BACKGROUND:Heterozygous de novo variants in the transcription factor Activity-Dependent Neuroprotective Protein (ADNP) cause a severe neurodevelopmental disorder, termed Helsmoortel-Van der Aa syndrome (HVDAS), characterised by autism, intellectual disability, and multisystem involvement. The ADNP gene is essential for embryonic development and interacts with components of several chromatin remodelling complexes. However, the precise pathophysiological mechanisms underlying the disorder remain incompletely understood. METHODS:We used CRISPR/Cas9 genome editing to create a 14-base pair deletion c.2463_2476del (p.Leu822Hisfs∗6) in the murine Adnp gene to establish a disease-relevant mouse model. Only male mice were used in this study to reduce variability associated with sex-specific differences. Molecular, transcriptomic (RNA-seq), chromatin accessibility (ATAC-seq), proteomic (mass spectrometry), and 3D genome architecture (Hi-C) analyses were performed in cortical brain tissue. Neuroanatomical and behavioural phenotyping, including Morris water maze, elevated plus maze, marble burying, social interaction testing, and the Live Mouse Tracker were conducted to assess cognitive and autism-related phenotypes. FINDINGS:Heterozygous mice are viable and fertile. Introduction of the 14-base pair deletion reduced cellular Adnp levels in the brain (p = 0.0008) and decreased its chromatin association (p = 0.001), parallelled by a genome-wide increase in chromatin accessibility. Morphological analyses revealed mild neuroanatomical alterations in regions associated with cognition, memory, learning, and motor function (p < 0.05). Behavioural testing confirmed cognitive impairment in the Morris water maze (p < 0.05), increased anxiety-like behaviour in the elevated plus maze (p = 0.0035), repetitive behaviour in the marble burying assay (p = 0.045), and impaired social interactions (p < 0.05). Transcriptome sequencing of the frontal cortex, an essential region involved in executive functions, cognition, and motor control, revealed predominant downregulation of the Wnt signalling pathway (p = 0.03). Cytoskeletal abnormalities were further coupled to synaptic plasticity deficits, dysregulation of transcription factors implicated in lineage specification, and alterations in neuronal cell numbers. Adnp directly regulated mechanisms of synaptic plasticity through interaction with Camk2a and Dbn1. In heterozygous mice, these protein interactions were disrupted, resulting in aberrant Camk2a phosphorylation at synapses (p = 0.012). Mass spectrometry identified changes in multiple chromatin-interacting proteins and cytoskeletal components, corroborating nuclear and cytoskeletal dysregulation observed at the transcriptomic level. Hi-C analysis detected locus-specific alterations in 3D genome architecture associated with transcriptional changes. INTERPRETATION:We generated a disease-relevant heterozygous Adnp mouse model that recapitulates key molecular and behavioural features observed in patients with Helsmoortel-Van der Aa syndrome. Our findings demonstrate a role for Adnp in chromatin regulation and Wnt signalling, coupled to aberrant expression of cytoskeletal components and synaptic dysfunction. This mouse model provides a mechanistic framework linking chromatin dysregulation to autism-related behaviours and represents a valuable platform for future preclinical studies. FUNDING:This work was supported by the Marguerite-Marie Delacroix Foundation (FFP240064 to C.P.D.), the Research Fund of the University of Antwerp (Methusalem grant "GENOMED" to R.F.K.), ERA-NET NEURON ("ADNPinMED"), and the European E-RARE programme ("IMPACT"). Additional support was provided by a crowdfunding initiative from the German ADNP parent community to support C.P.D.; E.P. was supported by the FWO grant G0A1Z24N. C.A.M. received support from the Marguerite-Marie Delacroix fellowship. D.S. was supported by an EMBO fellowship (ALTF 1564-2025). F.P. was supported by a Human Frontier Science Program Long-Term Fellowship (LT000111/2021-L) and an EMBO Long-Term Fellowship (ATLF 716-2020). W.V.B. acknowledges support from the University of Antwerp (ID46420/48076/50799) and COST Action CA18127 (International Nucleosome Consortium 5INC). W.V.B., F.K., E.P., and C.P.D. acknowledge the IMPULS BOF 2024. Infrastructure support was provided by FWO (including IRI grant I000123N, GOH4216N).

