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Introduction: Vocabulary knowledge is an essential element in language development. There is evidence of significant differences in vocabulary knowledge between deaf children and hearing peers of the same age. These differences put deaf students at a disadvantage when compared to their hearing counterparts. The aim of this study was to investigate whether certain types of words characterise the lexical difficulties of deaf students. Our starting point is that this knowledge is needed to design interventions that are adapted to the particular needs of these students for their lexical development., Methods: We evaluated the lexical comprehension of 90 Spanish students. The sample comprised 45 hearing students and 45 students with severe or profound hearing loss. Both groups were attending the later years of primary school (8 to -12-year-olds). They were tested using the Spanish Peabody Picture Vocabulary Test-III. The performance of both groups was statistically compared using percentiles and standard scores as well as a selected set of words from the test., Results: No significant differences between hearing and deaf groups were found by age, sex, and sociocultural level. Significant differences were found between groups in their percentile and standard scores. There were no significant differences in performance between the two groups on only five of the 25 words with the highest error rate in the deaf group. The qualitative analysis of the remaining 20 words that were especially challenging for deaf students reveals results of interest which could help guide interventions., Conclusions: The results indicate the need for lexical intervention for deaf students in the final years of primary school. The specialised support service for deaf learners should approach the intervention by targeting a specific type of vocabulary, making the semantic relationships between these words more transparent and promoting a deeper understanding of them., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

Fracture-related infections (FRIs), particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA), are challenging to treat. This study designed and evaluated a hydrogel loaded with a cocktail of bacteriophages and vancomycin (1.2 mg/mL). The co-delivery hydrogel showed 99.72% reduction in MRSA biofilm in vitro. The hydrogel released 54% of phages and 82% of vancomycin within 72 h and maintained activity for eight days, in vivo the co-delivery hydrogel with systemic antibiotic significantly reduced bacterial load by 0.99 log10 CFU compared to controls, with active phages detected in tissues at euthanasia (2 × 10 3 PFU/mL). No phage resistance was detected in the phage treatment groups, and serum neutralization resulted in only a 20% reduction in phage count. In this work, we show that a phage-antibiotic co-delivery system via CMC hydrogel is a promising adjunct to systemic antibiotic therapy for MRSA-induced FRI, highlighting its potential for localized, sustained delivery and improved treatment outcomes., (© 2024. The Author(s).)

Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Staphylococcus aureus ´ biofilm-forming ability and rapid resistance development pose a significant challenge to successful treatment, particularly in postoperative complications, emphasizing the need for enhanced therapeutic strategies. Bacteriophage (phage) therapy has reemerged as a promising and safe option to combat multidrug-resistant bacteria. However, questions regarding the efficacy of phages against biofilms and the development of phage resistance require further evaluation. Expanding on the adaptable and evolutionary characteristics of phages, we introduce an evolutionary approach to enhance the activity of S. aureus phages against biofilms. Unlike other in vitro directed evolution methods performed in planktonic cultures, we employed pre-stablished biofilms to do a serial-passage assay to evolve phages monitored by real-time isothermal microcalorimetry (IMC). The evolved phages demonstrated an expanded host range, with the CUB_MRSA-COL_R9 phage infecting 83% of strains in the collection ( n  = 72), surpassing the ISP phage, which represented the widest host range (44%) among the ancestral phages. In terms of antimicrobial efficacy, IMC data revealed superior suppression of bacterial growth by the evolved phages compared to the ancestral CUB-M and/or ISP phages against the respective bacterial strain. The phage cocktail exhibited higher efficacy, achieving over 90% suppression relative to the growth control even after 72 h of monitoring. Biofilm cell-counts, determined by RT-qPCR, confirmed the enhanced antibiofilm performance of evolved phages with no biofilm regrowth up to 48 h in treated MRSA15 and MRSA-COL strains. Overall, our results underscore the potential of biofilm-adapted phage cocktails to improve clinical outcomes in biofilm-associated infections, minimizing the emergence of resistance and lowering the risk of infection relapse. However, further investigation is necessary to evaluate the translatability of our results from in vitro to in vivo models, especially in the context of combination therapy with the current standard of care treatment., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ponce Benavente, Wagemans, Hinkel, Aguerri Lajusticia, Lavigne, Trampuz and Gonzalez Moreno.)

