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Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a Delta pA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.

The present study aims to address a gap of knowledge by evaluating the in-situ ability of edible wild fish (Liza aurata, Sparus aurata and Sarpa salpa) and mussel Mytilus galloprovincialis to serve as environmental vectors of MPs along the eastern Algerian coastline (namely; Sidi Salem, Ain Achir and Saint Cloud). Our data showed the widespread accumulation of MPs in highly consumed fish species and mussels in Annaba coastal area. MPs were found in all investigated organisms from the three studied areas. The most common size range of MPs was between 0,45 μm and 1,22 μm. Six polymers of MPs were identified from the sampling areas; polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polyethylene-vinyl acetate (PEVA), high-density polyethylene (HDPE), and low-density polyethylene (LDPE). The data provided by this study can serve as a support for assessing the status of MPs pollution in the Algerian coast areas and the associated human health risks., 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 Elsevier Ltd. All rights reserved.)

Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.

Seventh-pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the nontarget DNA strand. In vitro studies indicate that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N 4 AH and N 5 HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N 3 VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing., (© 2024. The Author(s).)

Two types of alginates, AlgLF and AlgP, were used in this study to produce alginate beads by electro-vibratory extrusion. AlgLF and AlgP exhibited different Mannuronate/Guluronate (M/G) ratios and molecular weights as measured by NMR and SEC-MALS. The calcium chloride concentration was found to have the greatest effect on bead size. Higher concentrations resulted in smaller beads. AlgLF with a higher molecular weight and a lower proportion of G blocks showed smaller beads. For both alginates, the bead size was also influenced by the flow rate and vibration frequency. Alginate solution aging showed a minimal effect. Alginate reticulation was modeled using a mathematical equation. The study provides insights for the optimization of alginate-based materials in different applications by shedding light on the main factors influencing bead size. The importance of the molecular weight, M/G ratio and calcium ion concentration in the gelling process is highlighted, providing opportunities for the tailoring of alginate materials through a phenomenological model.

Oligosaccharides derived from λ-carrageenan (λ-COs) are gaining interest in the cancer field. They have been recently reported to regulate heparanase (HPSE) activity, a protumor enzyme involved in cancer cell migration and invasion, making them very promising molecules for new therapeutic applications. However, one of the specific features of commercial λ-carrageenan (λ-CAR) is that they are heterogeneous mixtures of different CAR families, and are named according to the thickening-purpose final-product viscosity which does not reflect the real composition. Consequently, this can limit their use in a clinical applications. To address this issue, six commercial λ-CARs were compared and differences in their physiochemical properties were analyzed and shown. Then, a H 2 O 2 -assisted depolymerization was applied to each commercial source, and number- and weight-averaged molar masses (M n and M w ) and sulfation degree (DS) of the λ-COs produced over time were determined. By adjusting the depolymerization time for each product, almost comparable λ-CO formulations could be obtained in terms of molar masses and DS, which ranged within previously reported values suitable for antitumor properties. However, when the anti-HPSE activity of these new λ-COs was screened, small changes that could not be attributed only to their small length or DS changes between them were found, suggesting a role of other features, such as differences in the initial mixture composition. Further structural MS and NMR analysis revealed qualitative and semi-quantitative differences between the molecular species, especially in the proportion of the anti-HPSE λ-type, other CARs types and adjuvants, and it also showed that H 2 O 2 -based hydrolysis induced sugar degradation. Finally, when the effects of λ-COs were assessed in an in vitro migration cell-based model, they seemed more related to the proportion of other CAR types in the formulation than to their λ-type-dependent anti-HPSE activity.

Encapsulation is used in various industries to protect active molecules and control the release of the encapsulated materials. One of the structures that can be obtained using coextrusion encapsulation methods is the core-shell capsule. This review focuses on coextrusion encapsulation applications for the preservation of oils and essential oils, probiotics, and other bioactives. This technology isolates actives from the external environment, enhances their stability, and allows their controlled release. Coextrusion offers a valuable means of preserving active molecules by reducing oxidation processes, limiting the evaporation of volatile compounds, isolating some nutrients or drugs with undesired taste, or stabilizing probiotics to increase their shelf life. Being environmentally friendly, coextrusion offers significant application opportunities for the pharmaceutical, food, and agriculture sectors.

