THSD7A-associated membranous nephropathy involves both complement-mediated and autonomous podocyte injury.

Membranous nephropathy (MN) continues to be a leading cause of nephrotic syndrome in non-diabetic adults. As a unique subtype in the serology-based classification of MN, thrombospondin type 1 domain containing 7A (THSD7A)-associated MN has attracted increasing interest, because, unlike other autoantigens, THSD7A is also expressed in preclinical species, facilitating the study of its role in MN. A heterologous mouse model of THSD7A-associated MN was previously established using a proprietary in-house antibody that was unfortunately not available to the research community. Here, we developed a mouse model of THSD7A-associated MN by administering a commercially available antibody targeting the most N-terminal part of THSD7A. Our model was characterized by heavy proteinuria and pathological features of human MN without sex differences. Complement depletion with cobra venom factor only partially attenuated proteinuria and glomerular injury in this model, entailing that complement-independent pathomechanisms also contribute. Consistently, in vitro in primary podocytes, exposure to the anti-THSD7A antibody caused evident podocytopathic changes, including disruption of actin cytoskeleton integrity, podocyte hypermobility, oxidative stress, and apoptotic cell death. These signs of podocytopathy were preserved, albeit to a lesser extent, after complement inactivation, indicating autonomous podocyte injury. Furthermore, as the first FDA-approved treatment for primary MN, adrenocorticotropic hormone therapy with repository corticotropin injection (Purified Cortrophin Gel®) appeared to be beneficial and significantly attenuated proteinuria and glomerular injury, suggesting that this model may be useful for developing novel treatments or understanding the pathogenesis of MN. Collectively, our model, based on the use of a commercially available anti-THSD7A antibody, will be an important tool for MN research.

Methylome-wide analysis in systemic microbial-induced experimental periodontal disease in mice with different susceptibility.

Objective: The study delved into the epigenetic factors associated with periodontal disease in two lineages of mice, namely C57bl/6 and Balb/c. Its primary objective was to elucidate alterations in the methylome of mice with distinct genetic backgrounds following systemic microbial challenge, employing high-throughput DNA methylation analysis as the investigative tool. Methods: Porphyromonas gingivalis (Pg)was orally administered to induce periodontitis in both Balb/c and C57bl/6 lineage. After euthanasia, genomic DNA from both maxilla and blood were subjected to bisulfite conversion, PCR amplification and genome-wide DNA methylation analysis using the Ovation RRBS Methyl-Seq System coupled with the Illumina Infinium Mouse Methylation BeadChip. Results: Of particular significance was the distinct methylation profile observed within the Pg-induced group of the Balb/c lineage, contrasting with both the control and Pg-induced groups of the C57bl/6 lineage. Utilizing rigorous filtering criteria, we successfully identified a substantial number of differentially methylated regions (DMRs) across various tissues and comparison groups, shedding light on the prevailing hypermethylation in non-induced cohorts and hypomethylation in induced groups. The comparison between blood and maxilla samples underscored the unique methylation patterns specific to the jaw tissue. Our comprehensive methylome analysis further unveiled statistically significant disparities, particularly within promoter regions, in several comparison groups. Conclusion: The differential DNA methylation patterns observed between C57bl/6 and Balb/c mouse lines suggest that epigenetic factors contribute to the variations in disease susceptibility. The identified differentially methylated regions associated with immune regulation and inflammatory response provide potential targets for further investigation. These findings emphasize the importance of considering epigenetic mechanisms in the development and progression of periodontitis.

Loss of the long form of Plod2 phenocopies contractures of Bruck syndrome - osteogenesis imperfecta.