Background: Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics., Methods: The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS)., Results: The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions., Conclusion: These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies., (Copyright 2024, Mary Ann Liebert, Inc., publishers.)

In contrast to the many reports of successful real-world cases of personalized bacteriophage therapy (BT), randomized controlled trials of non-personalized bacteriophage products have not produced the expected results. Here we present the outcomes of a retrospective observational analysis of the first 100 consecutive cases of personalized BT of difficult-to-treat infections facilitated by a Belgian consortium in 35 hospitals, 29 cities and 12 countries during the period from 1 January 2008 to 30 April 2022. We assessed how often personalized BT produced a positive clinical outcome (general efficacy) and performed a regression analysis to identify functional relationships. The most common indications were lower respiratory tract, skin and soft tissue, and bone infections, and involved combinations of 26 bacteriophages and 6 defined bacteriophage cocktails, individually selected and sometimes pre-adapted to target the causative bacterial pathogens. Clinical improvement and eradication of the targeted bacteria were reported for 77.2% and 61.3% of infections, respectively. In our dataset of 100 cases, eradication was 70% less probable when no concomitant antibiotics were used (odds ratio = 0.3; 95% confidence interval = 0.127-0.749). In vivo selection of bacteriophage resistance and in vitro bacteriophage-antibiotic synergy were documented in 43.8% (7/16 patients) and 90% (9/10) of evaluated patients, respectively. We observed a combination of antibiotic re-sensitization and reduced virulence in bacteriophage-resistant bacterial isolates that emerged during BT. Bacteriophage immune neutralization was observed in 38.5% (5/13) of screened patients. Fifteen adverse events were reported, including seven non-serious adverse drug reactions suspected to be linked to BT. While our analysis is limited by the uncontrolled nature of these data, it indicates that BT can be effective in combination with antibiotics and can inform the design of future controlled clinical trials. BT100 study, ClinicalTrials.gov registration: NCT05498363 ., (© 2024. The Author(s).)

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability., (© 2024. The Author(s).)

The specificity of phages and their ability to evolve and overcome bacterial resistance make them potentially useful as adjuncts in the treatment of antibiotic-resistant bacterial infections. The goal of this study was to mimic a natural grouping of phages of interest and to evaluate the nature of their proliferation dynamics with bacteria. We have, for the first time, transferred naturally occurring phage groups directly from their sources of isolation to in vitro and identified 13 P. aeruginosa and 11 K. pneumoniae phages of 18 different genera, whose host range was grouped as 1.2-17%, 28-48% and 60-87%, using a large collection of P. aeruginosa (n = 102) and K. pneumoniae (n = 155) strains carrying different virulence factors and phage binding receptors. We introduced the interpretation model curve for phage liquid culturing, which allows easy and quick analysis of bacterial and phage co-proliferation and growth of phage-resistant mutants (PRM) based on qualitative and partially quantitative evaluations. We assayed phage lytic activities both individually and in 14 different cocktails on planktonic bacterial cultures, including three resistotypes of P. aeruginosa (PAO1, PA14 and PA7) and seven K. pneumoniae strains of different capsular serotypes. Based on the results, the natural phage cocktails designed and tested in this study largely performed well and inhibited PRM growth either synergistically or in proto-cooperation. This study contributes to the knowledge of phage behavior in cocktails and the formulation of therapeutic phage preparations. The paper also provides a detailed description of the methods of working with phages.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (10 8 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins., (© 2024. The Author(s).)

Leuconostoc carnosum is a bacterial species commonly associated with meat spoilage. However, some strains exhibit preservative effects due to bacteriocin production. Here, we report the complete genome sequences for two strains, L. carnosum 4010 and AMS1. Bacteriocin-related gene clusters were found on the plasmids of both strains., Competing Interests: The authors declare no conflict of interest.