This paper provides an overview of coextrusion methods for encapsulation. Encapsulation involves the coating or entrapment of a core material such as food ingredients, enzymes, cells, or bioactives. Encapsulation can help compounds add to other matrices, stabilize compounds during storage, or enable controlled delivery. This review explores the principal l coextrusion methods available that can be used to produce core-shell capsules through the use of coaxial nozzles. Four methods for encapsulation by coextrusion are examined in detail, including dripping, jet cutting, centrifugal, and electrohydrodynamic systems. The targeted capsule size determines the appropriate parameters for each method. Coextrusion technology is a promising encapsulation technique able to generate core-shell capsules in a controlled manner, which can be applied to cosmetic, food, pharmaceutical, agriculture, and textile industries. Coextrusion is an excellent way to preserve active molecules and present a significant economic interest.

Epstein-Barr virus (EBV) infects 95% of the world's population and persists latently in the body. It immortalizes B-cells and is associated with lymphomas. LCLs (lymphoblastoid cell lines, EBV latency III B-cells) inhibit anti-tumoral T-cell response following PD-L1 overexpression (programmed death-ligand 1 immune checkpoint). Many cancer cells, including some DLBCLs (diffuse large B-cell lymphomas), also overexpress PD-L1. Immunotherapies are based on inhibition of PD-L1/PD-1 interactions but present some dose-dependent toxicities. We aim to find new strategies to improve their efficiency by decreasing PD-L1 expression. Fucoidan, a polysaccharide extracted from brown seaweed, exhibits immunomodulatory and anti-tumor activities depending on its polymerization degree, but data are scarce on lymphoma cells or immune checkpoints. LCLs and DLBCLs cells were treated with native fucoidan ( Fucus vesiculosus ) or original very-low-molecular-weight fucoidan formulas (vLMW-F). We observed cell proliferation decrease and apoptosis induction increase with vLMW-F and no toxicity on normal B- and T-cells. We highlighted a decrease in transcriptional and PD-L1 surface expression, even more efficient for vLMW than native fucoidan. This can be explained by actin network alteration, suggesting lower fusion of secretory vesicles carrying PD-L1 with the plasma membrane. We propose vLMW-F as potential adjuvants to immunotherapy due to their anti-proliferative and proapoptotic effects and ability to decrease PD-L1 membrane expression.

CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads., Competing Interests: Declaration of interests M.S., I.Q., and M.J. are named inventors on a related patent application., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Iron-nitrogen-carbon (FeNC) catalysts derived from zeolitic-imidazolate frameworks (ZIFs) are worldwide accepted to be the most promising candidates for the oxygen reduction reaction (ORR), but the insufficient stability, the low FeN x exposure and poor density restrict their ORR activity. Here, we demonstrate a strategy to synthesize FeN x sites embedded in a micro/mesoporous N, S co-doped graphitic carbon (FeNC/MUS) by tuning the ligand linkers via the addition of 2-undecylimidazole as a co-ligand in ZIF precursors, and optimizing the electronic structure of Fe center by an in-situ addition of thiourea molecules as sulfur (S) source. 2-undecylimidazole offered an open porous structure to incorporate more FeN x , while the S-doping increased the density of FeN x . Besides, 2-undeclyimidazole cooperatively with S-doping caused favorable changes into the catalyst structure, particularly improved the exposure and density of FeN x sites and doubled the Brunauer-Emmetter-Teller surface area to 1132 m 2 g -1 contrasted to the pristine FeNC/M (544 m 2 g -1 ). FeNC/MUS displayed an accelerated ORR activity with a higher half-wave potential of 0.86 V (vs. reversible hydrogen electrode (RHE)) than that of Pt/C (0.84 V) in addition of a longer durability with a 11 % of activity decay after 30000 s in alkaline media. This work offers a new insight to design optimal ZIFs precursor and a facile electron withdrawing S-doping strategy for efficient electrocatalysis., 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 © 2022 Elsevier Inc. All rights reserved.)

