The lung microbiome has recently gained attention for potentially affecting respiratory viral infections, including influenza A virus, respiratory syncytial virus (RSV) and SARS-CoV-2. We will discuss the complexities of the lung microenvironment in the context of viral infections and the use of organ-on-chip (OoC) models in replicating the respiratory tract milieu to aid in understanding the role of temporary microbial colonization. Leveraging the innovative capabilities of OoC, particularly through integrating gut and lung models, opens new avenues to understand the mechanisms linking inter-organ crosstalk and respiratory infections. We will discuss technical aspects of OoC lung models, ranging from the selection of cell substrates for extracellular matrix mimicry, mechanical strain, breathing mechanisms and air-liquid interface to the integration of immune cells and use of microscopy tools for algorithm-based image analysis and systems biology to study viral infection . OoC offers exciting new options to study viral infections across host species and to investigate human cellular physiology at a personalized level. This review bridges the gap between complex biological phenomena and the technical prowess of OoC models, providing a comprehensive roadmap for researchers in the field.

Insects can survive harsh conditions, including Arctic winters, by entering a hormonally induced state of dormancy, known as diapause. Diapause is triggered by environmental cues such as shortening of the photoperiod (lengthening of the night). The time of entry into diapause depends on the latitude of the insects' habitat, and this applies even within a species: populations living at higher latitudes enter diapause earlier in the year than populations living at lower latitudes. A long-standing question in biology is whether the internal circadian clock, which governs daily behaviour and serves as a reference clock to measure night length, shows similar latitudinal adaptations. To address this question, we examined the onset of diapause and various behavioural and molecular parameters of the circadian clock in the cosmopolitan fly, , a species distributed throughout Europe from the Black Sea (41° N) to Arctic regions (69° N). We found that all clock parameters examined showed the same correlation with latitude as the critical night length for diapause induction. We conclude that the circadian clock has adapted to the latitude and that this may result in the observed latitudinal differences in the onset of diapause.

Pigment-dispersing factors (PDFs) are neuropeptides that play key roles in controlling the circadian rhythms in various insects, whereas their function remains elusive in other protostomes including tardigrades (water bears). Here we show that the three PDFs of the tardigrade are co-localized in two pairs of inner lobe cells in the brain, whereas only one PDF occurs in four additional cerebral and two extracerebral cells. The axons of the inner lobe cells pass through the contralateral brain hemisphere, descend to the ventral nerve cord and terminate in two pairs of potential release sites in the posteriormost trunk ganglion. Using assays, we demonstrate that all three PDFs and their deorphanized receptor (PDFR) are functional. Widespread localization of PDFR suggests that tardigrade PDFs may act as multifunctional hormones and neuromodulators that control major functions including light detection, neural processing, locomotion, feeding, digestion, osmoregulation, growth, embryonic development and oogenesis/reproduction.

Metabolic processes in fetuses can significantly influence piglet weight at birth. Understanding the genetic determinants of systemic metabolism is crucial for uncovering how genetic and molecular pathways impact biological mechanisms, particularly during the fetal phase. We present data on 1112 plasma metabolites using untargeted ultra-high performance liquid chromatography-tandem mass spectrometry methods, of 260 backcross (BC) fetuses from two sires' breeds at 63 days post-conception. Eight chemical superclasses have been identified, with lipids accounting for the majority of metabolites. Genomic heritability (h²) was estimated for each metabolite, revealing that 50% had h² values below 0.2, with a higher average in the amino acid class compared with the lipid. We annotated 448 significant metabolite quantitative trait loci associated with 10 metabolites, primarily lipids, indicating strong genetic regulation. Additionally, metabolite associations with sex, fetal weight and sire's breed were explored, revealing significant associations for 354 metabolites. Fetal weight influenced the largest number of metabolites, particularly glycerophospholipids and sphingolipids, emphasizing the genetic and metabolic complexity underlying fetal development. These findings enhance our understanding of the genetic regulation of metabolite levels and their associations with key phenotypic traits in fetuses, providing insights into metabolic pathways, potential biomarkers and serving as a baseline dataset for metabolomics studies of fetuses.