Bacteriophages must seize control of the host gene expression machinery to replicate. To bypass bacterial anti-phage defence systems, this host takeover occurs immediately upon infection. A general understanding of phage mechanisms for immediate targeting of host transcription and translation processes is lacking. Here we introduce an integrative high-throughput approach to uncover phage-encoded proteins that target the gene expression machinery of Pseudomonas aeruginosa immediately upon infection with the jumbo phage ΦKZ. By integrating biochemical, genetic and structural analyses, we identify an abundant and conserved phage factor ΦKZ014 that targets the large ribosomal subunit by binding the 5S ribosomal RNA, and rapidly promotes replication in several clinical isolates. ΦKZ014 is among the earliest ΦKZ proteins expressed after infection and remains bound to ribosomes during the entire translation cycle. Our study provides a strategy to decipher molecular components of phage-mediated host takeover and argues that phage genomes represent an untapped discovery space for proteins that modulate the host gene expression machinery., (© 2024. The Author(s).)

The introduction of high-throughput sequencing has resulted in a surge of available bacteriophage genomes, unveiling their tremendous genomic diversity. However, our current understanding of the complex transcriptional mechanisms that dictate their gene expression during infection is limited to a handful of model phages. Here, we applied ONT-cappable-seq to reveal the transcriptional architecture of six different clades of virulent phages infecting Pseudomonas aeruginosa . This long-read microbial transcriptomics approach is tailored to globally map transcription start and termination sites, transcription units, and putative RNA-based regulators on dense phage genomes. Specifically, the full-length transcriptomes of LUZ19, LUZ24, 14-1, YuA, PAK_P3, and giant phage phiKZ during early, middle, and late infection were collectively charted. Beyond pinpointing traditional promoter and terminator elements and transcription units, these transcriptional profiles provide insights in transcriptional attenuation and splicing events and allow straightforward validation of Group I intron activity. In addition, ONT-cappable-seq data can guide genome-wide discovery of novel regulatory element candidates, including noncoding RNAs and riboswitches. This work substantially expands the number of annotated phage-encoded transcriptional elements identified to date, shedding light on the intricate and diverse gene expression regulation mechanisms in Pseudomonas phages, which can ultimately be sourced as tools for biotechnological applications in phage and bacterial engineering., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

During phage infection, both virus and bacteria attempt to gain and/or maintain control over critical bacterial functions, through a plethora of strategies. These strategies include posttranslational modifications (PTMs, including phosphorylation, ribosylation, and acetylation), as rapid and dynamic regulators of protein behavior. However, to date, knowledge on the topic remains scarce and fragmented, while a more systematic investigation lies within reach. The release of AlphaFold, which advances PTM enzyme discovery and functional elucidation, and the increasing inclusivity and scale of mass spectrometry applications to new PTM types, could significantly accelerate research in the field. In this review, we highlight the current knowledge on PTMs during phage infection, and conceive a possible pipeline for future research, following an enzyme-target-function scheme., 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 Ltd.. All rights reserved.)

In the last decade, powerful high-throughput sequencing approaches have emerged to analyse microbial transcriptomes at a global scale. However, to date, applications of these approaches to microbial viruses such as phages remain scarce. Tailoring these techniques to virus-infected bacteria promises to obtain a detailed picture of the underexplored RNA biology and molecular processes during infection. In addition, transcriptome study of stress and perturbations induced by phages in their infected bacterial hosts is likely to reveal new fundamental mechanisms of bacterial metabolism and gene regulation. Here, we provide references and blueprints to implement emerging transcriptomic approaches towards addressing transcriptome architecture, RNA-RNA and RNA-protein interactions, RNA modifications, structures and heterogeneity of transcription profiles in infected cells that will provide guides for future directions in phage-centric therapeutic applications and microbial synthetic biology., 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 Ltd.. All rights reserved.)

Importance: Biofilm-related infections are among the most difficult-to-treat infections in all fields of medicine due to their antibiotic tolerance and persistent character. In the field of orthopedics, these biofilms often lead to therapeutic failure of medical implantable devices and urgently need novel treatment strategies. This forthcoming article aims to explore the dynamic interplay between newly isolated bacteriophages and routinely used antibiotics and clearly indicates synergetic patterns when used as a dual treatment modality. Biofilms were drastically more reduced when both active agents were combined, thereby providing additional evidence that phage-antibiotic combinations lead to synergism and could potentially improve clinical outcome for affected patients., Competing Interests: The authors declare no conflict of interest.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species., (© 2024. The Author(s).)

