses (LVs), the transition from (fed-)batch to perfusion processes is a key strategy to meet the increasing demands for cell and gene therapy applications. However, perfusion processes are associated with higher medium consumption. Therefore, it is necessary to develop appropriate small-scale models to reduce development costs. In this work, we present the use of the acoustic wave separation technology in combination with the Ambr 250 high throughput bioreactor system for intensified perfusion process development using stable LV producer cells. The intensified perfusion process developed in the Ambr 250 model, performed at a harvest rate of 3 vessel volumes per day (VVD) and high cell densities, resulted in a 1.4-fold higher cell-specific functional virus yield and 2.8-fold higher volumetric virus yield compared to the control process at a harvest rate of 1 VVD. The findings were verified at bench scale after optimizing the bioreactor set-up, resulting in a 1.4-fold higher cell-specific functional virus yield and 3.1-fold higher volumetric virus yield., (© 2024 CSL Innovation and The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore, new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion, proliferation stop, diminished tumor-initiating potential in immunodeficient mice, and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa, we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Indeed, inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue, distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation, indicating that targeted differentiation approaches may be therapeutically valuable. Moreover, we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection., (© 2024. The Author(s).)

Plasmids are a key vector of antibiotic resistance, but the current bioinformatics toolkit is not well suited to tracking them. The rapid structural changes seen in plasmid genomes present considerable challenges to evolutionary and epidemiological analysis. Typical approaches are either low resolution (replicon typing) or use shared k-mer content to define a genetic distance. However, this distance can both overestimate plasmid relatedness by ignoring rearrangements, and underestimate by over-penalizing gene gain/loss. Therefore a model is needed which captures the key components of how plasmid genomes evolve structurally - through gene/block gain or loss, and rearrangement. A secondary requirement is to prevent promiscuous transposable elements (TEs) leading to over-clustering of unrelated plasmids. We choose the 'Double Cut and Join Indel' (DCJ-Indel) model, in which plasmids are studied at a coarse level, as a sequence of signed integers (representing genes or aligned blocks), and the distance between two plasmids is the minimum number of rearrangement events or indels needed to transform one into the other. We show how this gives much more meaningful distances between plasmids. We introduce a software workflow pling (https://github.com/iqbal-lab-org/pling), which uses the DCJ-Indel model, to calculate distances between plasmids and then cluster them. In our approach, we combine containment distances and DCJ-Indel distances to build a TE-aware plasmid network. We demonstrate superior performance and interpretability to other plasmid clustering tools on the 'Russian Doll' dataset and a hospital transmission dataset.

Tritrichomonas foetus is a parasitic protist responsible for bovine trichomonosis, a reproductive disease associated with significant economic burden to the livestock industry throughout the world. Here, we present a chromosome-level reference genome of T. foetus -KV-1 (ATCC 30924) using short-read (Illumina Miseq), long-read (Oxford Nanopore) and chromatin-linked (Hi-C) sequencing. This is the first chromosome-level genome of a parasitic protist of the order Tritrichomonadida and the second within the Parabasalia lineage, after Trichomonas vaginalis, the human-associated causative agent of the sexually transmitted infection in humans. Our constructed genome is 148 Mb in size, with a N50 length of the scaffolds of 22.9 Mb. The contigs are anchored in five super-scaffolds, corresponding to the expected five chromosomes of the species and covering 78% of the genome assembly. We predict 41,341 protein-coding genes, of which 95.10% have been functionally annotated. This high-quality genome assembly serves as a valuable reference genome for T. foetus to support future studies in functional genomics, genetic conservation and taxonomy., (© 2024. The Author(s).)