Lactobacillus crispatus is a dominant member of the healthy vaginal microbiota, yet the mechanisms by which it modulates host immunity remain poorly defined, in part due to the lack of tractable in vivo models. Here, we integrate bacterial genetics, in vitro epithelial systems, human-derived data and proteomic approach (Olink®) to uncover a critical role for L. crispatus exopolysaccharides (EPS) in shaping the bacteria-vagina interactions. Comparative genomics identified a conserved EPS biosynthetic locus, with the priming glycosyltransferase gene epsE emerging as a regulatory node, in line with its distinct expression in human vaginal samples. Functional disruption of epsE abrogated L. crispatus EPS production and revealed its role for immune modulation. In human vaginal epithelial monolayers, EPS presence enhanced immune-regulatory (LAP TGF-beta-1) and anti-inflammatory (CST5) responses, whereas its absence triggered elevated pro-inflammatory cytokines (IL1β, IL6, IL8) and matrix metalloproteinase (MMP10). In a 3D vaginal organotypic model, EPS increased chemokines (CXCL5, CXCL6) linked to immune surveillance and the presence of the markers was validated in vaginal samples of healthy volunteers. These findings position EPS as a key immunomodulatory structure of L. crispatus, advancing our mechanistic understanding of host-commensal interactions and informing microbiome-based strategies to promote vaginal health.

Background & Aims: Data on changes in liver sinusoidal endothelial cells (LSECs) in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and their response to treatment are limited. This study aimed at determining (i) features associated with LSEC capillarisation in patients with MASLD; (ii) whether LSEC changes can regress with the pan-peroxisome proliferator-activated receptor (PPAR) agonist lanifibranor; (iii) the role of the different PPAR isotypes on LSEC changes in MASLD. Methods: We analysed CD34 expression, a marker of LSEC capillarisation, on liver biopsies from patients considered for inclusion in the NATIVE trial at baseline (n = 249), and after 24 weeks of placebo or lanifibranor (n = 173). Two rat models of MASLD were used to investigate the effect of lanifibranor or of mono-PPAR agonists on LSECs. Results: Lobular CD34 staining was more intense in patients with isolated steatosis than in those with no MASLD (52% vs. 10%; p = 0.03). In the overall cohort, this staining was more intense in patients with metabolic dysfunction-associated steatohepatitis (MASH) than in those without (63% vs. 41%; p = 0.01) and strongly correlated with liver fibrosis and to a lesser extent with liver inflammation. Lanifibranor treatment was associated with more common improvement in CD34 periportal staining (p = 0.025), and less frequent worsening of lobular staining (p = 0.028). Compared with healthy rats, rats with MASLD had higher CD34 staining, portal venous pressure, intrahepatic vascular resistance, and impaired liver endothelial function. Lanifibranor normalised or strongly improved these abnormalities, whereas mono-PPAR agonists caused partial improvements. Conclusions: In patients, LSEC capillarisation was increased at the earliest stages of MASLD and was associated with liver fibrosis and inflammation. In both patients and rats with MASLD, lanifibranor treatment was associated with improvement in liver endothelial phenotype. Impact and implications: Data on changes in liver sinusoidal endothelial cells (LSECs) in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and their response to treatment are limited. This study demonstrates that LSEC capillarisation is already present in the lobular zone of the liver of patients and rats at the stage of isolated steatosis, before metabolic dysfunction-associated steatohepatitis (MASH) onset, and progresses with liver fibrosis, and to a lesser extent with liver inflammation. Lanifibranor treatment, a pan-peroxisome proliferator-activated receptor agonist currently tested in a phase III clinical trial, improves LSEC capillarisation but also intrahepatic vascular resistance and portal pressure in MASLD. Targeting LSECs appears to be a promising approach to improve MASH.

The spore-forming capacity of Bacillus spp. enables environmental persistence and stable product formulations, yet the interactions of environmental Bacillus spores and vegetative cells with the human immune system are not fully understood. We investigated the immunostimulatory potential of seven environmental Bacillus isolates (B. subtilis, B. velezensis, B. licheniformis, B. pumilus) and optimized their inactivation methods to preserve surface integrity and immunostimulatory activity. Inactivation was evaluated using heat/UV-C and 10-100% formalin, followed by scanning electron microscopy and human reporter cell assays (THP1-Dual monocytes, HEK-Blue hTLR2/6 and hTLR4). Heat/UV-C treatment retained Bacillus morphology and enhanced immune activation compared to 10-100% formalin treatment. Spores and vegetative cells activated key immune transcription factors (nuclear factor kappa B and interferon regulatory factors) in human monocytes, with spores inducing 1.5- to 8-fold lower responses compared to vegetative cells, and strain- and species-dependent effects observed. All tested Bacillus isolates significantly activated Toll-like receptor TLR2/6, but not TLR4. Genome analysis identified Bacillus spore envelope components (SpsAEIK, CotBCGQ) that may influence differences in immune responses. Our findings improve understanding of Bacillus-human immune interactions, revealing strain- and species-specific immunostimulatory activity. These results support further exploration of Bacillus isolates for immune-related and environmental applications.

