Antimicrobial resistance has emerged as a global threat to the treatment of infectious diseases. Antibacterial photodynamic therapy (aPDT) is a promising alternative approach and is highly suitable for the treatment of cutaneous bacterial infections through topical applications. aPDT relies on light-responsive compounds called photosensitizer (PS) dyes, which generate reactive oxygen species (ROS) when induced by light, thereby killing bacterial cells. Despite several previous studies in this area, the molecular details of targeting and cell death mediated by PS dyes are poorly understood. In this study, we further investigate the antibacterial properties of two water-soluble Sn(IV) tetrapyridylporphyrins that were quaternized with methyl and hexyl groups (1 and 2). In this follow-up study, we demonstrate that Sn(IV)-porphyrins can be photoexcited by blue light (a 427 nm LED) and exhibit various levels of bactericidal activity against both Gram-(+) and Gram-(−) strains of bacteria. Using localization studies through fluorescence microscopy, we show that 2 targets the bacterial membrane more effectively than 1 and exhibits comparatively higher aPDT activity. Using multiple fluorescence reporters, we demonstrate that photoactivation of 1 and 2 results in extensive collateral damage to the bacterial cells including DNA cleavage, membrane damage, and delocalization of central systems necessary for bacterial growth and division. In summary, this investigation provides deep insights into the mechanism of bacterial killing mediated by the Sn(IV)-porphyrins. Moreover, our approach offers a new method for evaluating the activity of PS, which may inspire the discovery of new PS with enhanced aPDT activity.

Metabolites are intermediate products formed during metabolism. Metabolites play different roles, including providing energy, supporting structure, transmitting signals, catalyzing reactions, enhancing defense, and interacting with other species. Plant metabolomics research aims to detect precisely all metabolites found within tissues of plants through GC-MS. This chapter primarily focuses on extracting metabolites using chemicals such as methanol, chloroform, ribitol, MSTFA, and TMCS. The metabolic analysis method is frequently used according to the specific kind of sample or matrix being investigated and the analysis objective. Chromatography (LC, GC, and CE) with mass spectrometry and NMR spectroscopy is used in modern metabolomics to analyze metabolites from plant samples. The most frequently used method for metabolites analysis is the GC-MS. It is a powerful technique that combines gas chromatography’s separation capabilities with mass spectrometry, offering detailed information, including structural identification of each metabolite. This chapter contains an easy-to-follow guide to extract plant-based metabolites. The current protocol provides all the information needed for extracting metabolites from a plant, precautions, and troubleshooting.

Plant volatile organic compounds (VOCs) are organic chemicals that plants release as part of their natural biological processes. Various plant tissues produce VOCs, including leaves, stems, flowers, and roots. VOCs are essential in plant communication, defense against pests and pathogens, aroma and flavor, and attracting pollinators. The study of plant volatiles has become an increasingly important area of research in recent years, as scientists have recognized these compounds’ important roles in plant physiology. As a result, there has been a growing interest in developing methods for collecting and analyzing plant VOCs. HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography-mass spectrometry) is commonly used for plant volatile analysis due to its high sensitivity and selectivity. This chapter describes an efficient method for extracting and identifying volatile compounds by HS-SPME coupled with GC-MS in tomato fruits.

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation is known to completely repress fruit ripening. The heterozygous (RIN/rin) fruits have extended shelf life, ripen normally, but have inferior taste/flavour. To address this, we used genome editing to generate newer alleles of RIN (rinCR) by targeting the K-domain. Unlike previously reported CRISPR alleles, the rinCR alleles displayed delayed onset of ripening, suggesting that the mutated K-domain represses the onset of ripening. The rinCR fruits had extended shelf life and accumulated carotenoids at an intermediate level between rin and progenitor line. Besides, the metabolites and hormonal levels in rinCR fruits were more akin to rin. To overcome the negative attributes of rin, we crossed the rinCR alleles with Nps1, a dominant-negative phototropin1 mutant, which enhances carotenoid levels in tomato fruits. The resulting Nps1/rinCR hybrids had extended shelf life and 4.4–7.1-fold higher carotenoid levels than the wild-type parent. The metabolome of Nps1/rinCR fruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatiles in Nps1/rinCR were only observed in fruits bearing the homozygous Nps1 mutation. The Nps1 introgression into tomato provides a promising strategy for developing cultivars with extended shelf life, improved taste, and flavour.