Bruck syndrome is an autosomal recessive form of osteogenesis imperfecta (OI) caused by biallelic variants in PLOD2 or FKBP10 and is characterized by joint contractures, bone fragility, short stature, and scoliosis. PLOD2 encodes LH2, which hydroxylates type I collagen telopeptide lysines, a critical step for collagen crosslinking. The Plod2 global knockout mouse model is limited by early embryonic lethality, thus the role of PLOD2 in skeletogenesis is not well understood. We generated a novel Plod2 mouse line modeling a variant identified in two unrelated individuals with Bruck syndrome: PLOD2 c.1559dupC, predicting a frameshift and loss of the long isoform LH2b. In the mouse, the duplication led to loss of LH2b mRNA as well as significantly reduced total LH2 protein. This model, Plod2fs/fs, survived up to E18.5 although in non-Mendelian genotype frequencies. The homozygous frameshift model recapitulated the joint contractures seen in Bruck syndrome and had indications of absent type I collagen telopeptide lysine hydroxylation in bone. Genetically labeling tendons with Scleraxis-GFP in Plod2fs/fs mice revealed the loss of extensor tendons in the forelimb by E18.5 and developmental studies showed extensor tendons developed through E14.5 but were absent starting at E16.5. Second harmonic generation showed abnormal tendon type I collagen fiber organization, suggesting structurally abnormal tendons. Characterization of the skeleton by µCT and Raman spectroscopy showed normal bone mineralization levels. This work highlights the importance of properly crosslinked type I collagen in tendon and bone, providing a promising new mouse model to further our understanding of Bruck syndrome.

Bruck syndrome is a rare disease where individuals have brittle bone as well as contracted or stiff joints. Mutations in two genes are associated with Bruck syndrome and, in this work, we focus on PLOD2. Mice without Plod2 die at an early embryonic stage, before they have a chance to fully develop. In this work, we created a mouse with a PLOD2 mutation seen in people with Bruck syndrome. Some of these new Bruck syndrome model mice survived to a later gestational age, but all died at birth. The Bruck syndrome mice were small and had contracted joints. We found they were missing tendons in their arms and had structurally abnormal tendons in their knees. Bone mineralization was normal, but there were indications that the modifications needed for normal type I collagen structure were absent. Overall, this is an advantageous new mouse model of Bruck syndrome that can be used to study this rare disease and highlights the importance of Plod2 in tendon.

Transposon-based oncogene integration in Abcb4(Mdr2)-/- mice recapitulates high susceptibility to cholangiocarcinoma in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a dreaded complication of primary sclerosing cholangitis (PSC), difficult to diagnose and associated with high mortality. Lack of animal models of CCA recapitulating the hepatic microenvironment of sclerosing cholangitis hinders development of novel treatments. Here we sought to develop such PSC-associated CCA model in mice. METHODS: Ten-week-old Mdr2-/- mice with congenital PSC-like disease, and healthy wild-type littermates were subjected to either modified retrograde biliary instillation or hydrodynamic tail vein injection of sleeping beauty transposon-transposase plasmid system with activated AKT (myr-AKT) and Yap (YapS127A) protooncogenes (SB AKT/YAP1). The role of TGFß was interrogated via ALK5 inhibitor (SB-525334) administration. Tumor phenotype, burden and desmoplastic reaction were analyzed histologically and via RNA-seq. RESULTS: While SB AKT/YAP1 plasmids via retrograde biliary injection caused tumors in Mdr2-/-, only 26.67% (4/15) of these tumors were CCA. Alternatively, hydrodynamic tail vein injection of SB AKT/YAP1 resulted in robust tumorigenesis in all fibrotic Mdr2-/- mice with high CCA burden compared to healthy mice. Tumors phenotypically resembled human CCA, expressed multiple CCA (but not hepatocellular carcinoma) markers, and exhibited a profound desmoplastic reaction. RNA-seq analysis revealed profound transcriptional changes in CCA evolving in PSC-like context, with specific alterations in multiple immune pathways. Pharmacological TGFß inhibition led to enhanced immune cell tumor infiltration, reduced tumor burden and suppressed desmoplastic collagen accumulation compared to placebo CONCLUSION: We established a new high-fidelity cholangiocarcinoma model in mice, termed SB CCA.Mdr2-/-, which recapitulates the increased susceptibility to CCA in the setting of biliary injury and fibrosis observed in PSC. Through transcriptomics and pharmacological studies, we show dysregulation of multiple immune pathways and TGFß signaling as potential drivers of CCA in PSC-like microenvironment. IMPACT AND IMPLICATIONS: There is a lack of animal models for primary sclerosing cholangitis (PSC) related cholangiocarcinoma (PSC-CCA). We have developed and characterized a new mouse model of PSC-CCA, termed SB CCA.Mdr2-/-, which features reliable tumor induction in PSC-like background of biliary injury and fibrosis. Global gene expression alterations were identified and standardized tools, including automated whole slide image analysis methodology for tumor burden and feature analysis, were established to enable systematic research into PSC-CCA biology and formal pre-clinical drug testing.