In the peripheral nervous system, glial cells, known as Schwann cells (SCs), are responsible for supporting and maintaining nerves. One of the most important characteristics of SCs is their remarkable plasticity. In various injury contexts, SCs undergo a reprogramming process that generates specialized cells to promote tissue regeneration and repair. However, in pathological conditions, this same plasticity and regenerative potential can be hijacked. Different studies highlight the activation of the epithelial-mesenchymal transition (EMT) as a driver of SC phenotypic plasticity. Although SCs are not epithelial, their neural crest origin makes EMT activation crucial for their ability to adopt repair phenotypes, mirroring the plasticity observed during development. These adaptive processes are essential for regeneration. However, EMT activation in SCs-derived tumours enhances cancer progression and aggressiveness. Furthermore, in the tumour microenvironment (TME), SCs also acquire activated phenotypes that contribute to tumour migration and invasion by activating EMT in cancer cells. In this review, we will discuss how EMT impacts SC plasticity and function from development and tissue regeneration to pathological conditions, such as cancer.

Biotin identification (BioID) is an interactomics approach that utilizes proximity labelling to map the local interactome or proxeome of proteins within a cell. This study applies BioID to investigate proteins proximal to NAA60 (N-alpha-acetyltransferase 60), an N-terminal acetyltransferase (NAT) of pathological significance in human disease, characterized by its unique Golgi localization. NAA60 is known to N-terminally acetylate transmembrane proteins that present their N-terminus on the cytosolic face of the membrane, and its involvement in maintaining Golgi structure has previously been established. Using a stable cell-line expressing an NAA60-BirA* fusion protein, we isolated biotinylated proteins through streptavidin affinity purification. Mass spectrometry analysis revealed over 100 proximal partners of NAA60, enriched in proteins localized on the -side of the Golgi apparatus. High-confidence proximity interactors included golgins and GRASP proteins, essential for Golgi integrity. Considering the transmembrane nature of NAA60, the identification of biotinylated peptides inferred the topology of transmembrane protein interactors within the secretory pathway. Subsequent suborganellar localization analysis revealed a more prominent /-Golgi localization of NAA60. Our findings underscore the role of NAA60 and its interactors in maintaining Golgi structural integrity and highlight the effectiveness of BioID in generating critical protein topology data, invaluable for enhancing the prediction of protein topology within cellular compartments.

The human HELQ helicase is a superfamily 2, 3'-5 helicase homologous to POLQ and RNA helicases of the Ski2-like subfamily. It is involved in diverse aspects of DNA repair and is an emerging prognosis biomarker and novel drug target for cancer therapy. HELQ interacts with RPA through its inherently disordered N-HELQ domain and hence is recruited to RPA-bound DNA substrates. Our study reveals a novel role for HELQ in R-loop resolution. We show in cells and that HELQ is recruited by RPA at R-loops, which are then resolved if HELQ is catalytically active as an ATPase/helicase. Furthermore, we identify a functional interaction of HELQ with XRN2, a nuclear 5' to 3' exoribonuclease, which we suggest coordinates R-loop unwinding by HELQ with RNA digestion by XRN2. Collectively, we assign a new biological function for HELQ in genome stability in metazoans through its involvement with XRN2 in R-loop metabolism.

High cholesterol levels are associated with an increased risk of cardiovascular disease, specifically atherosclerosis, a leading cause of death worldwide. Atherosclerosis occurs when cholesterol and fat build up in plaques along blood vessel walls, restricting blood flow and preventing nutrients and oxygen from diffusing in and out of the bloodstream. High-density lipoprotein cholesterol (HDL) particles prevent the build-up of such plaques, removing excess cholesterol from the peripheral tissues and delivering it to the liver, where it can be removed from the body. This pathway is known as reverse cholesterol transport (RCT). Because HDL plays a key role in preventing plaque buildup, understanding how this molecule and RCT function in the body could help us develop much-needed new atherosclerosis therapies and prevention strategies. However, HDL metabolism is complex, and research on HDL has been less favoured than research investigating a much better-understood molecule, low-density lipoprotein cholesterol, as a treatment target. More specifically, the receptors involved in the process of taking up HDL within the liver and their relationships to one another, along with the mechanism of whole, or holoparticle uptake of HDL remain to be clarified. In this review, we discuss several outstanding mysteries in HDL metabolism, consider why previous clinical trials to improve cardiovascular health by modulating HDL levels have been unsuccessful and argue that understanding HDL metabolism is essential for crafting interventions to reduce cardiovascular disease risk.