Fibroblast activation protein alpha is a postprolyl proteolytic enzyme highly expressed in the tumor microenvironment, particularly in cancer-associated fibroblasts. Although previously thought to be restricted to cancer-associated fibroblasts, malignant cells, and pathological fibroblasts, recent studies have identified fibroblast activation protein expression in natural killer cells. However, its expression and activity in natural killer cells remain poorly characterized. Here, we investigated fibroblast activation protein expression and activity in resting and cytokine-stimulated (interleukin-2 and interleukin-15) primary human natural killer cells and NK92 cells. Natural killer cell activation resulted in a significant decrease in fibroblast activation protein expression and enzymatic activity. Treatment with the fibroblast activation protein inhibitor UAMC-1110 altered the expression of activating and inhibitory natural killer cell receptors and reduced perforin expression, though it did not impact degranulation or cytotoxic function. Culturing natural killer cells in cancer-associated fibroblast-conditioned medium or direct co-culture with cancer-associated fibroblasts increased fibroblast activation protein expression and activity in NK92 cells, with donor-dependent effects observed in primary natural killer cells. These conditions also led to a reduction in natural killer activating and inhibitory receptor expression. Furthermore, hypoxia upregulated fibroblast activation protein in both NK92 and primary natural killer cells. Overall, our findings demonstrate that fibroblast activation protein is downregulated in/on natural killer cells upon activation with interleukin-2 and interleukin-15, whereas it is upregulated under tumor microenvironment-mimicking conditions. This may suggest that fibroblast activation protein contributes to the phenotype formation of natural killer cells, particularly within the tumor microenvironment, where we hypothesize that natural killer cells might prioritize invasive capacity over cytotoxic capacity, thus upregulating fibroblast activation protein.

Fibroblast activation protein alpha (FAP) is a post-prolyl proteolytic enzyme highly expressed in the tumor microenvironment (TME), particularly in cancer-associated fibroblasts (CAFs). Although previously thought to be restricted to CAFs, malignant cells and pathological fibroblasts, recent studies have identified FAP expression in natural killer (NK) cells. However, its expression and activity in NK cells remain poorly characterized. Here, we investigated FAP expression and activity in resting and cytokine-stimulated (IL-2 and IL-15) primary human NK cells and NK92 cells. NK cell activation resulted in a significant decrease in FAP protein expression and enzymatic activity. Treatment with the FAP inhibitor UAMC-1110 altered the expression of activating and inhibitory NK cell receptors and reduced perforin expression, though it did not impact degranulation or cytotoxic function. Culturing NK cells in CAF-conditioned medium or direct co-culture with CAFs increased FAP expression and activity in NK92 cells, with donor-dependent effects observed in primary NK cells. These conditions also led to a reduction in NK activating and inhibitory receptor expression. Furthermore, hypoxia upregulated FAP in both NK92 and primary NK cells. Overall, our findings demonstrate that FAP is downregulated in/on NK cells upon activation with IL-2 and IL-15, whereas it is upregulated under TME-mimicking conditions. This may suggest that FAP contributes to the phenotype formation of NK cells, particularly within the TME, where we hypothesize that NK cells might prioritize invasive capacity over cytotoxic capacity, thus upregulating FAP.

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.

Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.