The gut microbiota-immune-brain axis is a feedback network which influences diverse physiological processes and plays a pivotal role in overall health and wellbeing. Although research in humans and laboratory mice has shed light into the associations and mechanisms governing this communication network, evidence of such interactions in wild, especially in young animals, is lacking. We therefore investigated these interactions during early development in a population of common buzzards ( Buteo buteo ) and their effects on individual condition. In a longitudinal study, we used a multi-marker approach to establish potential links between the bacterial and eukaryotic gut microbiota, a panel of immune assays and feather corticosterone measurements as a proxy for long-term stress. Using Bayesian structural equation modeling, we found no support for feedback between gut microbial diversity and immune or stress parameters. However, we did find strong relationships in the feedback network. Immunity was negatively correlated with corticosterone levels, and microbial diversity was positively associated with nestling body condition. Furthermore, corticosterone levels and eukaryotic microbiota diversity decreased with age while immune activity increased. The absence of conclusive support for the microbiota-immune-brain axis in common buzzard nestlings, coupled with the evidence for stress mediated immunosuppression, suggests a dominating role of stress-dominated maturation of the immune system during early development. Confounding factors inherent to wild systems and developing animals might override associations known from adult laboratory model subjects. The positive association between microbial diversity and body condition indicates the potential health benefits of possessing a diverse and stable microbiota., 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 Pereira, Hoffman, Krüger, Czirják, Rinaud, Ottensmann, Gladow, Caspers, Maraci, Kaiser and Chakarov.)

Novel, white-pigmented, Gram-negative bacterial strains (K-M0706 T , K-M0228, K-M0252, K-M0260) were isolated from clinical samples. With a similarity of up to 69.7 % to Serratia nevei S15 T and up to 63.8 % to Serratia marcescens ATCC 13880 T , as determined by digital DNA-DNA hybridization, the strains were assigned as novel species of the genus Serratia. The species can easily be differentiated from the red colored Serratia marcescens sensu stricto by its white phenotype. Discrimination between this strain and Serratia nevei is possible due to alpha-glucosidase activity and O/129-resistance, as shown for strain K-M0706 T . The major fatty acids were determined as myristate, palmitate, cis-9,10-methylenehexadecanoate, linoleate, and (all cis-9,10)-methyleneoctadecanoate. These phenotypical and genomic data support the assignment of a novel species within the genus Serratia, named S. sarumanii due to its pathogenicity and white phenotype, with strain K-M0706 T as the type strain., 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 © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Polar offshore environments are considered the last pristine soundscapes, but accelerating climate change and increasing human activity threaten their integrity. In order to assess the acoustic state of polar oceans, there is the need to investigate their soundscape characteristics more holistically. We apply a set of 14 ecoacoustic metrics (EAMs) to identify which metrics are best suited to reflect the characteristics of disturbed and naturally intact polar offshore soundscapes. We used two soundscape datasets: (i) the Arctic eastern Fram Strait (FS), which is already impacted by anthropogenic noise, and (ii) the quasi-pristine Antarctic Weddell Sea (WS). Our results show that EAMs when applied in concert can be used to quantitatively assess soundscape variability, enabling the appraisal of marine soundscapes over broad spatiotemporal scales. The tested set of EAMs was able to show that the eastern FS, which is virtually free from sea ice, lacks seasonal soundscape dynamics and exhibits low acoustic complexity owing to year-round wind-mediated sounds and anthropogenic noise. By contrast, the WS exhibits pronounced seasonal soundscape dynamics with greater soundscape heterogeneity driven in large part by the vocal activity of marine mammal communities, whose composition in turn varies with the prevailing seasonal sea ice conditions., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

