Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.

Bacterial species able to produce proteins that are toxic against insects have been discovered at the beginning of the last century. However, up to date only two of them have been used as pesticides in mosquito control strategies targeting larval breeding sites: Bacillus thuringensis var. israelensis and Lysinibacillus sphaericus. Aiming to expand the arsenal of biopesticides, bacterial cultures from 44 soil samples were assayed for their ability to kill larvae of Aedes albopictus. A method to select, grow and test the larvicidal capability of spore-forming bacteria from each soil sample was developed. This allowed identifying 13 soil samples containing strains capable of killing Ae. albopictus larvae. Among the active isolates, one strain with high toxicity was identified as Brevibacillus laterosporus by 16S rRNA gene sequencing and by morphological characterization using transmission electron microscopy. The new isolate showed a larvicidal activity significantly higher than the B. laterosporus LMG 15441 reference strain. Its genome was phylogenomically characterized and compared to the available Brevibacillus genomes. Thus, the new isolate can be considered as a candidate adjuvant to biopesticides formulations that would help preventing the insurgence of resistance.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Decades of investigation on genomic DNA have brought us deeper insights into its organization within the nucleus and its metabolic mechanisms. This was fueled by the parallel development of experimental techniques and has stimulated model building to simulate genome conformation in agreement with the experimental data. Here, we will discuss our recent discoveries on the chromatin units of DNA replication and DNA damage response. We will highlight their remarkable structural similarities and how both revealed themselves as clusters of nanofocal structures each on the hundred thousand base pair size range corresponding well with chromatin loop sizes. We propose that the function of these two global genomic processes is determined by the loop level organization of chromatin structure with structure dictating function. Abbreviations: 3D-SIM: 3D-structured illumination microscopy; 3C: chromosome conformation capture; DDR: DNA damage response; FISH: fluorescent in situ hybridization; Hi-C: high conformation capture; HiP-HoP: highly predictive heteromorphic polymer model; IOD: inter-origin distance; LAD: lamina associated domain; STED: stimulated emission depletion microscopy; STORM: stochastic optical reconstruction microscopy; SBS: strings and binders switch model; TAD: topologically associated domain Graphical abstract Landmarks of structural organization of chromatin and their relationship with metabolic functions.

Lipid nanoparticles (LNP) have been extensively studied for their ability to encapsulate and protect RNA molecules from degradation. More recently, a few studies have begun to explore their applications as carriers for brain drug delivery via various administration routes. Nose-to-brain delivery represents a promising alternative to both invasive local injections and systemic administration, offering the possibility to bypass the blood-brain barrier and directly access the brain, achieve rapid absorption, reduce systemic exposure, and allow for ease of administration. In order to evaluate the viability of this alternative route, it is essential to acquire a better understanding of the intraneuronal mass transport of LNP, particularly in terms of how effectively and efficiently they deliver their payloads from the periphery to neuronal cell bodies. However, most previous studies have focused primarily on the delivery vector itself rather than on the fate of the transported cargo. In this study, we investigate the retrograde trafficking of nucleic acid-loaded LNP in primary cortical neurons, focusing on the transport of both the particle and the payload. Three distinct LNP were formulated to characterize different aspects of their interaction with the cells, with the major LNP player of this study containing a red-fluorescent Rhodamine B-tagged lipid and a green fluorescently FAM-tagged RNA. Flow cytometry was used to document LNP uptake by primary cortical neurons over time. Additionally, confocal microscopy was then used to investigate the colocalization of LNP and RNA after a conventional 2D culture treatment. As a final step, a compartmentalized chip that separates the somal and the axonal regions of cortical neurons was used to study the intraneuronal dynamics of LNP and their cargo. In this second setup, LNP were selectively administered at the axonal compartment, and the fluorescent signals from the vector (red) and the payload (green) were imaged through time-lapse microscopy. The progressive accumulation of RNA found at cellular bodies also in the absence of the red signal suggested an efficient retrograde transport of the LNP payload toward the soma. Comprehensively, this work demonstrates that primary cortical neurons are capable of efficiently uptaking LNP and of intracellularly transporting both LNP and their RNA cargo. Interestingly, a different colocalization trend (LNP-RNA) emerged depending on the followed setup. Localized axonal transfection appeared to favor dissociation of RNA from the LNP and subsequent accumulation at the soma. Overall, our work provides a fundamental in vitro proof of concept of the RNA delivery to the cellular bodies of primary cortical neurons via the retrograde transport of LNP vectors administered at the axonal termini. This finding, together with the image-analysis-based quantification of the RNA accumulation described in our work, paves the way for future studies aimed at designing lipid-based nanoparticles for RNA therapeutic delivery to the brain via peripheral administration.

Recombinant tissue-type plasminogen activator (tPA) is the only approved thrombolytic drug for acute ischemic stroke, a condition associated with severe disabilities and high mortality. However, when the blood-brain barrier (BBB) is damaged, tPA can exacerbate cerebral injury and increase the risk of hemorrhagic transformation, limiting its use to a small subset of patients. To address this challenge and minimize extravascular accumulation, we combined tPA with micrometer-sized particles (DPN). We then tested their safety and neuroprotective effects. After a 1 h transient occlusion of the middle cerebral artery, free tPA, tPA-DPN, or saline was administered to assess mice survival, neurological behavior, and infarcted area extent. Free-tPA exacerbated brain damage, resulting in a modest 10% survival rate at 24 h post intervention. Conversely, tPA-DPN displayed a far better prognosis, with a 75% survival rate comparable to that of saline. No statistical differences were documented between tPA-DPN and saline for the Activity Score and the Neurological Severity Score. tPA-DPN did not increase lesion volume or BBB permeability, unlike free-tPA, which led to an over 2-fold enlarged lesion volume and 50% higher BBB permeability. The safety profile of tPA-DPN is attributed to the robust conjugation of tPA onto DPN and the lack of DPN extravasation, resulting in negligible cerebrovascular damage of free tPA and glial and neuron impairment. The vascular confinement of tPA linked to microscopic particles reduces drug side effects and represents a valuable strategy for safe and effective tPA delivery, even in the postacute stroke phase.