Acinetobacter (A.) baumannii has emerged as a difficult-to-treat nosocomial bacterial human pathogen. A. baumannii is to be dealt with under the “One Health” approach, and its surveillance in human, animal, and environmental settings assumes paramount importance in understanding its plausible transmission dynamics. Accurate identification of A. baumannii, its clonal complexes, and sequence types is important for understanding the epidemiological distribution, evolutionary relationships, and transmission dynamics. A wide range of genotyping techniques are applied for the differentiation of the Acinetobacter calcoaceticus-baumannii (ACB) complex. However, there is no single straight-forward genotype method applied for rapid assays. Currently, two multilocus sequence typing (MLST) Oxford and Pasture schemes exist; though considered a gold standard for sequence typing, harmonizing the schemes is not a straightforward process. The whole genome sequencing-based core-genome multilocus sequence typing (cgMLST) and core single nucleotide polymorphism (cgSNP) are robust and precise sequence typing; however, they are expensive, depending on the quality of sequencing and demand a higher level of computational skills. In the past decade, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based species identification has been successfully employed for rapid discrimination of the ACB complex. MALDI typing is rapid, easier, cheaper, and as reliable as molecular methods. Strain level A. baumannii identification confidence improved upon augmentation of existing databases with in-house reference spectra of well-defined isolates. The application of artificial intelligence and machine learning might be useful in clonal sequence types (ST)-level identification. The genus Acinetobacter’s taxonomic classification is evolving, and newer STs are being described; hence, the establishment of a central repository of A. baumannii reference spectra will help in harmonizing across the laboratories and help in the global level surveillance program on A. baumannii in “One Health” perspective. This review sheds light on the challenges related to techniques employed for the identification of Acinetobacter and the potential application and future perspectives of MALDI-TOF MS.

Surface Enhanced Raman Scattering (SERS) is emerging as a potent analytical tool for the detection of various pollutants in complex environments due to its distinctive vibrational fingerprint ability and pronounced detection sensitivity. Precautious of adverse blue-green economies and ecological impacts, sustainable generation of SERS active substrates and analyte casting matrices are getting prioritized. Herein, gold nanoflowers (AuNFs) of ∼75 ± 15 nm were initially biofabricated using an expended cell culture medium as a one-step synthesis cum stabilization strategy. Then the heavy architecture of multi-faceted AuNFs with deep pits and edges, that acted as hotspots for enhancing the localized electromagnetic fields, was utilized for the direct SERS detection of commonly used carcinogenic herbicides collected from agro-farms at nanomolar regimes with 0.44 ppm and 0.27 ppm for Glyphosate and amino methyl phosphonic acid, respectively. Such a low level detection is superior by 8.33% when compared to the reported values. Computational finite-difference time-domain (FDTD) simulations affirmed the enhanced SERS effect from the multi-faceted nanostructure of AuNFs with structural heterogeneities that provide numerous hotspots to amplify the localized electromagnetic field. More eminently, fish scale derived biotemplate through AuNF-analyte drop casting contributed to the exceptional intensities, attributed to the naturally grooved hierarchically porous hydrophilic lamellar structures contact angle of 73°. Overall, the adapted bioengineering of SERS substrate is safe, robust, affordable and reproducible, fostered by bioderived durable biomatrix offering potent sustainable SERS detection of various biomedically and environmentally relevant molecules.