Inbreeding, the mating of individuals that are related through common ancestry, is of central importance in evolutionary and conservation biology due to its impacts on individual fitness and population dynamics. However, while advanced genomic approaches have revolutionised the study of inbreeding in animals, genomic studies of inbreeding are rare in plants and lacking in fungi. We investigated global patterns of inbreeding in the prized edible porcini mushroom Boletus edulis using 225 whole genomes from seven lineages distributed across the northern hemisphere. Genomic inbreeding was quantified using runs of homozygosity (ROHs). We found appreciable variation both among and within lineages, with some individuals having over 20% of their genomes in ROHs. Much of this variation could be explained by a combination of elevation and latitude, and to a lesser extent by predicted habitat suitability during the last glacial maximum. In line with this, the majority of ROHs were short, reflecting ancient common ancestry dating back approximately 200-1700 generations ago, while longer ROHs indicative of recent common ancestry (less than approximately 50 generations ago) were infrequent. Our study reveals the inbreeding legacy of major climatic events in a widely distributed forest mutualist, aligning with prevailing theories and empirical studies of the impacts of historical glaciation events on the dominant forest tree species of the northern hemisphere., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

The accelerated spread of antimicrobial-resistant bacteria has caused a serious health problem and rendered antimicrobial treatments ineffective. Innovative approaches are crucial to overcome the health threat posed by resistant pathogens and prevent the emergence of untreatable infections. Triggering stress responses in bacteria can diminish susceptibility to various antimicrobials by inducing resistance mechanisms. Therefore, a thorough understanding of stress response control, especially in relation to antimicrobial resistance, offers valuable perspectives for innovative and efficient therapeutic approaches to combat antimicrobial resistance. The aim of this study was to evaluate the stress responses of 8 different bacteria by analyzing reporter metabolites, around which significant alterations were observed, using a pathway-driven computational approach. For this purpose, the transcriptomic data that the bacterial pathogens were grown under 11 different stress conditions mimicking the human host environments were integrated with the genome-scale metabolic models of 8 pathogenic species (Enterococcus faecalis OG1R, Escherichia coli EPEC O127:H6 E2348/69, Escherichia coli ETEC H10407, Escherichia coli UPEC 536, Klebsiella pneumoniae MGH 78578, Pseudomonas aeruginosa PAO1, Staphylococcus aureus MRSA252, and Staphylococcus aureus MSSA476). The resulting reporter metabolites were enriched in multiple metabolic pathways, with cofactor biosynthesis being the most important. The results of this study will serve as a guide for the development of antimicrobial agents as they provide a first insight into potential drug targets., (© 2024. The Author(s).)

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease, and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed, and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells, we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions, including irregularly enlarged cell morphology, cell cycle arrest with downregulation of Ki67, and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation, and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable., (© 2024. The Author(s).)

Oranges are the most processed fruit in the world-it is therefore apparent that the industrial production of orange juice generates large quantities of orange peel as a by-product. Unfortunately, the management of the orange peel waste leads to economic and environmental problems. Meanwhile, the use of sustainable raw materials for the production of bulk chemicals, such as amino acids, is becoming increasingly attractive. To address both issues, this study focused on the use of orange peel waste as a raw material for media preparation for the production of amino acids by engineered Corynebacterium glutamicum . C. glutamicum grew on pure orange peel hydrolysate (OPH) and growth was enhanced by the addition of a nitrogen source and a pH buffer. Inhibitory effects by the combination of high concentrations of OPH, (NH 4 ) 2 SO 4 , and MOPS buffer in the wild-type strain (WT), were overcome in the tyrosine-producing engineered C. glutamicum strain AROM3. Genetic modifications that we identified to allow for improved growth rates under these conditions included the deletions of the vanillin dehydrogenase gene vdh , the ʟ-lactate dehydrogenase gene ldhA and the 19 genes comprising cluster cg2663-cg2686. A growth inhibiting compound present in high concentrations in the OPH is 5-(hydroxymethyl)furfural (HMF). We identified vdh as being primarily responsible for the oxidation of HMF to its acid 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), as the formation of HMFCA was reduced by 97% upon deletion of vdh in C. glutamicum WT. In addition, we showed that growth limitations could be overcome by adjusting the media preparation, using a combination of cheap ammonia water and KOH for pH neutralization after acidic hydrolysis. Overall, we developed a sustainable medium based on orange peel waste for the cultivation of C. glutamicum and demonstrated the successful production of the exemplary amino acids ʟ-arginine, ʟ-lysine, ʟ-serine, ʟ-valine and ʟ-tyrosine., 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 Junker, Sariyar Akbulut and Wendisch.)

Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50&#95;0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σH Sc . Therefore, the protein encoded by ACSP50&#95;0507 was named σH As . Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50&#95;0507 ( sigH As frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its As frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σH As was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σH As to the upstream sequence of a strongly upregulated gene. Autoregulation of σH As was observed, and binding to its own gene promoter region was also confirmed.

The use of bacteriocins is a promising approach for addressing the immense threat of food-borne and drug-resistant pathogens. In recent years screening platforms for novel bacteriocins using whole-cell biosensors have been established. During screening cell-to-cell heterogeneity is currently neglected but might play a crucial role in signal development of the whole-cell biosensor after bacteriocin exposure. In this study, we explored the temporal dynamics of the signal heterogeneity of the biosensor Listeria innocua LMG2785/pNZpHin2 Lm after nisin exposure using microfluidic single-cell analysis. The results provided novel and detailed insights into the dynamics of cell-to-cell heterogeneity in L. innocua LMG2785/pNZpHin2 Lm at different nisin concentrations with a high spatio-temporal resolution. Furthermore, the formation of subpopulations during bacteriocin exposure was observed. In-depth single-cell tracking even revealed the regeneration of disrupted cells and recovery of pH homeostasis in rare instances. These findings are highly important for the future design and execution of bacteriocin assays and for the interpretation of fluorescence signal development at the population level after exposure to different concentrations of bacteriocins (here, nisin), as well as for obtaining deeper insights into single-cell persistence strategies to quantify the efficacy and efficiency of novel bacteriocins., 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 Fante, Desiderato, Riedel and Grünberger.)

Inbreeding depression, the loss of offspring fitness due to consanguineous mating, is generally detrimental for individual performance and population viability. We investigated inbreeding effects in a declining population of Antarctic fur seals (Arctocephalus gazella) at Bird Island, South Georgia. Here, localised warming has reduced the availability of the seal's staple diet, Antarctic krill, leading to a temporal increase in the strength of selection against inbred offspring, which are increasingly failing to recruit into the adult breeding population. However, it remains unclear whether selection operates before or after nutritional independence at weaning. We therefore used microsatellite data from 885 pups and their mothers, and SNP array data from 98 mother-offspring pairs, to quantify the effects of individual and maternal inbreeding on three important neonatal fitness traits: birth mass, survival and growth. We did not find any clear or consistent effects of offspring or maternal inbreeding on any of these traits. This suggests that selection filters inbred individuals out of the population as juveniles during the time window between weaning and recruitment. Our study brings into focus a poorly understood life-history stage and emphasises the importance of understanding the ecology and threats facing juvenile pinnipeds., (© 2024. The Author(s).)

Microalgae are regarded as sustainable and potent chassis for biotechnology. Their capacity for efficient photosynthesis fuels dynamic growth independent from organic carbon sources and converts atmospheric CO 2 directly into various valuable hydrocarbon-based metabolites. However, approaches to gene expression and metabolic regulation have been inferior to those in more established heterotrophs (e.g., prokaryotes or yeast) since the genetic tools and insights in expression regulation have been distinctly less advanced. In recent years, however, these tools and their efficiency have dramatically improved. Various examples have demonstrated new trends in microalgal biotechnology and the potential of microalgae for the transition towards a sustainable bioeconomy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L -1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The Great Pacific Garbage Patch, a significant collection of plastic introduced by human activities, provides an ideal environment to study bacterial lifestyles on plastic substrates. We proposed that bacteria colonizing the floating plastic debris would develop strategies to deal with the ultraviolet-exposed substrate, such as the production of antioxidant pigments. We observed a variety of pigmentation in 67 strains that were directly cultivated from plastic pieces sampled from the Garbage Patch. The genomic analysis of four representative strains, each distinct in taxonomy, revealed multiple pathways for carotenoid production. These pathways include those that produce less common carotenoids and a cluster of photosynthetic genes. This cluster appears to originate from a potentially new species of the Rhodobacteraceae family. This represents the first report of an aerobic anoxygenic photoheterotrophic bacterium from plastic biofilms. Spectral analysis showed that the bacteria actively produce carotenoids, such as beta-carotene and beta-cryptoxanthin, and bacteriochlorophyll a. Furthermore, we discovered that the genetic ability to synthesize carotenoids is more common in plastic biofilms than in the surrounding water communities. Our findings suggest that plastic biofilms could be an overlooked source of bacteria-produced carotenoids, including rare forms. It also suggests that photoreactive molecules might play a crucial role in bacterial biofilm communities in surface water., (© 2024 The Authors. Environmental Microbiology published by John Wiley & Sons Ltd.)