Summary: Today, hundreds of post-translational modification (PTM) sites are routinely identified at once, but the comparison of new experimental datasets to already existing ones is hampered by the current inability to search most PTM databases at the protein residue level. We present FLAMS (Find Lysine Acylations and other Modification Sites), a Python3-based command line and web-tool that enables researchers to compare their PTM sites to the contents of the CPLM, the largest dedicated protein lysine modification database, and dbPTM, the most comprehensive general PTM database, at the residue level. FLAMS can be integrated into PTM analysis pipelines, allowing researchers to quickly assess the novelty and conservation of PTM sites across species in newly generated datasets, aiding in the functional assessment of sites and the prioritization of sites for further experimental characterization., Availability and Implementation: FLAMS is implemented in Python3, and freely available under an MIT license. It can be found as a command line tool at https://github.com/hannelorelongin/FLAMS, pip and conda; and as a web service at https://www.biw.kuleuven.be/m2s/cmpg/research/CSB/tools/flams/., (© The Author(s) 2024. Published by Oxford University Press.)

Detailed transcription maps of bacteriophages are not usually explored, limiting our understanding of molecular phage biology and restricting their exploitation and engineering. The ONT-cappable-seq method described here brings phage transcriptomics to the accessible nanopore sequencing platform and provides an affordable and more detailed overview of transcriptional features compared to traditional RNA-seq experiments. With ONT-cappable-seq, primary transcripts are specifically capped, enriched, and prepared for long-read sequencing on the nanopore sequencing platform. This enables end-to-end sequencing of unprocessed transcripts covering both phage and host genome, thus providing insight on their operons. The subsequent analysis pipeline makes it possible to rapidly identify the most important transcriptional features such as transcription start and stop sites. The obtained data can thus provide a comprehensive overview of the transcription by your phage of interest., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter describes the workflow to implement deep sequencing into standard phage display experiments on protein libraries. By harvesting the power of high throughput of these techniques, it allows for comprehensive analysis of the naïve library and library evolution in response to selection by ligand binding. The mutagenized target region of the protein variants encoded by the phage pool is analyzed by Illumina paired-end sequencing. Sequence data are processed to extract selection-enriched amino acid motifs. In addition, a complementary long-read sequencing approach is proposed enabling the monitoring of display vector stability., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Engineered lysins are considered as highly promising alternatives for antibiotics. Our previous screening study using VersaTile technology identified 1D10 as a possible lead compound with activity against Acinetobacter baumannii strains under elevated human serum concentrations. In this manuscript, we reveal an unexpected mode of action and exceptional thermoresistance for lysin 1D10. Our findings shed new light on the development of engineered lysins, providing valuable insights for future research in this field., Competing Interests: Y.B. acted as a scientific advisor of Lisando GmbH. H.G., R.L., and Y.B. are co-inventors on a patent application related to this work (VersaTile technique) filed by Ghent University and the University of Leuven (no. WO/2018/114980, filed on 19 February 2017, published on 28 June 2018).

Importance: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS., Competing Interests: The authors declare no conflict of interest.

Importance: The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria., Competing Interests: The authors declare no conflict of interest.

Importance: As antimicrobial resistance becomes more prevalent, the application of (bacterio)phage therapy as an alternative treatment for difficult-to-treat infections is (re)gaining popularity. Over the past decade, numerous promising case reports and series have been published demonstrating the therapeutic potential of phage therapy. However, important questions remain regarding the optimal treatment protocol and, unlike for medicinal products, there are currently no predefined quality standards for the stability of phage preparations. Phage titers can be influenced by several factors which could lead to reduced titers after preparation and storage and, ultimately, subtherapeutic applications. Determining the stability of different phages in different recipients according to the route of administration is therefore one of the first important steps in establishing a standardized protocol for phage therapy., Competing Interests: The authors declare no conflict of interest.