Scriptaid is a chemical compound with anti-tumoural effects due to its role as a histone deacetylase inhibitor. Despite sharing part of the chemical structure with other ligands of G-quadruplexes (G4s), the interaction of Scriptaid with G4s has not been explored before. We synthesized Scriptaid and screened its cytotoxic activity in cellular models of colorectal cancer (CRC). We extensively evaluated its biological activity by cell cycle, immunofluorescence, qRT-PCR and Western blot experiments. To identify the G4 targets of Scriptaid, we conducted a panel of binding assays. Here, we show that Scriptaid induced cytotoxicity, cell cycle arrest and nucleolar stress in CRC cells. Moreover, Scriptaid impaired RNA polymerase I (Pol I) transcription, stabilized G4s and caused DNA damage. Finally, we disclose that these effects were attributable to the binding of Scriptaid to G4s in ribosomal DNA. In conclusion, our work reveals that a primary impact of Scriptaid on human cells is the interaction with G4s.

Glycolysis, present in most organisms, is evolutionarily one of the oldest metabolic pathways. It has great relevance at a physiological level because it is responsible for generating ATP in the cell through the conversion of glucose into pyruvate and reducing nicotinamide adenine dinucleotide (NADH) (that may be fed into the electron chain in the mitochondria to produce additional ATP by oxidative phosphorylation), as well as for producing intermediates that can serve as substrates for other metabolic processes. Glycolysis takes place through 10 consecutive chemical reactions, each of which is catalysed by a specific enzyme. Although energy transduction by glucose metabolism is the main function of this pathway, involvement in virulence, growth, pathogen-host interactions, immunomodulation and adaptation to environmental conditions are other functions attributed to this metabolic pathway. In humans, where glycolysis occurs mainly in the cytosol, the mislocalization of some glycolytic enzymes in various other subcellular locations, as well as alterations in their expression and regulation, has been associated with the development and progression of various diseases. In this review, we describe the role of glycolytic enzymes in the pathogenesis of diseases of clinical interest. In addition, the potential role of these enzymes as targets for drug development and their potential for use as diagnostic and prognostic markers of some pathologies are also discussed.

Migration is a widely observed phenomenon supported by morphological, physiological and behavioural traits that vary with season and sex in many species. Recently, the genetic components underpinning migration in the marmalade hoverfly (Diptera: Syrphidae) have been unpacked through detection of differentially expressed genes between migrant and non-migrant females. Males also migrate, but changing sex ratios during autumn migration, from around 50% female in northern Europe to around 90% in southern Europe, suggests males are poor long-distance fliers. To elucidate the mechanisms underpinning this sex difference, we performed morphological, physiological and transcriptomic characterization of actively migrating females and males. Both sexes show similar physiological adaptations including hyperphagia and starvation resistance, but females display higher tolerance to cold, have lower wing loading values and display a greater flight capacity. In addition, females modulate the expression of genes involved in immunity, hypoxia and longevity while suppressing hormonal pathways involved in maintaining reproductive diapause. These traits contribute to the success of female migrants and underlie the diminishing pool of males, influencing population dynamics across huge geographic areas and through the whole migratory and overwintering period.

Antimicrobial resistance (AMR) is one of the top global health threats. In 2019, AMR was associated with 4.95 million deaths, of which 1.97 million were caused by drug-resistant infections directly. The main subset of AMR is antibiotic resistance, that is, the resistance of bacteria to antibiotic treatment. Traditional and most commonly used antibiotic susceptibility tests are based on the detection of bacterial growth and its inhibition in the presence of an antimicrobial. These tests typically take over 1-2 days to perform, so empirical therapy schemes are often administered before proper testing. Rapid tests for AMR are necessary to optimize the treatment of bacterial infection. Here, we combine the MTT test with Raman spectroscopy to provide a 1.5 h long test for minimal inhibitory concentration determination. Several and strains were tested with three types of antibiotics, including ampicillin from penicillin family, kanamycin from aminoglycoside family and levofloxacin from fluoroquinolone family. The test provided the same minimal inhibitory concentrations as traditional Etest confirming its robustness.