Glyphosate (Gly) is a massively utilized toxic herbicide exceeding its statutory restrictions, causing adverse environmental and health impacts. Engineered nanomaterials, even though are integral to remediate Gly, their practical use is limited due to time and energy driven purifications, and negative environmental impacts. Here, a 3D wide area (~1.6 ± 0.4 cm2) Cu2O nanoparticle supported biotemplate is designed using fish-scale wastes as a sustainable approach for the ultra-efficient and selective hand-remediation of Gly from real-time samples from agro-farms. While the innate metal binding and reducing ability of collagenous scales aided self-synthesis cum grafting of Cu2O, the selective binding potential of Cu2O to Gly facilitated its hand-retrieval; as assessed using optical characterizations, Fourier transform infrared spectroscopy, thermogravimetric analysis and liquid chromatography mass spectrometry. Optimization studies revealed extractions of diverse pay-loads of Gly between 0.1 μg/mL to 40 μg/mL per 80 mg biotemplate grafted with ~6.354 μg of sub-5 nm Cu2O and was exponential to the number of Cu2O@biotemplates. Even though pH and surfactant didn't have any impact on the adsorption of Gly to the Cu2O@biotemplates, increase in the ionic strength led to a drastic increase in the adsorption. Density function theory simulations unveiled the involvement of phosphonic and carboxylic groups of Gly for interaction with Cu2O with a bond length of 1.826 Å and 1.833 Å, respectively. Overall, our sustainably generated, cost-efficient, hand-retrievable Cu2O supported biotemplate can be generalized to extract diverse organophosphorus toxins from agro-farms and other sewage embodiments. Synopsis: Glyphosate is an excessively applied herbicide with potent health hazards and carcinogenicity. Thus, a hand removable Cu2O-supported biotemplate to selectively and efficiently remediate glyphosate from irrigation water is developed.

Nanocatalysts are extremely crucial for the expedited synthesis of various chemicals, fuels, and pharmaceutical molecules both in academia and industry. To overcome the limitations of nanocatalysts and or microstructure supported catalysts such as agglomeration (due to inter-particle dipolar forces preventing longer shelf-lives), compromised catalytic activity (e.g., nickel-titanium dioxide bimetallic catalyst, showed high selectivity to hydrogenate 3-nitrostyrene into 3-vinylaniline (90.2 %) compared to unmodified nickel (55.3 %), due to metal-plane formation by titanium dioxide), cytotoxicity (with over 90 % cell killing in the presence of the nanocatalysts above ∼ 0.2 mg/mL), catalyst retrieval (demanding energy intensive procedures such as centrifugation (∼10,000 g and above), membrane filtrations (∼0.2 µm), magnetic separations (0.9–1.1 T) and absurd practical implementation there is a tremendous development of 3-dimensional wide-area supported catalysts. This review update the readers on the evolution of highly catalytic nanoparticles for various heterogeneous catalysis. Uniquely, wide-area supported catalysts wherein the nanoparticles are grafted to 3-dimensional nature-inspired or pristine natural materials as sustainable strategies are discussed. The role of wide-area of the support in overcoming the limitations of nanocatalysts and microstructures by enabling bidirectional reactant access, catalyst efficiency, reusability, stability and sustainability are highlighted. Next, we focus on the metal-affinity and redox-potential of the natural support that aid autogenic biosynthesis and self-assembly of nanocatalysts. Followed by discussions on supplementary properties of the support such as type, structural-hierarchy, surface-area, absorption, porosity and rigidity in tuning the stability, biodegradability, compatibility, functionality and performance of the catalyst. Accentuated, with the impact of support in dictating the choice of fixed batch vs continuous flow reactors, co-relative to modulating the catalytic efficiency and turnover frequencies. Finally, the exclusive role of wide-area of the support and its biological nature in allowing the extraction of noble precursor off the support after catalyst poisoning is emphasized. These discussions, for the first time, spotlight the versatility, resilient nature of the emerging ultra-efficient wide-area supported catalysts that are generated using sustainable procedures for diverse large-volume heterogeneous catalysis.