Bacteriophage-derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic-resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high-throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram-positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time-kill assays in phosphate-buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta-lactam penicillin used to intramammarily treat bovine Gram-positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add-on in the treatment of bovine streptococcal mastitis., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9-30 and MK.R3-15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis, responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics., Competing Interests: Declaration of competing interest All authors declare that there is no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Phage therapy is an emerging antimicrobial treatment for critical multidrug-resistant pathogens. In this review, the specific potential and challenges of phage therapy for patients with hidradenitis suppurativa (HS) are discussed. This represents a unique challenge as HS is a chronic inflammatory disease, but presenting with acute exacerbations, which have an enormous negative impact on patient's quality of life. The therapeutic arsenal for HS has expanded in the past decade, for example, with adalimumab and several other biologicals that are currently under investigation. However, treatment of HS remains challenging for dermatologists because there are individuals who do not respond to any classes of the current treatment options when used for a first or second time. Furthermore, after several courses of treatment, a patient may lose their response to therapy, meaning long-term use is not always an option. Culturing studies and 16S ribosomal RNA profiling highlight the complex polymicrobial nature of HS lesions. Despite the detection of various bacterial species in lesion samples, several key pathogens, including Staphylococcus, Corynebacterium and Streptococcus, may be potential targets for phage therapy. Using phage therapy for the treatment of a chronic inflammatory disease could potentially provide new insights into the role of bacteria and the immune system in HS development. In addition, it is possible more details on the immunomodulatory effects of phages may come to light., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The current threat of multidrug resistant strains necessitates development of alternatives to antibiotics such as bacteriophages. This study describes the isolation and characterization of a novel Salmonella Typhimurium phage 'Arash' from hospital wastewater in Leuven, Belgium. Arash has a myovirus morphology with a 95 nm capsid and a 140 nm tail. The host range of Arash is restricted to its isolation host. Approximately 86% of the phage particles are adsorbed to a host cell within 10 min. Arash has latent period of 65 min and burst size of 425 PFU/cell. Arash has a dsDNA genome of 180,819 bp with GC content of 53.02% with no similarities to any characterized phages, suggesting Arash as a novel species in the novel 'Arashvirus' genus. Arash carries no apparent lysogeny-, antibiotic resistance- nor virulence-related genes. Proteome analysis revealed 116 proteins as part of the mature phage particles of which 27 could be assigned a function. Therefore, the present findings shed light on the morphological, microbiological and genomic characteristics of Arash and suggest its potential application as therapeutic and/or biocontrol agent., (© 2023. BioMed Central Ltd., part of Springer Nature.)

In recent decades, there has been a notable increase in antibiotic-resistant Pseudomonas aeruginosa isolates, necessitating the development of innovative treatments to combat this pathogen. This manuscript explores the potential of different phage proteins to attenuate virulence factors of P. aeruginosa , particularly the type II secretion system (T2SS). PIT2, a protein derived from the lytic Pseudomonas phage LMA2 inhibits the T2SS effectors PrpL and LasA and attenuates the bacterial virulence toward HeLa cells and Galleria mellonella . Using RNAseq-based differential gene expression analysis, PIT2's impact on the LasR regulatory network is revealed, which plays a key role in bacterial quorum sensing. This discovery expands our knowledge on phage-encoded modulators of the bacterial metabolism and offers a promising anti-virulence target in P. aeruginosa . As such, it lays the foundation for a new phage-inspired anti-virulence strategy to combat multidrug resistant pathogens and opens the door for SynBio applications., Competing Interests: 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., (© 2023 The Authors.)

Proteus mirabilis is an opportunistic pathogen and is responsible for more than 40% of all cases of catheter-associated urinary tract infections (CAUTIs). Healthcare-associated infections have been aggravated by the constant emergence of antibiotic-resistant bacterial strains. Because of this, the use of phages to combat bacterial infections gained renewed interest. In this study, we describe the biological and genomic features of two P. mirabilis phages, named BigMira and MidiMira. These phages belong to the Acadevirus genus (family Autographiviridae ). BigMira and MidiMira are highly similar, differing only in four missense mutations in their phage tail fiber. These mutations are sufficient to impact the phages' depolymerase activity. Subsequently, the comparative genomic analysis of ten clinical P. mirabilis strains revealed differences in their antibiotic resistance profiles and lipopolysaccharide locus, with the latter potentially explaining the host range data of the phages. The massive presence of antimicrobial resistance genes, especially in the phages' isolation strain P. mirabilis MCS, highlights the challenges in treating infections caused by multidrug-resistant bacteria. The findings reinforce BigMira and MidiMira phages as candidates for phage therapy purposes.

