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Intravital 2P-microscopy enables the longitudinal study of brain tumor biology in superficial mouse cortex layers. Intravital microscopy of the white matter, an important route of glioblastoma invasion and recurrence, has not been feasible, due to low signal-to-noise ratios and insufficient spatiotemporal resolution. Here, we present an intravital microscopy and artificial intelligence-based analysis workflow (Deep3P) that enables longitudinal deep imaging of glioblastoma up to a depth of 1.2 mm. We find that perivascular invasion is the preferred invasion route into the corpus callosum and uncover two vascular mechanisms of glioblastoma migration in the white matter. Furthermore, we observe morphological changes after white matter infiltration, a potential basis of an imaging biomarker during early glioblastoma colonization. Taken together, Deep3P allows for a non-invasive intravital investigation of brain tumor biology and its tumor microenvironment at subcortical depths explored, opening up opportunities for studying the neuroscience of brain tumors and other model systems. Observation of glioblastoma invasion in subcortical brain regions is challenging. Here the authors develop a Deep3P workflow to enable longitudinal deep imaging of glioblastoma and its microenvironment by combining three-photon microscopy and deep learning-based analysis, unraveling the invasion routes of these tumor cells into and within the white matter. [ABSTRACT FROM AUTHOR]

Comprehensive analysis of tissue cell type composition using microscopic techniques has primarily been confined to ex vivo approaches. Here, we introduce NuCLear (Nucleus-instructed tissue composition using deep learning), an approach combining in vivo two-photon imaging of histone 2B-eGFP-labeled cell nuclei with subsequent deep learning-based identification of cell types from structural features of the respective cell nuclei. Using NuCLear, we were able to classify almost all cells per imaging volume in the secondary motor cortex of the mouse brain (0.25 mm3 containing approximately 25,000 cells) and to identify their position in 3D space in a noninvasive manner using only a single label throughout multiple imaging sessions. Twelve weeks after baseline, cell numbers did not change yet astrocytic nuclei significantly decreased in size. NuCLear opens a window to study changes in relative density and location of different cell types in the brains of individual mice over extended time periods, enabling comprehensive studies of changes in cell type composition in physiological and pathophysiological conditions. This study presents NuCLear (Nucleus-instructed tissue composition using deep learning), a novel method enabling in vivo analysis of cell types in the mouse brain. It combines two-photon imaging of labeled cell nuclei with deep learning to classify and track cells over time, allowing the study of cellular changes in physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Locomotion is a complex task involving excitatory and inhibitory circuitry in spinal gray matter. While genetic knockouts examine the function of individual spinal interneuron (SpIN) subtypes, the phenotype of combined SpIN loss remains to be explored. We modified a kainic acid lesion to damage intermediate gray matter (laminae V-VIII) in the lumbar spinal enlargement (spinal L2-L4) in female rats. A thorough, tailored behavioral evaluation revealed deficits in gross hindlimb function, skilled walking, coordination, balance and gait two weeks post-injury. Using a Random Forest algorithm, we combined these behavioral assessments into a highly predictive binary classification system that strongly correlated with structural deficits in the rostro-caudal axis. Machine-learning quantification confirmed interneuronal damage to laminae V-VIII in spinal L2-L4 correlates with hindlimb dysfunction. White matter alterations and lower motoneuron loss were not observed with this KA lesion. Animals did not regain lost sensorimotor function three months after injury, indicating that natural recovery mechanisms of the spinal cord cannot compensate for loss of laminae V-VIII neurons. As gray matter damage accounts for neurological/walking dysfunction in instances of spinal cord injury affecting the cervical or lumbar enlargement, this research lays the groundwork for new neuroregenerative therapies to replace these lost neuronal pools vital to sensorimotor function. [ABSTRACT FROM AUTHOR]

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes' expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called 'Quanty-cFOS', with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells. [ABSTRACT FROM AUTHOR]