Biological materials with unique surface properties provide a new avenue for fabricating green and sensitive SERS-active substrates. Herein, we present a simple but efficient method to prepare surface-enhanced Raman scattering (SERS) substrates by depositing silver nanoparticles (AgNPs) on fish scale substrates using an evaporation-induced self-assembly method (EISA). Characterization of the formed flexible Ag-impregnated substrate proved outstanding SERS sensitivity, uniformity, and reproducibility properties, with a Raman enhancement factor of 1.3 × 106 and a relative standard deviation of 6.4 %. Using this powerful fish scale substrate, a toxic environmental pollutant perfluorooctane sulfonamide (PFOSA) was indirectly detected in lake water, soil, and human urine samples. Due to its chemical structure, it is difficult to detect low concentrations of PFOSA in real samples. Interestingly, malachite green (MG) was smartly used as the Raman label for PFOSA detection in real samples. One of the main appeals is that the concentration of PFOSA can be correlated with a decrease in the SERS signal of MG in real samples. In conclusion, the strategy employed and reproducible SERS substrates may have diverse applications in clinical and environmental analyses.

Treating sewage waters contaminated with persistent organic pollutants (POPs) presents a pressing environmental concern, mandating, affordable, implementable and sustainable remediations. Supported catalysts, wherein metal nanoparticles are grafted onto inert supports to endow porosity, reactant access, performance and catalyst re-use are emerging as sustainable catalytic platforms. Herein, size-controllable, mechanically recyclable 3D-Cu2O@megacatalyst of ∼81 ± 5 cm2, ∼37 ± 3 cm2 and ∼1 ± 0.6 cm2 were biofabricated by exploiting the innate metal binding feature of pristine eggshells. The as-fabricated Cu2O@megacatalyst was utilized for the Fenton-like treatment of POPs, with exceptional activities against diverse molecules: antibiotic (tetracycline (TC)), textile dye (methylene blue) and pharmaceutical precursor (4-nitrophenol) with the degradation efficiencies of 95.6 %, 96.8 % and 93.4 %, respectively. Optimization studies revealed that our megacatalyst can function consistently in the presence of various oxidising agents, free radical scavengers, wide pH, temperatures and inorganic and organic contaminants. The catalyst demonstrated stability and catalytic efficiency in different real-time water matrices: ultrapure water-95.6 %, tap water-84 %, lake water-86 %, and river water-91 %. Furthermore, plausible reaction mechanism and decomposition pathways for TC degradation were assessed using GC-MS, while evaluating the toxicity using ECOSAR and oxygen uptake assay, which revealed less toxic reaction intermediates and end products. Overall, our results provide new insight into the sustainable development of a generalized highly stable, scalable, ultra-efficient and mechanically recyclable Fenton-like supported catalyst for the detoxification of POPs in sewage waters.

One of the major constituents of mitochondrial membranes is the phospholipids, which play a key role in maintaining the structure and the functions of the mitochondria. However, mitochondria do not synthesize most of the phospholipids in situ, necessitating the presence of phospholipid import pathways. Even for the phospholipids, which are synthesized within the inner mitochondrial membrane (IMM), the phospholipid precursors must be imported from outside the mitochondria. Therefore, the mitochondria heavily rely on the phospholipid transport pathways for its proper functioning. Since, mitochondria are not part of a vesicular trafficking network, the molecular mechanisms of how mitochondria receive its phospholipids remain a relevant question. One of the major ways that hydrophobic phospholipids can cross the aqueous barrier of inter or intraorganellar spaces is by apposing membranes, thereby decreasing the distance of transport, or by being sequestered by lipid transport proteins (LTPs). Therefore, with the discovery of LTPs and membrane contact sites (MCSs), we are beginning to understand the molecular mechanisms of phospholipid transport pathways in the mitochondria. In this review, we will present a brief overview of the recent findings on the molecular architecture and the importance of the MCSs, both the intraorganellar and interorganellar contact sites, in facilitating the mitochondrial phospholipid transport. In addition, we will also discuss the role of LTPs for trafficking phospholipids through the intermembrane space (IMS) of the mitochondria. Mechanistic insights into different phospholipid transport pathways of mitochondria could be exploited to vary the composition of membrane phospholipids and gain a better understanding of their precise role in membrane homeostasis and mitochondrial bioenergetics.

