Tissue-specific transcriptional programming of macrophages controls the microRNA transcriptome targeting multiple functional pathways.

Recent interest in the biology and function of peritoneal tissue resident macrophages (pMΦ) has led to a better understanding of their cellular origin, programming, and renewal. The programming of pMΦ is dependent on microenvironmental cues and tissue-specific transcription factors, including GATA6. However, the contribution of microRNAs remains poorly defined. We conducted a detailed analysis of the impact of GATA6 deficiency on microRNA expression in mouse pMΦ. Our data suggest that for many of the pMΦ, microRNA composition may be established during tissue specialization and that the effect of GATA6 knockout is largely unable to be rescued in the adult by exogenous GATA6. The data are consistent with GATA6 modulating the expression pattern of specific microRNAs, directly or indirectly, and including miR-146a, miR-223, and miR-203 established by the lineage-determining transcription factor PU.1, to achieve a differentiated pMΦ phenotype. Lastly, we showed a significant dysregulation of miR-708 in pMΦ in the absence of GATA6 during homeostasis and in response to LPS/IFN-γ stimulation. Overexpression of miR-708 in mouse pMΦ in vivo altered 167 mRNA species demonstrating functional downregulation of predicted targets, including cell immune responses and cell cycle regulation. In conclusion, we demonstrate dependence of the microRNA transcriptome on tissue-specific programming of tissue macrophages as exemplified by the role of GATA6 in pMΦ specialization.

Improved DiOlistic labelling technique for neurons in situ: Detailed visualisation of dendritic spines and concurrent histochemical-detection in fixed tissue.

DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research.

A Synoptic Review of Plant Disease Epidemics and Outbreaks Published in 2022.

This scientometric study reviews the scientific literature and CABI distribution records published in 2022 to find evidence of major disease outbreaks and first reports of pathogens in new locations or on new hosts. This is the second time we have done this, and this study builds on our work documenting and analyzing reports from 2021. Pathogens with three or more articles identified in 2022 literature were Xylella fastidiosa, Bursaphelenchus xylophilus, Meloidogyne species complexes, 'Candidatus Liberibacter asiaticus', Raffaelea lauricola, Fusarium oxysporum formae specialis, and Puccinia graminis f. sp. tritici. Our review of CABI distribution records found 29 pathogens with confirmed first reports in 2022. Pathogens with four or more first reports were Meloidogyne species complexes, Pantoea ananatis, grapevine red globe virus, and Thekopsora minima. Analysis of the proportion of new distribution records from 2022 indicated that grapevine red globe virus, sweet potato chlorotic stunt virus, and 'Ca. Phytoplasma vitis' may have been actively spreading. As we saw last year, there was little overlap between the pathogens identified by reviewing scientific literature versus distribution records. We hypothesize that this lack of concordance is because of the unavoidable lag between first reports of the type reported in the CABI database of a pathogen in a new location and any subsequent major disease outbreaks being reported in the scientific literature, particularly because the latter depends on the journal policy on types of papers to be considered, whether the affected crop is major or minor, and whether the pathogen is of current scientific interest. Strikingly, too, there was also no overlap between species assessed to be actively spreading in this year's study and those identified last year. We hypothesize that this is because of inconsistencies in sampling coverage and effort over time and delays between the first arrival of a pathogen in a new location and its first report, particularly for certain classes of pathogens causing only minor or non-economically damaging symptoms, which may have been endemic for some time before being reported. In general, introduction of new pathogens and outbreaks of extant pathogens threaten food security and ecosystem services. Continued monitoring of these threats is essential to support phytosanitary measures intended to prevent pathogen introductions and management of threats within a country.

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

Review of per- and poly-fluoroalkyl treatment in combustion-based thermal waste systems in the United States.

Per- and poly-fluoroalkyl substances (PFAS) are a class of synthetic chemicals known for their widespread presence and environmental persistence. Carbon-fluorine (C-F) bonds are major components among PFAS and among the strongest organic bonds, thus destroying PFAS may present significant challenge. Thermal treatment such as incineration is an effective and approved method for destroying many halogenated organic chemicals. Here, we present the results of existing studies and testing at combustion-based thermal treatment facilities and summarize what is known regarding PFAS destruction and mineralization at such units. Available results suggest the temperature and residence times reached by some thermal treatment systems are generally favorable to the destruction of PFAS, but the possibility for PFAS or fluorinated organic byproducts to escape destruction and adequate mineralization and be released into the air cannot be ruled out. Few studies have been conducted at full-scale operating facilities, and none to date have attempted to characterize possible fluorinated organic products of incomplete combustion (PICs). Further, the ability of existing air pollution control (APC) systems, designed primarily for particulate and acid gas control, to reduce PFAS air emissions has not been determined. These data gaps remain primarily due to the previous lack of available methods to characterize PFAS destruction and PIC concentrations in facility air emissions. However, newly developed stack testing methods offer an improved understanding of the extent to which thermal waste treatment technologies successfully destroy and mineralize PFAS in these waste streams.

Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors.

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

Pre-migratory flights in migrant songbirds: the ecological and evolutionary importance of understudied exploratory movements.

Across the animal kingdom, from honeybees to cranes to beavers, exploratory movements to exploit resources, scout prospective territories, or otherwise gain valuable experiences and information that promote fitness have been documented. For example, exploratory movements to investigate potential dispersal targets have been observed in roe deer, Northern cardinals, and tigers alike. However, despite how widespread these movements are, a cohesive definition of exploratory movements has been lacking. We first provide a clear definition of exploratory movements, and use one particular group-migratory songbirds-to catalogue exploratory movements across the annual cycle. The exceptional mobility of migratory songbirds results in exploratory movements not only at a local scale, but also on a regional scale, both in and out of the breeding season. We review the extent to which these movements are made within this group, paying particular attention to how such movements confer fitness benefits, as by securing high-quality territories, prospecting for extra-pair paternity, or even exploiting ephemeral resources. We then zoom in one step further to a particular exploratory movement that has been, to date, almost completely overlooked within this group: that of pre-migratory flights. These flights, which occur during the transitional period between the stationary breeding period and the onset of migration, occur at night and may not be made by all individuals in a population-reasons why these flights have been heretofore critically understudied. We provide the first definition for this behaviour, summarise the current knowledge of this cryptic movement, and hypothesise what evolutionary/ecological advantages conducting it may confer to the individuals that undertake it. As these flights provide experience to the individuals that undertake them, we expect that birds that make pre-migratory flights are better equipped to survive migration (direct fitness benefits) and, due to orientation/navigation abilities, may also reach preferred territories on breeding and wintering grounds faster (indirect fitness benefits). We hope to encourage ecologists to consider such hidden movements in their research concepts and to enhance the framework of movement ecology by this behaviour due to its presumed high biological importance to the annual cycle of birds.