The objectives of this work were to address the fundamental characteristics of ansa-zirconocene catalyzed E/diene copolymerization and E/diene/1-hexene and E/diene/propylene terpolymerizations, and the quantitative relationship between diene structure and polymer chain propagation rate constant in term of quantifiable catalytic active sites. One of the most important but unknown factors in olefins ansa-zirconocene complexes is the distribution of the catalyst between sites actively participating in polymer chain formation and dormant sites. A set of ethylene/dienes copolymerizations, and ethylene/dienes/1-hexene and ethylene/dienes/1-hexene terpolymerizations catalyzed with ansa -zirconocenes/borate/triisobutylaluminium ( rac -Et(Ind) 2 ZrCl 2 /[Ph 3 C][B(C 6 F 5 ) 4 ]/triisobutylaluminium (TIBA) were performed in toluene at 50 °C To determine the active center [C*]/[Zr] ratio variation in the copolymerization of E with different dienes and their terpolymerization with 1-hexene and propylene, each polymer propagation chain ends were quenched with 2-thiophenecarbonyl, which selectively quenches the metal-polymer bonds through acyl chloride. The ethylene, propylene, 1-hexene, and diene composition-based propagation rate constants ( k p E, k p P, k p 1-H, and k p diene), thermal (melting and crystalline) properties, composition (mol% of ethylene, propylene, 1-hexene, and diene), molecular weight, and polydispersity were also studied in this work. Systematic comparisons of the proportion of catalytically [Zr]/[C*] active sites and polymerization rate constant ( k p ) for ansa -zirconocenes catalyzed E/diene, E/diene/1-hexene, and E/diene/propylene polymerization have not been reported before. We evaluated the addition of 1-hexene and propylene as termonomers in the copolymerization with E/diene. To make a comparison for each diene under identical conditions, we started the polymerization by introducing an 80/20 mole ratio of E/P and 0.12 mol/L of 1-hexene in the system. The catalyst behavior against different dienes, 1-hexene, and propylene is very interesting, including changes in thermal properties, cyclization of 1-hexene, and decreased incorporation of isoprene and butadiene, changes in the diffusion barriers in the system, and its effect on k p .

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses., (© 2022. The Author(s).)

Background: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome., Findings: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20-30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding., Conclusions: The phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Employing a suitable cocatalyst is very important to improve photocatalytic H 2 evolution activity. Herein, two plasmonic cocatalysts, Au nanoparticles and TiN nanoparticles were in-situ coupled over the g-C 3 N 4 nanotube to form a ternary 0D/0D/1D Au/TiN/g-C 3 N 4 composite via a successive thermal polycondensation and chemical reduction method. The g-C 3 N 4 nanotube acted as a support for the growth of Au and TiN nanoparticles, leading to intimate contact between g-C 3 N 4 nanotube with Au nanoparticles and TiN nanoparticles. As a result, multiple interfaces and dual-junctions of Au/g-C 3 N 4 Schottky-junction and TiN/g-C 3 N 4 ohmic-junction were constructed, which helped to promote the charged carriers' separation and enhanced the photocatalytic performance. Furthermore, loading plasmonic cocatalysts of Au nanoparticles and TiN nanoparticles can enhance the light absorption capacity. Consequently, the Au/TiN/g-C 3 N 4 composite exhibited significantly enhanced photocatalytic H 2 evolution activity (596 μmol g -1 h -1 ) compared to g-C 3 N 4 or binary composites of Au/g-C 3 N 4 and TiN/g-C 3 N 4 . This work highlights the significant role of cocatalysts in photocatalysis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements 1 . However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion 2-7 . Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ 7 , but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)