Reactive oxygen species (ROS) contain at least one oxygen atom and one or more unpaired electrons and include singlet oxygen, superoxide anion radical, hydroxyl radical, hydroperoxyl radical, and free nitrogen radicals. Intracellular ROS can be formed as a consequence of several factors, including ultra-violet (UV) radiation, electron leakage during aerobic respiration, inflammatory responses mediated by macrophages, and other external stimuli or stress. The enhanced production of ROS is termed oxidative stress and this leads to cellular damage, such as protein carbonylation, lipid peroxidation, deoxyribonucleic acid (DNA) damage, and base modifications. This damage may manifest in various pathological states, including ageing, cancer, neurological diseases, and metabolic disorders like diabetes. On the other hand, the optimum levels of ROS have been implicated in the regulation of many important physiological processes. For example, the ROS generated in the mitochondria (mitochondrial ROS or mt-ROS), as a byproduct of the electron transport chain (ETC), participate in a plethora of physiological functions, which include ageing, cell growth, cell proliferation, and immune response and regulation. In this current review, we will focus on the mechanisms by which mt-ROS regulate different pathways of host immune responses in the context of infection by bacteria, protozoan parasites, viruses, and fungi. We will also discuss how these pathogens, in turn, modulate mt-ROS to evade host immunity. We will conclude by briefly giving an overview of the potential therapeutic approaches involving mt-ROS in infectious diseases.

This paper presents a comprehensive overview of the potential applications for Photo-Acoustic (PA) imaging employing functional nanoparticles. The exploration begins with an introduction to nanotechnology and nanomaterials, highlighting the advancements in these fields and their crucial role in shaping the future. A detailed discussion of the various types of nanomaterials and their functional properties sets the stage for a thorough examination of the fundamentals of the PA effect. This includes a thorough chronological review of advancements, experimental methodologies, and the intricacies of the source and detection of PA signals. The utilization of amplitude and frequency modulation, design of PA cells, pressure sensor-based signal detection, and quantification methods are explored in-depth, along with additional mechanisms induced by PA signals. The paper then delves into the versatile applications of photoacoustic imaging facilitated by functional nanomaterials. It investigates the influence of nanomaterial shape, size variation, and the role of composition, alloys, and hybrid materials in harnessing the potential of PA imaging. The paper culminates with an insightful discussion on the future scope of this field, focusing specifically on the potential applications of photoacoustic (PA) effect in the domain of biomedical imaging and nanomedicine. Finally, by providing the comprehensive overview, the current work provides a valuable resource underscoring the transformative potential of PA imaging technique in biomedical research and clinical practice.

The substitution of semiconductor quantum dots (QDs) by a small number of transition-metal ions with magnetic properties gives rise to magnetic-doped semiconductors. With a balance of optical and magnetic properties, these magnetic semiconductors are widely used in spintronics, bioimaging and magnetic resonance imaging (MRI) applications. To facilitate their usage in bio-applications, it is critical to synthesize water-soluble magnetic QDs with a stabilized structure while maintaining their optical and magnetic properties. Here in our work, we have developed a facile substituted synthetic route to achieve Cr-doped CdSe (Cr-CdSe) via hydrothermal method. The effects of doping on the structural, optical, and magnetic properties of Cr-CdSe were studied using X-ray diffraction, UV-visible spectroscopy, and photoluminescence lifetime. We then explored their chemical nature and change in morphology with an increase in doping concentration via X-ray photoelectron spectroscopy and transmission electron microscopy. Water-soluble QDs have been used as bioimaging probes for the past few decades due to their strong fluorescence, photostability and improved tissue or cellular penetration. However, incorporating magnetic material into a fluorescent entity harnesses the ability to control the strengths of both modalities, which enhances diagnostic accuracy and facilitates its application in bio-systems, especially in early accurate diagnosis. Finally, we demonstrate the competency of Cr-CdSe as a dual-imaging probe with fluorescent cellular imaging and MRI applications.