Plant growth is ultimately driven by cell division and expansion, but how these processes are regulated to mediate a wide range of genotypic variation in organ size is still poorly understood. To address this, we screened an EMS maize mutant population to identify a new EMS maize dwarf mutant with small, pale-yellow leaves (dpl). The mutation was mapped to a region of 11.58 Mb at the 3’ end of chromosome 7. We identified Zm00001d022394 as a potential causal gene for the dpl phenotype, encoding a pentatricopeptide repeat-containing (PPR) family protein involved in chloroplast gene expression and function, explaining the pale color of dpl. Mature dpl leaves are thinner and shorter due to a reduced number of cells of approximately normal length. The chloroplasts of dpl are reduced in size and number, correlating with a decreased chlorophyll content, however chloroplast ultrastructure was not affected. Consistent with the reduced chlorophyll content photosynthetic rate of dpl were reduced by 50 % and a 30 reduction of Fv/Fm suggests photoinhibition. As a consequence, soluble and insoluble sugar levels are severely reduced throughout the leaf growth zone. At the cell level reduced cell division rates and size of the division zone, explain the reduced leaf elongation rate (LER). The growth of dpl leaves can be restored by supplying growing leaves with sucrose through their cut tips, which also restores sucrose levels in the division zone of maize leaf, demonstrating that limited sugar availability explains the reduced growth phenotype. Inversely, we phenocopied the mutant growth phenotype by inhibiting photosynthetic electron transport in wild type plants with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Our study of dpl provides a functional link between inhibition of photosynthesis, soluble sugar flux to the leaf growth zone, the regulation of cell division and whole leaf growth.

Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.

Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.

Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the Nmu promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions, in situ hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c + monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206 + MhcII hi and Timp2 + MhcII lo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206 + Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206 + Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

Approximately 20% of sleeping sickness patients exhibit respiratory complications, however, with a largely unknown role of the parasite. Here we show that tsetse fly-transmitted Trypanosoma brucei parasites rapidly and permanently colonize the lungs and occupy the extravascular spaces surrounding the blood vessels of the alveoli and bronchi. They are present as nests of multiplying parasites exhibiting close interactions with collagen and active secretion of extracellular vesicles. The local immune response shows a substantial increase of monocytes, macrophages, dendritic cells and γδ and activated αβ T cells and a later influx of neutrophils. Interestingly, parasite presence results in a significant reduction of B cells, eosinophils and natural killer cells. T. brucei infected mice show no infection-associated pulmonary dysfunction, mirroring the limited pulmonary clinical complications during sleeping sickness. However, the substantial reduction of the various immune cells may render individuals more susceptible to opportunistic infections, as evident by a co-infection experiment with respiratory syncytial virus. Collectively, these observations provide insights into a largely overlooked target organ, and may trigger new diagnostic and supportive therapeutic approaches for sleeping sickness.

Objective: sensorineural hearing loss (SNHL) and bilateral vestibulopathy (BV) have been associated with cognitive decline and incident dementia. Our aim was to investigate the combined effect of profound SNHL and BV on spatial cognition and hippocampal neurogenesis in adult mice. Methods: Single oral intake of allylnitrile produces otovestibular failure in less than a week. Behavioral assessment included recording of spontaneous activity, motor activity, spatial cognition, etc. Evaluation of hippocampal neurogenesis was performed 8 weeks after treatment by quantification of neural precursor cells and proliferating cells in the dentate gyrus by staining with doublecortin (Dcx) and Ki67, respectively. Results: Profound SNHL and BV were confirmed in the allylnitrile-treated mice respectively by means of auditory brainstem response (ABR) and acoustic startle response, and several vestibular tests. Spatial cognitive deficits, i.e. higher latency to target, were observed with the Barnes maze. In the right hemisphere, no statistically significant difference was observed between groups. In the left hemisphere, the difference in mean cell densities of Dcx positive cells was statistically significant when compared to the control group, whereas the difference in mean cell density of Ki67 positive cells did not differ significantly. Conclusion: Spatial cognitive deficits and decreased immunoreactivity to DCX in the left hippocampus were observed 8 weeks after adult mice acquired profound SNHL and BV.