Clinicians are increasingly confronted with the limitations of antibiotics to clear bacterial infections in patients. It has long been assumed that only antibiotic resistance plays a pivotal role in this phenomenon. Indeed, the worldwide emergence of antibiotic resistance is considered one of the major health threats of the 21st century. However, the presence of persister cells also has a significant influence on treatment outcomes. These antibiotic-tolerant cells are present in every bacterial population and are the result of the phenotypic switching of normal, antibiotic-sensitive cells. Persister cells complicate current antibiotic therapies and contribute to the development of resistance. In the past, extensive research has been performed to investigate persistence in laboratory settings; however, antibiotic tolerance under conditions that mimic the clinical setting remain poorly understood. In this study, we optimized a mouse model for lung infections with the opportunistic pathogen Pseudomonas aeruginosa. In this model, mice are intratracheally infected with P. aeruginosa embedded in seaweed alginate beads and subsequently treated with tobramycin via nasal droplets. A diverse panel of 18 P. aeruginosa strains originating from environmental, human, and animal clinical sources was selected to assess survival in the animal model. Survival levels were positively correlated with the survival levels determined via time-kill assays, a common method to study persistence in the laboratory. We showed that survival levels are comparable and thus that the classical persister assays are indicative of antibiotic tolerance in a clinical setting. The optimized animal model also enables us to test potential antipersister therapies and study persistence in relevant settings. IMPORTANCE The importance of targeting persister cells in antibiotic therapies is becoming more evident, as these antibiotic-tolerant cells underlie relapsing infections and resistance development. Here, we studied persistence in a clinically relevant pathogen, Pseudomonas aeruginosa. It is one of the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.), which are considered major health threats. P. aeruginosa is mostly known to cause chronic lung infections in cystic fibrosis patients. We mimicked these lung infections in a mouse model to study persistence under more clinical conditions. It was shown that the survival levels of natural P. aeruginosa isolates in this model are positively correlated with the survival levels measured in classical persistence assays in vitro . These results not only validate the use of our current techniques to study persistence but also open opportunities to study new persistence mechanisms or evaluate new antipersister strategies in vivo ., Competing Interests: The authors declare no conflict of interest.

The increase in antibiotic-resistant avian-pathogenic Escherichia coli (APEC), the causative agent of colibacillosis in poultry, warrants urgent research and the development of alternative therapies. This study describes the isolation and characterization of 19 genetically diverse, lytic coliphages, 8 of which were tested in combination for their efficacy in controlling in ovo APEC infections. Genome homology analysis revealed that the phages belong to nine different genera, one of them being a novel genus ( Nouzillyvirus ). One phage, REC, was derived from a recombination event between two Phapecoctavirus phages (ESCO5 and ESCO37) isolated in this study. Twenty-six of the 30 APEC strains tested were lysed by at least one phage. Phages exhibited varying infectious capacities, with narrow to broad host ranges. The broad host range of some phages could be partially explained by the presence of receptor-binding protein carrying a polysaccharidase domain. To demonstrate their therapeutic potential, a phage cocktail consisting of eight phages belonging to eight different genera was tested against BEN4358, an APEC O2 strain. In vitro , this phage cocktail fully inhibited the growth of BEN4358. In a chicken lethality embryo assay, the phage cocktail enabled 90% of phage-treated embryos to survive infection with BEN4358, compared with 0% of nontreated embryos, indicating that these novel phages are good candidates to successfully treat colibacillosis in poultry. IMPORTANCE Colibacillosis, the most common bacterial disease affecting poultry, is mainly treated by antibiotics. Due to the increased prevalence of multidrug-resistant avian-pathogenic Escherichia coli, there is an urgent need to assess the efficacy of alternatives to antibiotherapy, such as phage therapy. Here, we have isolated and characterized 19 coliphages that belong to nine phage genera. We showed that a combination of 8 of these phages was efficacious in vitro to control the growth of a clinical isolate of E. coli. Used in ovo , this phage combination allowed embryos to survive APEC infection. Thus, this phage combination represents a promising treatment for avian colibacillosis., Competing Interests: The authors declare no conflict of interest.