Xylazine is an agonist at kappa opioid receptors and exhibits sex-specific responses to opioid antagonism.

Xylazine is in the unregulated drug supply at increasing rates, usually combined with fentanyl, necessitating understanding of its pharmacology. Despite commentary from politicians, and public health officials, it is unknown how xylazine impacts naloxone efficacy, and. few studies have examined it alone. Here, we examine the impact of xylazine alone and in combination with fentanyl on several behaviors in mice. Surprisingly, naloxone precipitates withdrawal from xylazine and fentanyl/xylazine coadministration, with enhanced sensitivity in females. Further, xylazine is a full agonist at kappa opioid receptors, a potential mechanism for its naloxone sensitivity. Finally, we demonstrate surprising effects of xylazine to kappa opioid antagonism, which are relevant for public health considerations. These data address an ongoing health crisis and will help inform critical policy and healthcare decisions.

Bidirectional fear modulation by discrete anterior insular circuits in male mice.

The brain's ability to appraise threats and execute appropriate defensive responses is essential for survival in a dynamic environment. Humans studies have implicated the anterior insular cortex (aIC) in subjective fear regulation and its abnormal activity in fear/anxiety disorders. However, the complex aIC connectivity patterns involved in regulating fear remain under investigated. To address this, we recorded single units in the aIC of freely moving male mice that had previously undergone auditory fear conditioning, assessed the effect of optogenetically activating specific aIC output structures in fear, and examined the organization of aIC neurons projecting to the specific structures with retrograde tracing. Single-unit recordings revealed that a balanced number of aIC pyramidal neurons' activity either positively or negatively correlated with a conditioned tone-induced freezing (fear) response. Optogenetic manipulations of aIC pyramidal neuronal activity during conditioned tone presentation altered the expression of conditioned freezing. Neural tracing showed that non-overlapping populations of aIC neurons project to the amygdala or the medial thalamus, and the pathway bidirectionally modulated conditioned fear. Specifically, optogenetic stimulation of the aIC-amygdala pathway increased conditioned freezing, while optogenetic stimulation of the aIC-medial thalamus pathway decreased it. Our findings suggest that the balance of freezing-excited and freezing-inhibited neuronal activity in the aIC and the distinct efferent circuits interact collectively to modulate fear behavior.

Slow misfolding of a molten globule form of a mutant prion protein variant into a ß-rich dimer.

Misfolding of the prion protein is linked to multiple neurodegenerative diseases. A better understanding of the process requires the identification and structural characterization of intermediate conformations via which misfolding proceeds. In this study, three conserved aromatic residues (Tyr168, Phe174, and Tyr217) located in the C-terminal domain of mouse PrP (wt moPrP) were mutated to Ala. The resultant mutant protein, 3A moPrP, is shown to adopt a molten globule (MG)-like native conformation. Hydrogen-deuterium exchange studies coupled with mass spectrometry revealed that for 3A moPrP, the free energy gap between the MG-like native conformation and misfolding-prone partially unfolded forms is reduced. Consequently, 3A moPrP misfolds in native conditions even in the absence of salt, unlike wt moPrP, which requires the addition of salt to misfold. 3A moPrP misfolds to a ß -rich dimer in the absence of salt, which can rapidly form an oligomer upon the addition of salt. In the presence of salt, 3A moPrP misfolds to a ß -rich oligomer about a thousand-fold faster than wt moPrP. Importantly, the misfolded structure of the dimer is similar to that of the salt-induced oligomer. Misfolding to oligomer seems to be induced at the level of the dimeric unit by monomer-monomer association, and the oligomer grows by accretion of misfolded dimeric units. Additionally, it is shown that the conserved aromatic residues collectively stabilize not only monomeric protein, but also the structural core of the ß-rich oligomers. Finally, it is also shown that 3A moPrP misfolds much faster to amyloid-fibrils than does the wt protein.

SIRT1-driven mechanism: sevoflurane's interference with mESC neural differentiation via PRRX1/DRD2 cascade.