Neuroepithelial bodies (NEBs) are clusters of pulmonary neuroendocrine cells (PNECs) that represent less than one percent of the airway epithelial cells. PNECs store and release neurotransmitters, are surrounded by a unique population of stem cells, and are contacted by extensive nerve terminals. Today, multidisciplinary approaches allow combining functional morphological investigations in cryosections, live cell imaging in lung vibratome slices and selective gene expression analysis after laser microdissection of genetically tagged NEBs. However, due to their relatively low number and widespread distribution, the information remains highly fragmentary and quantification of NEBs, associated nerve endings and stem cells is problematic.The present study aimed at acquiring the first whole lung visualization and quantification of the total population of NEBs. As a proof‐of‐concept, we used a challenging triple immunolabeling and four channel imaging protocol for simultaneous identification of the selective myelinated vagal sensory innervation of NEBs.Lungs of GAD67‐GFP mice, which harbor GFP fluorescent NEB cells, were subjected to multiple immunostaining and tissue clearing. The list of optical clearing techniques is quickly growing, and several were tested; for our goals the iDisco+ protocol appeared to result in both the most complete clearing of mouse lungs and the best preservation of immunofluorescence in NEB nerve fiber populations. After fixation, methanol treatment, and permeabilization, whole mouse lungs were simultaneously immunostained for GFP (NEB cells), calbindin D‐28k and P2X3 ATP receptors (selective markers for two subpopulations of myelinated vagal afferents in mouse NEBs), and myelin basic protein (marker for all myelinated nerve fibers).Immunostained whole lungs could be imaged using light sheet microscopy (Ultramicroscope II, LaVision Biotec; Olympus MVPLAPO 2× objective; lasers: 488, 561, 640 and 785nm; emission filters: 525/50, 620/60, 680/30 and 845/55nm; Andor Neo sCMOS camera) at a total magnification of 1.26× and a z‐step of 10 μm. Optical sections were recorded in a mosaic of two by two tiles, and images stitched and merged with a linear blending algorithm (ImageJ).It could be shown that the applied staining, clearing and imaging protocols allow for 3D rendering of the complete lungs, with all NEBs and associated myelinated nerve terminals, including possibilities for 3D quantification. Selected individual NEB/nerve terminal complexes can be further analyzed in detail using high resolution confocal imaging and 3D reconstruction (Leica TCS SP8 DLS).The presented methodology opens interesting opportunities for imaging and quantification of total populations of unevenly dispersed microscopic structures in whole lungs, even when visualization requires complex immunostaining. Future goals include dedicated automated 3D quantification and shortening of the protocol time, which for now is about four weeks.Support or Funding InformationSupport: UA grant GOA BOF 2015 (30729 to DA).

Although plant organ shapes are defined by spatio-temporal variations of directional tissue expansion, this is a little characterized aspect of organ growth regulation. Although it is well known that the plant hormone gibberellin increases the leaf length/with ratio, its effects on cell expansion in the growing leaf are largely unknown. To understand how variations in rate and anisotropy of growth establish the typical monocotelydonous leaf shape, we studied the leaf growth zone of maize (Zea mays) with a kinematic analysis of cell expansion in the three directions of growth: proximo-distal, medio-lateral, and dorso-ventral. To determine the effect of gibberellin, we compared a gibberellin-deficient dwarf3 mutant and the overproducing UBI::GA20OX-1 line with their wild types. We found that, as expected, longitudinal growth was dominant throughout the growth zone. The highest degree of anisotropy occurred in the division zone, where relative growth rates in width and thickness were almost zero. Growth anisotropy was smaller in the elongation zone, due to higher lateral and dorso-ventral growth rates. Growth in all directions stopped at the same position. Gibberellin increased the size of the growth zone and the degree of growth anisotropy by stimulating longitudinal growth rates. Inversely, the duration of expansion was negatively affected, so that mature cell length was unaffected, while width and height of cells were reduced. Our study provides a detailed insight in the dynamics of growth anisotropy in the maize leaf and demonstrates that gibberellin specifically stimulates longitudinal growth rates throughout the growth zone.

Although an increasing number of beneficial microbiome members are characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus complex are more prevalent in the healthy URT than in patients with chronic rhinosinusitis (CRS). Several URT-specific isolates are cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes are found in the identified lactobacilli, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contains fimbriae that enable strong adherence to URT epithelium, inhibit the growth and virulence of several URT pathogens, and successfully colonize nasal epithelium of healthy volunteers. This study thus demonstrates that specific lactobacilli are adapted to the URT and could have a beneficial keystone function in this habitat.

The intestinal wall has a complex topographical architecture. The multi‐layered network of the enteric nervous system and its intercellular interactions are difficult to map using traditional section‐based or whole‐mount histology. With the advent of optical clearing techniques, it has become feasible to visualize intact tissue and organs in 3D. However, as yet, a gap still needs to be filled in that no in‐depth analysis has been performed yet on the potential of different clearing techniques for the small intestine.