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. KEY POINTS: • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens., (© 2023. The Author(s).)

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In recent years, the food industry has expended considerable effort to design novel products that replace animal proteins with legumes; however, the actual environmental benefits of such products are often not quantified. Here, we performed Life Cycle Assessments (LCA) to evaluate the environmental performance of four new fermented food products based on different mixtures of animal (cow milk) and plant (pea) protein sources (100% Pea, 75% Pea-25% Milk, 50% Pea-50% Milk, 25% Pea-75% Milk). The system perimeter encompassed all stages from agricultural production of the ingredients to the creation of the final ready-to-eat products. Impacts were calculated for all environmental indicators included in the EF 3.0 Method in SimaPro software based on a functional unit of 1 kg of ready-to-eat product. Life cycle inventories included all of the flows analyzed by the LCA (raw materials, energy, water, cleaning products, packaging, transport, waste). Foreground data were acquired directly on the manufacturing site; background data were taken from the Ecoinvent 3.6 database. The dataset contains details on the products, processes, equipment, and infrastructure considered; mass and energy flows; Life Cycle Inventories (LCI); and Life Cycle Impact Assessment (LCIA). These data improve our understanding of the environmental impact of plant-based alternatives to dairy products, which is currently poorly documented., Competing Interests: 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., (© 2023 The Author(s).)

Strain MDTJ8 T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n -caproate. The strain grows and produces formate, acetate, n -butyrate, n -caproate and lactate from mono-, di- and polymeric saccharides at 37-60 °C (optimum, 50-55 °C) and at pH 5.0-7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3-0.5×1.0-3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8 T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae , being nearest to Caproicibacter fermentans EA1 T (94.8 %) and Caproiciproducens galactitolivorans BS-1 T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae . Pairwise average nucleotide identity and DNA-DNA hybridization values between strain MDJT8 T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8 T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8 T are C 14 : 0 , C 14 : 0 DMA (dimethyl acetal) and C 16 : 0 , while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8 T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8 T (=DSM 114174 T =LMG 32615 T =NCCB 100883 T ).

Autographiviridae is a diverse yet distinct family of bacterial viruses marked by a strictly lytic lifestyle and a generally conserved genome organization. Here, we characterized Pseudomonas aeruginosa phage LUZ100, a distant relative of type phage T7. LUZ100 is a podovirus with a limited host range which likely uses lipopolysaccharide (LPS) as a phage receptor. Interestingly, infection dynamics of LUZ100 indicated moderate adsorption rates and low virulence, hinting at temperate characteristics. This hypothesis was supported by genomic analysis, which showed that LUZ100 shares the conventional T7-like genome organization yet carries key genes associated with a temperate lifestyle. To unravel the peculiar characteristics of LUZ100, ONT-cappable-seq transcriptomics analysis was performed. These data provided a bird's-eye view of the LUZ100 transcriptome and enabled the discovery of key regulatory elements, antisense RNA, and transcriptional unit structures. The transcriptional map of LUZ100 also allowed us to identify new RNA polymerase (RNAP)-promoter pairs that can form the basis for biotechnological parts and tools for new synthetic transcription regulation circuitry. The ONT-cappable-seq data revealed that the LUZ100 integrase and a MarR-like regulator (proposed to be involved in the lytic/lysogeny decision) are actively cotranscribed in an operon. In addition, the presence of a phage-specific promoter transcribing the phage-encoded RNA polymerase raises questions on the regulation of this polymerase and suggests that it is interwoven with the MarR-based regulation. This transcriptomics-driven characterization of LUZ100 supports recent evidence that T7-like phages should not automatically be assumed to have a strictly lytic life cycle. IMPORTANCE Bacteriophage T7, considered the "model phage" of the Autographiviridae family, is marked by a strictly lytic life cycle and conserved genome organization. Recently, novel phages within this clade have emerged which display characteristics associated with a temperate life cycle. Screening for temperate behavior is of utmost importance in fields like phage therapy, where strictly lytic phages are generally required for therapeutic applications. In this study, we applied an omics-driven approach to characterize the T7-like Pseudomonas aeruginosa phage LUZ100. These results led to the identification of actively transcribed lysogeny-associated genes in the phage genome, pointing out that temperate T7-like phages are emerging more frequent than initially thought. In short, the combination of genomics and transcriptomics allowed us to obtain a better understanding of the biology of nonmodel Autographiviridae phages, which can be used to optimize the implementation of phages and their regulatory elements in phage therapy and biotechnological applications, respectively.