Investigating the sevoflurane-induced perturbation in the differentiation of mouse embryonic stem cells (mESCs) into neural stem cells (mNSCs), our study delineates a novel SIRT1/PRRX1/DRD2/PKM2/NRF2 axis as a key player in this intricate process. Sevoflurane treatment hindered mESC differentiation, evidenced by altered expression patterns of pluripotency and neural lineage markers. Mechanistically, sevoflurane downregulated Sirt1, setting in motion a signaling cascade. Sevoflurane may inhibit PKM2 dimerization and NRF2 signaling pathway activation by inhibiting the expression of SIRT1 and its downstream genes Prrx1 and DRD2, ultimately inhibiting mESCs differentiation into mNSCs. These findings contribute to our understanding of the molecular basis of sevoflurane-induced neural toxicity, presenting a potential avenue for therapeutic intervention in sevoflurane-induced perturbation in the differentiation of mESCs into mNSCs by modulating the SIRT1/PRRX1/DRD2/PKM2/NRF2 axis.

Post-COVID pulmonary injury in K18-hACE2 mice shows persistent neutrophils and neutrophil extracellular trap formation.

The involvement of neutrophils in the lungs during the recovery phase of coronavirus disease 2019 (COVID-19) is not well defined mainly due to the limited accessibility of lung tissues from COVID-19 survivors. The lack of an appropriate small animal model has affected the development of effective therapeutic strategies. We here developed a long COVID mouse model to study changes in neutrophil phenotype and association with lung injury. Our data shows persistent neutrophil recruitment and neutrophil extracellular trap formation in the lungs for up to 30 days post-infection which correlates with lung fibrosis and inflammation.

PAX5 functions as a tumor suppressor by RB-E2F-mediated cell cycle arrest in Kaposi sarcoma-associated herpesvirus-infected primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a malignant B-cell lymphoma attributable to Kaposi sarcoma-associated herpesvirus (KSHV) infection. PEL is characterized by invasive behavior, showing recurrent effusions in body cavities. The clinical outcome and typical prognosis in patients with PEL are poor and potentially lethal. Clarification of the pathogenesis in PEL is urgently needed in order to develop novel therapies. PEL cells generally lack B-cell surface markers, and we therefore hypothesized that the B-cell transcription factor, PAX5, would be down-regulated in PEL. The expression of PAX5 is detected from the pro-B to the mature B-cell stage and is indispensable for the differentiation of B-cells. PAX5 was silenced in PEL cells via its promoter methylation. Up-regulation of PAX5 induced several genes coding for B-cell surface marker mRNA, but not protein level. PAX5 inhibited cell growth via G1 cell cycle arrest. PAX5 bound to RB and increased its protein expression. RB/E2F-regulated genes were significantly down-regulated in microarray analysis and PCR experiments. To elucidate the in vivo role of PAX5, we examined the restoration of PAX5 in a PEL mouse model. The ascites volume and organ invasions were significantly suppressed by PAX5 restoration. Reduction of PAX5 has played a crucial role in the oncogenesis of PEL, and PAX5 is a tumor suppressor in PEL. Targeting PAX5 could represent a novel therapeutic strategy for patients with PEL.

The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Anatomy-Constrained Synthesis for Spleen Segmentation Improvement in Unpaired Mouse Micro-CT Scans with 3D CycleGAN.

OBJECTIVE: Auto-segmentation in mouse micro-CT enhances the efficiency and consistency of preclinical experiments but often struggles with low-native-contrast and morphologically complex organs, such as the spleen, resulting in poor segmentation performance. While CT contrast agents can improve organ conspicuity, their use complicates experimental protocols and reduces feasibility. We developed a 3D Cycle Generative Adversarial Network (CycleGAN) incorporating anatomy-constrained U-Net models to leverage contrast-enhanced CT (CECT) insights to improve unenhanced native CT (NACT) segmentation. Approach: We employed a standard CycleGAN with an anatomical loss function to synthesize virtual CECT images from unpaired NACT scans at two different resolutions. Prior to training, two U-Nets were trained to automatically segment six major organs in NACT and CECT datasets, respectively. These pretrained 3D U-Nets were integrated during the CycleGAN training, segmenting synthetic images, and comparing them against ground truth annotations. The compound loss within the CycleGAN maintained anatomical fidelity. Full image processing was achieved for low-resolution datasets, while high-resolution datasets employed a patch-based method due to GPU memory constraints. Automated segmentation was applied to original NACT and synthetic CECT scans to evaluate CycleGAN performance using the Dice Similarity Coefficient (DSC) and the 95th percentile Hausdorff Distance (HD95p). Main Results: High-resolution scans showed improved auto-segmentation, with an average DSC increase from 0.728 to 0.773 and a reduced HD95p from 1.19 mm to 0.94 mm. Low-resolution scans benefited more from synthetic contrast, showing a DSC increase from 0.586 to 0.682 and an HD95p reduction from 3.46 mm to 1.24 mm. Significance: Implementing CycleGAN to synthesize CECT scans substantially improved the visibility of the mouse spleen, leading to more precise auto-segmentation. This approach shows the potential in preclinical imaging studies where contrast agent use is impractical. .