Electric vehicles (EVs) energized with electricity derived from renewable energy power systems can aid in reducing carbon emissions from road transport. But to enable faster adoption of EVs, increasing the distance traveled when the battery is fully charged, and fast charging is necessary. At the same time, effective thermal management in battery systems plays a vital role in enhancing the performance, safety, and longevity of Li-ion batteries (LiBs). Thus, designing a cost-effective battery TM system is necessary for faster adoption of EVs. Among the various available TM systems for LiBs, the external thermal management technique was found to be more effective when compared to passive thermal management. The external LiB thermal management system incorporated with phase change material (PCM) can enable effective dissipation of heat from it with minimal energy requirement. However, the performance of these systems can be further enhanced by enhancing their thermal conductivity by suspending nanoparticles. However, the selection of appropriate PCM is essential to ensure effective thermal management. Hence, the central focus of this review is to identify the key parameters that affect the performance of PCM-based thermal management in LiBs. The paper also explores different battery thermal management (BTM) system architectures, encompassing carbon-based, metal-based, and hybrid solutions, delineating their respective strengths and limitations. The review integrates insights on thermal conductivity correlations established by previous research works. These correlations enable the prediction of thermal behavior in BTM materials, streamlining the design and optimization process. By addressing these limitations, the transition to sustainable and environmentally friendly transportation systems is a global imperative to combat climate change.

Jumbo phages are characterized by their remarkably large-sized genome and unique life cycles. Jumbo phages belonging to Chimalliviridae family protect the replicating phage DNA from host immune systems like CRISPR-Cas and restriction-modification system through a phage nucleus structure. Several recent studies have provided new insights into jumbo phage infection biology, but the pan-genome diversity of jumbo phages and their relationship with CRISPR-Cas targeting beyond Chimalliviridae are not well understood. In this study, we used pan-genome analysis to identify orthologous gene families shared among 331 jumbo phages with complete genomes. We show that jumbo phages lack a universally conserved set of core genes but identified seven “soft-core genes” conserved in over 50% of these phages. These genes primarily govern DNA-related activities, such as replication, repair, or nucleotide synthesis. Jumbo phages exhibit a wide array of accessory and unique genes, underscoring their genetic diversity. Phylogenetic analyses of the soft-core genes revealed frequent horizontal gene transfer events between jumbo phages, non-jumbo phages, and occasionally even giant eukaryotic viruses, indicating a polyphyletic evolutionary nature. We categorized jumbo phages into 11 major viral clusters (VCs) spanning 130 sub-clusters, with the majority being multi-genus jumbo phage clusters. Moreover, through the analysis of hallmark genes related to CRISPR-Cas targeting, we predict that many jumbo phages can evade host immune systems using both known and yet-to-be-identified mechanisms. In summary, our study enhances our understanding of jumbo phages, shedding light on their pan-genome diversity and remarkable genome protection capabilities. IMPORTANCE Jumbo phages are large bacterial viruses known for more than 50 years. However, only in recent years, a significant number of complete genome sequences of jumbo phages have become available. In this study, we employed comparative genomic approaches to investigate the genomic diversity and genome protection capabilities of the 331 jumbo phages. Our findings revealed that jumbo phages exhibit high genetic diversity, with only a few genes being relatively conserved across jumbo phages. Interestingly, our data suggest that jumbo phages employ yet-to-be-identified strategies to protect their DNA from the host immune system, such as CRISPR-Cas.

Successful development of phage-based therapeutics and their utility predominantly depend on the mode and route of phage administration. Topical and site-directed phage application evokes minimal immune clearance and allows more phage-host adsorption, thereby ensuring higher phage efficacy. However, a notable drawback of conventional topical phage applications is the absence of sustained release. Occlusive emollients guarantee the controlled release of active pharmaceutical ingredients (APIs), thereby facilitating administration, preventing moisture loss, and acting as a skin barrier. In this study, we developed phage and human platelet lysate (h-PL) incorporated cetomacrogol-based creams for combined phage therapy and wound healing. The base material for phage immobilization was formulated by emulsifying paraffin and sterile water with cetomacrogol (emulsifying agent). Specifically, we incorporated a Pseudomonas aeruginosa-infecting lytic phage vB_PaeM_M12PA in the formulation and characterized its genome in this study. Cetomacrogol, a nonionic PEG (polyethylene glycol) based ether, rendered phage stability and allowed initial burst release followed by continuous controlled release of phages from the embedding matrix in the initial 6-8 h. Rheological studies showed that the material has elastic properties with storage moduli (G′) values ranging from 109.51 ± 2.10 to 126.02 ± 3.13 kPa, indicating frequency-independent deformation. Platelet lysates in the cream acted as wound healing agents, and in vitro evaluation of cell migration and wound healing capacity of h-PL showed a significant enhancement by the sixth hour compared to untreated groups. The phage-incorporated cream showed sustained phage release in solid media and a significant reduction in bacterial growth in liquid cultures. In vivo wound healing studies in 6-week-old Wistar rats with full-thickness excision wounds and subsequent histopathological studies showed that the formulation enhanced wound healing and tissue restoration efficiency. In conclusion, the study unveils a promising approach for integrated phage therapy and wound healing strategies.