The phage T7 RNA polymerase (RNAP) and lysozyme form the basis of the widely used pET expression system for recombinant expression in the biotechnology field and as a tool in microbial synthetic biology. Attempts to transfer this genetic circuitry from Escherichia coli to non-model bacterial organisms with high potential have been restricted by the cytotoxicity of the T7 RNAP in the receiving hosts. We here explore the diversity of T7-like RNAPs mined directly from Pseudomonas phages for implementation in Pseudomonas species, thus relying on the co-evolution and natural adaptation of the system towards its host. By screening and characterizing different viral transcription machinery using a vector-based system in P. putida ., we identified a set of four non-toxic phage RNAPs from phages phi15, PPPL-1, Pf-10, and 67PfluR64PP, showing a broad activity range and orthogonality to each other and the T7 RNAP. In addition, we confirmed the transcription start sites of their predicted promoters and improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition. This set of viral RNAPs expands the adaption of T7-inspired circuitry towards Pseudomonas species and highlights the potential of mining tailored genetic parts and tools from phages for their non-model host.

The Chromosome-centric Human Proteome Project (C-HPP) aims at identifying the proteins as gene products encoded by the human genome, characterizing their isoforms and functions. The existence of products has now been confirmed for 93.2% of the genes at the protein level. The remaining mostly correspond to proteins of low abundance or difficult to access. Over the past years, we have significantly contributed to the identification of missing proteins in the human spermatozoa. We pursue our search in the reproductive sphere with a focus on early human embryonic development. Pluripotent cells, developing into the fetus, and trophoblast cells, giving rise to the placenta, emerge during the first weeks. This emergence is a focus of scientists working in the field of reproduction, placentation and regenerative medicine. Most knowledge has been harnessed by transcriptomic analysis. Interestingly, some genes are uniquely expressed in those cells, giving the opportunity to uncover new proteins that might play a crucial role in setting up the molecular events underlying early embryonic development. Here, we analyzed naive pluripotent and trophoblastic stem cells and discovered 4 new missing proteins, thus contributing to the C-HPP. The mass spectrometry proteomics data was deposited on ProteomeXchange under the data set identifier PXD035768.

Foodborne Salmonella enteritidis infections place human health at risk, driven by regular outbreaks and individual cases by different contaminated food materials. This study was conducted to characterize and employ a single bacteriophage as a potential biocontrol agent. Phage Rostam was isolated, characterized and then applied as biocontrol agent against S. enteritidis in liquid whole eggs and eggshell. Rostam is a novel myovirus belonging to the Rosemountvirus genus and active against Escherichia coli and Salmonella spp. Rostam is stable in a pH range from 4 to 10, a salt concentration of 1-9 %, whereas UV radiation gradually reduces phage stability, and its 53 kb genome sequence indicates this phage does not contain known toxins or lysogeny-associated genes. Its latent period is short with a burst size of 151 PFU/cell, under standard growth conditions. Killing curves indicate that at higher multiplicities of infection (MOI), the reduction in S. enteritidis count is more pronounced. Phage Rostam (MOI 10,000) reduces S. enteritidis growth to below the detection limit at 4 °C in both liquid whole eggs and on the eggshell within 24 h. Due to its high lytic activity and stability in relevant conditions, Rostam has the potential to be an efficient biopreservative for egg and egg products., Competing Interests: Declarations of interest None., (Copyright © 2023. Published by Elsevier B.V.)