Purpose: To define the impact of systematic biopsy (SB) cores directed in the same area of index lesion in patients undergoing targeted biopsy (TB) and SB for prostate cancer (PCa) suspicion. Methods: We retrospectively analyzed data of biopsy-naïve patients with one single suspicious lesion at mpMRI who underwent TB plus SB at our institution between January 2015 and September 2021. A convenient sample of 336 patients was available for our analyses. The primary outcome was to evaluate the impact of overlapping SB cores directed to the index lesion at mpMRI. The secondary outcome was to evaluate the SB cores concordance in terms of highest Gleason Score Detection with TB cores. Results: 56% of patients were found to have site-specific concordance. SB cores determined disease upgrade in 22.1% patients. Thirty-one (16.4%) site-concordant patients experienced upgrade through overlapping SB cores, while 149 (79.3%) had no benefit by SB cores, and 8 (4.3%) patients had the worst ISUP at TB cores. 50% of the patients with negative-TB were upgraded to insignificant PCa, and 17.5% was upgraded from negative to unfavorable-intermediate- or high-risk PCa. Overall, 14 (19.4%) patients were also upgraded from ISUP 1 on TB to csPCa, with 28.5% of these harboring high-risk PCa. In csPCas at TB, 9 (12.5%) patients were upgraded from intermediate- to high-risk disease by SB. Conclusions: TB alone consents to identify worst ISUP PCa in vast majority of patients scheduled for biopsy. A non-negligible number of patients are upgraded via-SB cores, including also index lesion overlapping cores. Omitting these cores might lead to a suboptimal patient management. [ABSTRACT FROM AUTHOR]

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis. Author summary: To survive in the human body, the malaria parasite invades and lives within human red blood cells. Once within the red blood cell, the parasite renovates the host cell to its own needs. Here we have studied which factors from both parasite and host cell are required for this renovation process, and discover that human chaperone proteins, referred to as HSP70, are required. It appears that a particular parasite-derived protein, PFA66, recruits and modifies the function of the human HSP70. As this interaction between a parasite and human protein is novel and essential for parasite survival, our study identifies a potential Achilles' Heel which may be targeted for development of new anti-malaria therapies. [ABSTRACT FROM AUTHOR]

Magnetic resonance imaging (MRI) of the brain combined with voxel-based morphometry (VBM) revealed changes in gray matter volume (GMV) in various disorders. However, the cellular basis of GMV changes has remained largely unclear. We correlated changes in GMV with cellular metrics by imaging mice with MRI and two-photon in vivo microscopy at three time points within 12 weeks, taking advantage of age-dependent changes in brain structure. Imaging fluorescent cell nuclei allowed inferences on (i) physical tissue volume as determined from reference spaces outlined by nuclei, (ii) cell density, (iii) the extent of cell clustering, and (iv) the volume of cell nuclei. Our data indicate that physical tissue volume alterations only account for 13.0% of the variance in GMV change. However, when including comprehensive measurements of nucleus volume and cell density, 35.6% of the GMV variance could be explained, highlighting the influence of distinct cellular mechanisms on VBM results. [ABSTRACT FROM AUTHOR]

Magnetic resonance imaging (MRI) of the brain combined with voxel-based morphometry (VBM) revealed changes in gray matter volume (GMV) in various disorders. However, the cellular basis of GMV changes has remained largely unclear. We correlated changes in GMV with cellular metrics by imaging mice with MRI and two-photon in vivo microscopy at three time points within 12 weeks, taking advantage of age-dependent changes in brain structure. Imaging fluorescent cell nuclei allowed inferences on (i) physical tissue volume as determined from reference spaces outlined by nuclei, (ii) cell density, (iii) the extent of cell clustering, and (iv) the volume of cell nuclei. Our data indicate that physical tissue volume alterations only account for 13.0% of the variance in GMV change. However, when including comprehensive measurements of nucleus volume and cell density, 35.6% of the GMV variance could be explained, highlighting the influence of distinct cellular mechanisms on VBM results. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor‐induced angiogenesis. Here, we describe a novel time‐resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose‐response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2‐dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival. [ABSTRACT FROM AUTHOR]

Several lines of evidence imply changes in inhibitory interneuron connectivity and subsequent alterations in oscillatory network activities in the pathogenesis of Alzheimer’s Disease (AD). Recently, we provided evidence for an increased immunoreactivity of both the postsynaptic scaffold protein gephyrin and the GABAA receptor γ2-subunit in the hippocampus of young (1 and 3 months of age), APPPS1 mice. These mice represent a well-established model of cerebral amyloidosis, which is a hallmark of human AD. In this study, we demonstrate a robust increase of parvalbumin immunoreactivity and accentuated projections of parvalbumin positive (PV+) interneurons, which target perisomatic regions of pyramidal cells within the hippocampal subregions CA1 and CA3 of 3-month-old APPPS1 mice. Colocalisation studies confirmed a significant increase in the density of PV+ projections labeled with antibodies against a presynaptic (vesicular GABA transporter) and a postsynaptic marker (gephyrin) of inhibitory synapses within the pyramidal cell layer of CA1 and CA3. As perisomatic inhibition by PV+-interneurons is crucial for the generation of hippocampal network oscillations involved in spatial processing, learning and memory formation we investigated the impact of the putative enhanced perisomatic inhibition on two types of fast neuronal network oscillations in acute hippocampal slices: 1. spontaneously occurring sharp wave-ripple complexes (SPW-R), and 2. cholinergic γ-oscillations. Interestingly, both network patterns were generally preserved in APPPS1 mice similar to WT mice. However, the comparison of simultaneous CA3 and CA1 recordings revealed that the incidence and amplitude of SPW-Rs were significantly lower in CA1 vs CA3 in APPPS1 slices, whereas the power of γ-oscillations was significantly higher in CA3 vs CA1 in WT-slices indicating an impaired communication between the CA3 and CA1 network activities in APPPS1 mice. Taken together, our data demonstrate an increased GABAergic synaptic output of PV+ interneurons impinging on pyramidal cells of CA1 and CA3, which might limit the coordinated cross-talk between these two hippocampal areas in young APPPS1 mice and mediate long-term changes in synaptic inhibition during progression of amyloidosis. [ABSTRACT FROM AUTHOR]