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii . This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of Cr BKT, Pa crtB, and Cr CHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

Background: Exploring the dynamics of gut microbiome colonisation during early-life stages is important for understanding the potential impact of microbes on host development and fitness. Evidence from model organisms suggests a crucial early-life phase when shifts in gut microbiota can lead to immune dysregulation and reduced host condition. However, our understanding of gut microbiota colonisation in long-lived vertebrates, especially during early development, remains limited. We therefore used a wild population of common buzzard nestlings (Buteo buteo) to investigate connections between the early-life gut microbiota colonisation, environmental and host factors., Results: We targeted both bacterial and eukaryotic microbiota using the 16S and 28S rRNA genes. We sampled the individuals during early developmental stages in a longitudinal design. Our data revealed that age significantly affected microbial diversity and composition. Nest environment was a notable predictor of microbiota composition, with particularly eukaryotic communities differing between habitats occupied by the hosts. Nestling condition and infection with the blood parasite Leucocytozoon predicted microbial community composition., Conclusion: Our findings emphasise the importance of studying microbiome dynamics to capture changes occurring during ontogeny. They highlight the role of microbial communities in reflecting host health and the importance of the nest environment for the developing nestling microbiome. Overall, this study contributes to understanding the complex interplay between microbial communities, host factors, and environmental variables, and sheds light on the ecological processes governing gut microbial colonisation during early-life stages., (© 2024. The Author(s).)

Background: Given a sequencing read, the broad goal of read mapping is to find the location(s) in the reference genome that have a "similar sequence". Traditionally, "similar sequence" was defined as having a high alignment score and read mappers were viewed as heuristic solutions to this well-defined problem. For sketch-based mappers, however, there has not been a problem formulation to capture what problem an exact sketch-based mapping algorithm should solve. Moreover, there is no sketch-based method that can find all possible mapping positions for a read above a certain score threshold., Results: In this paper, we formulate the problem of read mapping at the level of sequence sketches. We give an exact dynamic programming algorithm that finds all hits above a given similarity threshold. It runs in O ( | t | + | p | + ℓ 2 ) time and O ( ℓ log ℓ ) space, where |t| is the number of k -mers inside the sketch of the reference, |p| is the number of k -mers inside the read's sketch and ℓ is the number of times that k -mers from the pattern sketch occur in the sketch of the text. We evaluate our algorithm's performance in mapping long reads to the T2T assembly of human chromosome Y, where ampliconic regions make it desirable to find all good mapping positions. For an equivalent level of precision as minimap2, the recall of our algorithm is 0.88, compared to only 0.76 of minimap2., (© 2024. The Author(s).)

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 10 7  TU mL -1  in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 10 7  TU mL -1  in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 10 7  TU mL -1  day -1  for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34 + cells, reducing the LV quantities required for gene and cell therapy applications., (© 2024 CSL Innovation GmbH and The Authors. Biotechnology Journal published by Wiley‐VCH GmbH.)