Attenuated responses to attention-modulating drugs in the neuroligin-3R451C mouse model of autism.

Attention deficits are frequently reported within the clinical autism population. Despite not being a core diagnostic feature, some aetiological theories place atypical attention at the centre of autism development. Drugs used to treat attention dysfunction are therefore increasingly prescribed to autistic patients, though currently off-label with uncertain efficacy. We utilised a rodent-translated touchscreen test of sustained attention in mice carrying an autism-associated R451C mutation in the neuroligin-3 gene (Nlgn3R451C). In doing so, we replicated their cautious but accurate response profile and probed it using two widely prescribed attention-modulating drugs: methylphenidate (MPH) and atomoxetine (ATO). In wild-type mice, acute administration of MPH (3 mg/kg) promoted impulsive responding at the expense of accuracy, while ATO (3 mg/kg) broadly reduced impulsive responding. These drug effects were absent in Nlgn3R451C mice, other than a small reduction in blank touches to the screen following ATO administration. The absence of drug effects in Nlgn3R451C mice likely arises from their altered behavioural baseline and underlying neurobiology, highlighting caveats to the use of classic attention-modulating drugs across disorders and autism subsets. It further suggests that altered dopaminergic and/or norepinephrinergic systems may drive behavioural differences in the Nlgn3R451C mouse model of autism, supporting further targeted investigation.

AnNoBrainer, An Automated Annotation of Mouse Brain Images using Deep Learning.

Annotation of multiple regions of interest across the whole mouse brain is an indispensable process for quantitative evaluation of a multitude of study endpoints in neuroscience digital pathology. Prior experience and domain expert knowledge are the key aspects for image annotation quality and consistency. At present, image annotation is often achieved manually by certified pathologists or trained technicians, limiting the total throughput of studies performed at neuroscience digital pathology labs. It may also mean that simpler and quicker methods of examining tissue samples are used by non-pathologists, especially in the early stages of research and preclinical studies. To address these limitations and to meet the growing demand for image analysis in a pharmaceutical setting, we developed AnNoBrainer, an open-source software tool that leverages deep learning, image registration, and standard cortical brain templates to automatically annotate individual brain regions on 2D pathology slides. Application of AnNoBrainer to a published set of pathology slides from transgenic mice models of synucleinopathy revealed comparable accuracy, increased reproducibility, and a significant reduction (~ 50%) in time spent on brain annotation, quality control and labelling compared to trained scientists in pathology. Taken together, AnNoBrainer offers a rapid, accurate, and reproducible automated annotation of mouse brain images that largely meets the experts' histopathological assessment standards (> 85% of cases) and enables high-throughput image analysis workflows in digital pathology labs.

OMIP-105: A 30-color full-spectrum flow cytometry panel to characterize the immune cell landscape in spleen and tumor within a syngeneic MC-38 murine colon carcinoma model.

This panel was designed to characterize the immune cell landscape in the mouse tumor microenvironment as well as mouse lymphoid tissues (e.g., spleen). As an example, using the MC-38 mouse syngeneic tumor model, we demonstrated that we could measure the frequency and characterize the functional status of CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells, macrophages, granulocytes, monocytes, and dendritic cells. This panel is especially useful for understanding the immune landscape in "cold" preclinical tumor models with very low immune cell infiltration and for investigating how therapeutic treatments may modulate the immune landscape.

Investigating the anticancer properties of six benzothiazolopyrimidine derivatives on colon carcinoma cells, in vitro and in vivo.