The PACS (phosphofurin acidic cluster sorting protein) proteins are membrane trafficking regulators, required for maintaining cellular homeostasis and preventing disease states. Mutations in human and cause human neurodevelopmental disorders, characterized by epileptic seizures and neurodevelopmental delay. In vertebrates, functional analysis of PACS proteins is complicated by the presence of two paralogues which can compensate for the loss of each other. Here, we characterize the unique fly homologue of human PACS proteins. We demonstrate that Drosophila PACS (dPACS) is required for cell division in dividing spermatocytes and neuroblasts. In primary spermatocytes, dPACS colocalizes with GOLPH3 at the Golgi stacks and is essential for maintaining Golgi architecture. In dividing cells, dPACS is necessary for central spindle stability and contractile ring constriction. dPACS and GOLPH3 proteins form a complex and are mutually dependent for localization to the cleavage site. We propose that dPACS, by associating with GOLPH3, mediates the flow of vesicle trafficking that supports furrow ingression during cytokinesis. Furthermore, loss of dPACS leads to defects in tubulin acetylation and severe bang sensitivity, a phenotype associated with seizures in flies. Together our findings suggest that a Drosophila disease model may contribute to understanding the molecular mechanisms underpinning human PACS syndromes.

Emerging infectious diseases can have a major impact on fitness of novel hosts, thereby contributing to ongoing species declines. In social insects, collaborative brood care by workers and successful mating of male sexuals are key to colony fitness. The microsporidian endoparasite has spread almost globally, shifting across honeybee species and now to bumblebees. However, despite being linked to recent population declines, its possible impact on bumblebee colony fitness remains poorly understood. Here, we show that infections can significantly impact worker feeding glands, as well as longevity, sperm quality and mating abilities of drones. In the laboratory, workers and drones were either exposed to the parasite or not. Then, parasite infection rates and loads, as well as lethal and sublethal parameters, were assessed. Infected drones revealed higher parasite infection rates and spore titres, as well as mortality compared with female workers, suggesting sex-specific susceptibility. Furthermore, infections impaired feeding glands, affected sperm traits and altered mating behaviour, all of which are key to colony fitness. Our findings provide a mechanistic explanation on how contributes to the ongoing decline of wild bumblebee populations, calling for respective mitigation measures.

Social immunity-mediated sanitation behaviours occur in insects when microbially killed corpses are removed and/or dismembered by healthy nestmates. However, little is known concerning the chemical signals or receptor proteins that mediate these responses. Here, we identify cuticular components in the eusocial red important fire ant, : behenic acid, which induces dismemberment behaviour, and oleic and ,-9,12-linoleic acids, which inhibit dismemberment in a process mediated by odorant-binding protein-15 (SiOBP15). Yeast two-hybrid screening and protein-protein interaction analyses identified the ant immunity-related proteins apolipophorin-III (SiApoLp-III) and fatty acid binding protein-5 (SiFABP5) as SiOBP15 interacting partners. SiOBP15 and SiFABP5 bound all three dismemberment-related compounds, whereas interactions between SiOBP15 and SiApoLp-III narrowed binding to behenic acid. RNAi-mediated gene expression knockdown of , or revealed that behenic acid chemoreception determines dismemberment behaviour via SiApoLp-III/SiOBP15, whereas SiOBP15 or SiOBP15/SiFABP5 recognition of linoleic acid inhibits dismemberment behaviour. These data identify a host circuit linking olfactory proteins and proteins involved in innate immunity to control the degree of sanitation behaviour elicited in response to microbial infection. We identify specific chemical cues transduced by these proteins, providing a mechanism connecting olfaction-related processes to innate immunity, host-pathogen interactions and social immunity.

This open question research article highlights mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), which have emerged as crucial cellular structures that challenge our traditional understanding of organelle function. This review highlights the critical importance of MAMs as a frontier in cell biology with far-reaching implications for health, disease and ageing. MAMs serve as dynamic communication hubs between the ER and mitochondria, orchestrating essential processes such as calcium signalling, lipid metabolism and cellular stress responses. Recent research has implicated MAM dysfunction in a wide array of conditions, including neurodegenerative diseases, metabolic disorders, cardiovascular diseases and cancer. The significant lack of biological knowledge behind MAM function emphasizes the need to study these enigmatic subcellular sites in greater detail. Key open questions include the mechanisms controlling MAM formation and disassembly, the full complement of MAM-associated proteins and how MAMs contribute to cellular decision-making and ageing processes. Advancing our understanding of MAMs through interdisciplinary approaches and cutting-edge technologies promises to reveal new insights into fundamental cellular signalling pathways and potentially lead to innovative therapeutic strategies for a range of diseases. As such, MAM research represents a critical open question in biology with the potential to transform our understanding of cellular life and human health.