The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution. [ABSTRACT FROM AUTHOR]

Background: The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date. Results: To address these questions, we applied long-term 2-photon microscopy time-lapse analysis of habenular neural circuit development combined with depth color coding in a transgenic line, labeling all main components of the network. Additional laser ablations and cell tracking experiments using the photoconvertible PSmOrange system in GFP transgenic fish show that the ventral habenulae develop in prosomere 2, posterior and lateral to the dorsal habenulae in the dorsal thalamus. Mutant analysis demonstrates that the ventral habenular nuclei only develop in the presence of functional Tcf7l2, a downstream modulator of the Wnt signaling cascade. Consistently, photoconverted thalamic tcf7l2exl/exl mutant cells do not contribute to habenula formation. Conclusions: We show in vivo that dorsal and ventral habenulae develop in different regions of prosomere 2. In the process of ventral habenula formation, functional tcf7l2 gene activity is required and in its absence, ventral habenular neurons do not develop. Influenced by signals from parapineal cells, dorsal habenular neurons differentiate at a time at which ventral habenular cells are still on their way towards their final destination. Thus, our finding may provide a simple explanation as to why only neuronal populations of the dorsal habenulae differ in size across brain hemispheres. [ABSTRACT FROM AUTHOR]

Summary: Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis. [Copyright &y& Elsevier]

Abstract: Near-wall turbulent coherent structures, obtained by applying Jeong and Hussain''s vortex identification criterion to DNS flow fields of a dilute polymer channel flow, are compared with those observed in a Newtonian flow at the same Reynolds number. The mean vortex in the polymer flow is weaker, less tilted and inclined, while its length and radius are higher. Cross-flow velocity fluctuations near the wall drop consistently. The vortical structures orient and stretch the polymer chains near the wall, producing high additional stresses in the flow. The consistent result is a new pressure and stress distribution which acts in opposition to the redistribution of kinetic energy from the streamwise to the cross-flow direction. Although the basic features of near-wall turbulence are maintained, the whole turbulence cycle reaches an equilibrium state where Reynolds stresses and wall friction are lower. [Copyright &y& Elsevier]

Mesothelioma is often difficult to diagnose due to its rarity and its unusual histopathological features that could lend to diagnostic pitfalls and misdiagnosis. The WHO histological classification of pleural tumors in 2021 recommended a pathologic grading system for malignant pleural mesothelioma. Architectural aspects and cytological features, with nuclear grading, bent on a neoplastic score with fundamental prognostic and diagnostic value. Unusual features must be correctly assigned in the grading system to avoid misdiagnosis, especially toward metastatic lesions or reactive pleural processes. In this paper, we present two cases as examples of unusual morphological and architectural features with a brief literature review. [ABSTRACT FROM AUTHOR]

Simple Summary: The excessive desmoplasia is the hallmark of human pancreatic cancer that influences the local T-cell-based immune response. In the present work, the stromal collagen organization in normal and malignant pancreatic tissues as well as its relationsship to T-cell distribution in pancreatic cancer were studied. It was found that differences in collagen organization do not change the spatial orientation of T-cell migration and do not influence the availability of tumor cells for T-cells. The results of the study do not support the concept of use of stroma collagen organization for improvement of spatial T-cell distribution in the tumor. The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell–tumor cell contact in pancreatic ductal adenocarcinoma. [ABSTRACT FROM AUTHOR]

Background: The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest–derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction. Methods: Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin‐embedded individuals' specimens were analyzed accordingly. Key Results: Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles. Conclusions & Inferences: Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications. [ABSTRACT FROM AUTHOR]