Colorectal cancer (CRC) is the third most common cancer in the world. Despite considerable improvements in the treatment of this cancer, further research to discover novel and more effective agents is ongoing. In this study, possible cytotoxic and apoptotic properties of six benzothiazolopyrimidine derivatives were studied. To assess the IC50 values of these agents, MTT assay was performed on HCT 116, CT26, and NIH/3T3 cells. Moreover, cell death mechanism induced by studied compounds was evaluated by PI/annexin V staining. Then, based on molecular docking results and in vitro experiments, the compounds with the highest anticancer properties were further analyzed in vivo in a mouse model of CRC. MTT results indicated that BTP(1) and BTP(4) had the highest selective cytotoxicity on colorectal cancer cells. Furthermore, flow cytometry results demonstrated a considerable increase in the percentage of the early apoptotic cells in BTP(1)- and BTP(4)-treated groups. In vivo studies confirmed the antitumor properties of the two compounds by a significant regression in tumor size of BTP(1)- and BTP(4)-treated mice compared to control groups. Histopathological examination of tumor tissues showed an increased number of apoptotic cells in these two groups compared to the control animals. Additionally, hematoxylin and eosin staining of the spleen and liver of treated mice did not exhibit considerable tissue damage. Thus, BTP(1) and BTP(4) can be considered promising agents in the treatment of colorectal cancer, although further experiments are required to assess their mechanism of action before their application in clinical studies.

Syntaxin-6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates.

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.

Combining array tomography with electron tomography provides insights into leakiness of the blood-brain barrier in mouse cortex.

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

A novel mouse model for hepatocyte-specific apoptosis-induced myeloid cell-dominant sterile liver injury and repair.

Apoptosis, inflammation, and wound healing are critical pathophysiological events associated with various liver diseases. Currently, there is a lack of in vivo approaches to study hepatocyte apoptosis-induced liver injury and repair. To address this critical knowledge gap, we developed a unique genetically modified mouse model, namely, 3xTg-iHAP (3-Transgene with inducible Hepatocyte Apoptosis Phenotype) in this study. The 3xTg-iHAP mice possess three transgenes including Alb-Cre, Rosa26-rtTA, and tetO-Fasl on a B6 background. These mice are phenotypically normal, viable, and fertile. After subcutaneous administration of a single dose of doxycycline (5 mg/kg, Dox) to 3xTg-iHAP mice, we observed a complete histological spectrum of sterile liver wound-healing responses: asymptomatic hepatocyte apoptosis at 8 h, necrotic liver injury and sterile inflammation at 48 h, followed by hepatocyte mitosis and regeneration within 7 days. During the injury phase, the mice exhibited an increase in biomarkers of ALT, CXCL1, and IL-6 in peripheral blood and α-SMA protein in liver tissues. Conversely, the mice displayed a decrease in these markers in the recovery phase. Remarkably, this model shows that the sterile liver injury following elevated hepatocyte apoptosis is associated with an increase in myeloid cells in the liver. Within 7 days post-Dox administration, the liver of Dox-treated 3xTg-iHAP mice displays a normal histological structure, indicating completion of wound-healing. Together, we established a novel mouse model of injury and regeneration induced by hepatocyte apoptosis. This tool provides a robust in vivo platform for studying the pathophysiology of sterile liver inflammation, regeneration, and new therapeutic interventions for liver diseases.

The Effect of Fractional Carbon Dioxide Laser on Melanogenesis in Human Melanocytes and Vitiligo Mouse Models.

Introduction: Vitiligo is an acquired skin pigmentation disorder, the cause of which is poorly understood. Researchers in this field are dedicated to exploring novel treatments for achieving re-pigmentation. Methods: Mice were randomly selected and divided into control, model, and model+laser groups. Evaluate the impact of different levels of carbon dioxide laser irradiation on tyrosinase activity, melanocyte viability, and melanin content. Results: In this study, it was found that the cell viability and melanin content were significantly enhanced in human melanocytes after treatment with different energy densities of fractional carbon dioxide laser. In addition, laser-treated vitiligo mouse models showed mild pathological changes. Discussion: Therefore, we believe that fractional carbon dioxide laser may be a potential adjunctive modality for treating vitiligo.