Secretory cargos are exported from the ER via COPII-coated vesicles that have an inner matrix of Sec23/Sec24 heterotetramers and an outer cage of Sec13/Sec31 heterotetramers. In addition to COPII, Sec13 is part of the nuclear pore complex (NPC) and the regulatory SEA/GATOR complex in eukaryotes, which typically have one Sec13 orthologue. The kinetoplastid parasite has two paralogues: TbSec13.1, an accepted component of both COPII and the NPC, and TbSec13.2. Little is known about TbSec13.2, but others have proposed that it, and its orthologue in the distantly related diplonemid , operate exclusively in the SEA/GATOR complex, and that this represents an evolutionary diversification of function unique to the euglenozoan protists. Using RNAi silencing in trypanosomes, we show both TbSec13s are essential. Knockdown of each dramatically and equally delays transport of GPI-anchored secretory cargo, indicating roles for both in COPII-mediated trafficking from the ER. Immunofluorescence and proximity labelling studies confirm that both TbSec13.1 and TbSec13.2 co-localize with TbSec24.1 to ER exit sites, and thus are functional components of the COPII machinery. Our findings indicate that TbSec13.2 function is not restricted to the SEA/GATOR complex in trypanosomes.

The conserved process of centriole duplication requires the establishment of a Sas6-centred cartwheel initiated by Plk4's phosphorylation of Ana1/STIL. Subsequently, the centriole undergoes conversion to a centrosome requiring its radial expansion and elongation, mediated by a network requiring interactions between Cep135, Ana1/Cep295 and Asterless/Cep152. Here, we show that mutant alleles encoding overlapping N- and C-terminal parts of Ana1 are capable of intragenic complementation to rescue radial expansion. This permits the recruitment of Asl and thereby centriole duplication and mechanosensory cilia formation to restore the coordination defects of these mutants. This genetic combination also rescues centriole duplication in the male germ line but does not rescue the elongation of the triplet microtubule-containing centrioles of primary spermatocytes. Consequently, these males are coordinated but sterile. Such centriole elongation is rescued by the continuous, full-length Ana1 sequence. We define a region that when deleted within otherwise intact Ana1 does not permit primary spermatocyte centrioles to elongate but still allows recruitment of Asl. Our findings point to differing demands upon the physical organization of Ana1 for the distinct processes of radial expansion and elongation of centrioles.

Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six proteins and demonstrate (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.

Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenic models expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. We show that these reporters are colocalized with a peroxisomal marker and that they can reflect pexophagy induction by iron chelation and inhibition by depletion of the core autophagy protein Atg5. Using light sheet microscopy, we have been able to obtain a global overview of pexophagy levels across the entire organism at different stages of development. Tissue-specific control of pexophagy is exemplified by areas of peroxisome abundance but minimal pexophagy, observed in clusters of oenocytes surrounded by epithelial cells where pexophagy is much more evident. Enhancement of pexophagy was achieved by feeding flies with the iron chelator deferiprone, in line with past results using mammalian cells. Specific drivers were used to visualize pexophagy in neurons, and to demonstrate that specific depletion in the larval central nervous system of Hsc70-5, the homologue of the chaperone HSPA9/mortalin, led to a substantial elevation in pexophagy.

Antibodies are valuable biological reagents used in a wide range of discovery research, biotechnology, diagnostic and therapeutic applications. Currently, both commercial and laboratory-scale antibody production is reliant on expression from mammalian cells, which can be time-consuming and requires the use of specialist facilities and costly growth reagents. Here, we describe a simple, rapid and cheap method for producing and isolating functional monoclonal antibodies and antibody fragments from bacterial cells that can be used in a range of laboratory applications. This simple method only requires access to basic microbial cell culture and molecular biology equipment, making scalable in-house antibody production accessible to the global diagnostics, therapeutics and molecular bioscience research communities.