Actin is a major cytoskeletal system that mediates the intricate organization of macromolecules within cells. The bacterial cytoskeletal protein MreB is a prokaryotic actin-like protein governing the cell shape and intracellular organization in many rod-shaped bacteria, including pathogens. MreB stands as a target for antibiotic development, and compounds like A22 and its analogue, MP265, are identified as potent inhibitors of MreB. The bacterial actin MreB shares structural homology with eukaryotic actin despite lacking sequence similarity. It is currently not clear whether small molecules that inhibit MreB can act on eukaryotic actin due to their structural similarity. In this study, we investigate the molecular interactions between A22 and its analogue MP265 with MreB and eukaryotic actin through a molecular dynamics approach. Employing MD simulations and free energy calculations with an all-atom model, we unveil the robust interaction of A22 and MP265 with MreB, and substantial binding affinity is observed for A22 and MP265 with eukaryotic actin. Experimental assays reveal A22’s toxicity to eukaryotic cells, including yeast and human glioblastoma cells. Microscopy analysis demonstrates the profound effects of A22 on actin organization in human glioblastoma cells. This integrative computational and experimental study provides new insights into A22’s mode of action, highlighting its potential as a versatile tool for probing the dynamics of both prokaryotic and eukaryotic actins.

Bacteriophages, the natural predators of bacteria, are incredibly potent candidates to counteract antimicrobial resistance (AMR). However, the rapid development of phage-resistant mutants challenges the potential of phage therapy. Understanding the mechanisms of bacterial adaptations to phage predation is crucial for phage-based prognostic applications. Phage cocktails and combinatorial therapy, using optimized dosage patterns of antibiotics, can negate the development of phage-resistant mutations and prolong therapeutic efficacy. In this study, we describe the characterization of a novel bacteriophage and the physiology of phage-resistant mutant developed during infection. M12PA is a P. aeruginosa-infecting bacteriophage with Myoviridae morphology. We observed that prolonged exposure of P. aeruginosa to M12PA resulted in the selection of phage-resistant mutants. Among the resistant mutants, pyomelanin-producing mutants, named PA-M, were developed at a frequency of 1 in 16. Compared to the wild-type, we show that PA-M mutant is severely defective in virulence properties, with altered motility, biofilm formation, growth rate, and antibiotic resistance profile. The PA-M mutant exhibited reduced pathogenesis in an allantoic-infected chick embryo model system compared to the wild-type. Finally, we provide evidence that combinatory therapy, combining M12PA with antibiotics or other phages, significantly delayed the emergence of resistant mutants. In conclusion, our study highlights the potential of combinatory phage therapy to delay the development of phage-resistant mutants and enhance the efficacy of phage-based treatments against P. aeruginosa.

PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways. (Figure presented.) Mono-ADP-ribosylation has emerged as a crucial factor in innate immune responses, but is understudied due to the lack of sensitive detection methods. This study visualizes endogenous interferon-induced ADP-ribosylation and shows that PARP14 is a major enzyme responsible for this signalling event. Immunity responses induce PARP14-dependent ADP-ribosylation. SARS2-CoV2 Mac1 can remove PARP14-dependent ADP-ribosylation. PARP14, PARP9 and DTX3L regulate the formation of ubiquitin and ADPr foci in the cytoplasm. PARP14 activity is regulated by PARP9/DTX3L, through (1) the hydrolytic activity of PARP9 and (2) PARP14 interaction with DTX3L.