Probes for INS

The mammalian epidermis undergoes constant renewal, replenished by a pool of stem cells and terminal differentiation of their progeny. This is accompanied by changes in gene expression and morphology that are orchestrated, in part, by epigenetic modifiers. Here, we define the role of the histone acetyltransferase KAT2A in epidermal homeostasis and provide a comparative analysis that reveals key functional divergence with its paralog KAT2B. In contrast to the reported function of KAT2B in epidermal differentiation, KAT2A supports the undifferentiated state in keratinocytes. RNA-seq analysis of KAT2A- and KAT2B- depleted keratinocytes revealed dysregulated epidermal differentiation. Depletion of KAT2A led to premature expression of epidermal differentiation genes in the absence of inductive signals, whereas loss of KAT2B delayed differentiation. KAT2A acetyltransferase activity was indispensable in regulating epidermal differentiation gene expression. The metazoan-specific N terminus of KAT2A was also required to support its function in keratinocytes. We further showed that the interplay between KAT2A- and KAT2B-mediated regulation was important for normal cutaneous wound healing in vivo. Overall, these findings reveal a distinct mechanism in which keratinocytes use a pair of highly homologous histone acetyltransferases to support divergent functions in self-renewal and differentiation processes.

RESULTS : Uniform Manifold Approximation and Projection clustering identified distinct expression signatures from the ganglion cell layer(GCL), inner nuclear layer(INL), retinal pigment epithelium (RPE)/choroid/sclera, optic nerve, and ciliary body (Fig, 1) but not the outer nuclear layer(ONL) which was contaminated with expression from other layers. Our findings highlight Clu, C4b, Apoe, and C1qa genes (z-score 3.0, 2.4, 2.3, and 2.2) as potential markers of disease in the RPE. Gene Set Enrichment analysis between rd6 and WT eyes showed upregulation of glycolysis and carbon metabolism pathways in the GCL and Rap1, Hippo and lysosome pathways in the RPE/Choroid/sclera. The ribosomal pathway was downregulated in these layers. No significant pathways were found in the INL, ciliary body or optic nerve.

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling. We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities.

Ureteric bud induction and branching morphogenesis is fundamental to the establishment of the renal architecture and is a key determinant of nephron number. Defective ureteric bud morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor RET and coreceptor GFRA1 appears to be particularly important in ureteric bud development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT.To investigate whether and how inactivation of Ash2l, which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the ureteric bud lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with CUT&Tag-seq.Ureteric bud-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the ureteric bud tip, delaying budding and impairing ureteric bud branching morphogenesis. These effects were associated with downregulation of Ret, Gfra1, and Wnt11, which participate in RET/GFRA1 signaling.These experiments identify ASH2L-dependent H3K4 methylation in the ureteric bud lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in ureteric bud morphogenesis, which, if deficient, may lead to CAKUT.

Cholangiocarcinoma is the second most common primary liver malignant neoplasm. It usually affects older individuals in their seventh decade of life with no gender predilection. Recently, a distinct subtype of cholangiocarcinoma has emerged with 2 proposed names: "cholangioblastic" and "solid tubulocystic." This variant predominantly occurs in younger women who lack the common risk factors for patients diagnosed with cholangiocarcinomas, such as older age and chronic liver disease or cirrhosis. We describe 3 new patients with a cholangioblastic variant of intrahepatic cholangiocarcinoma. At the time of diagnosis, the patients were aged 19-, 46-, and 28-year-old; 2 females and 1 male (the 46-year-old). None of our patients had a history of chronic liver disease or known predisposing factors for liver tumors. Tumor size ranged from 2.3 to 23 cm in greatest dimension. Histological examination of these tumors demonstrated reproducible morphology characterized by trabecular, nested, and multicystic patterns with micro and macro follicles filled with eosinophilic material. The immunohistochemical profile showed that the tumor cells were positive for keratin 7, inhibin, synaptophysin, and albumin in situ hybridization, while negative for HepPar1, arginase, and INSM1. All tumors lacked conventional intrahepatic cholangiocarcinoma/adenocarcinoma morphology. We also review the literature and emphasize that neuroendocrine tumors should be recognized as a major diagnostic pitfall of this variant.

Background: The standard graft material for alveolar cleft repair (ACR) is autogenous iliac crest. However, a promising alternative potential graft adjunct - newborn human umbilical cord mesenchymal stem cells (h-UCMSC) - has yet to be explored in vivo. Their capacity for selfrenewal, multipotent differentiation, and proliferation allows h-UCMSC to be harnessed for regenerative medicine. Our study seeks to evaluate the efficacy of using tissue-derived hUCMSC and their osteogenic capabilities in a murine model to improve ACR. Methods: Foxn1 mice were separated into three groups with the following calvarial defects: (1) no-treatment (empty defect; n=6), (2) poly (D,L-lactide-co-glycolide) (PLGA) scaffold (n=6), and (3) h-UCMSC with PLGA (n=4). Bilateral 2-mm diameter parietal bone critical-sized defects were created using a dental drill. Micro-CT imaging occurred at 1, 2, 3, and 4 weeks postoperatively. The mice were euthanized 4 weeks postoperatively for RNAscope analysis, immunohistochemistry, and histology. Results: No mice experienced complications during the follow-up period. Micro-CT and histology demonstrated that the no-treatment (1) and PLGA-only (2) defects remained patent without significant defect size differences across groups. In contrast, the h-UCMSC with PLGA group (3) had significantly greater bone fill on micro-CT and histology. Conclusions: We demonstrate a successful calvarial defect model for the investigation of hUCMSC-mediated osteogenesis and bone repair. Furthermore, evidence reveals that PLGA alone has neither short-term effects on bone formation nor any unwanted side effects, making it an 3 attractive scaffold. Further investigation using h-UCMSC with PLGA in larger animals is warranted to advance future translation to patients requiring ACR. Clinical Relevance Statement: Our results demonstrate a successful murine calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair and provide preliminary evidence for the safe and efficacious use of this graft adjunct in alveolar cleft repair.

PTEN-induced kinase 1 (PINK1) is a short-lived protein required for the removal of damaged mitochondria through Parkin translocation and mitophagy. Because the short half-life of PINK1 limits its ability to be trafficked into neurites, local translation is required for this mitophagy pathway to be active far from the soma. The Pink1 transcript is associated and cotransported with neuronal mitochondria. In concert with translation, the mitochondrial outer membrane proteins synaptojanin 2 binding protein (SYNJ2BP) and synaptojanin 2 (SYNJ2) are required for tethering Pink1 mRNA to mitochondria via an RNA-binding domain in SYNJ2. This neuron-specific adaptation for the local translation of PINK1 provides distal mitochondria with a continuous supply of PINK1 for the activation of mitophagy.

Usual Interstitial Pneumonia (UIP) is a histological pattern characteristic of Idiopathic Pulmonary Fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control. Furthermore, the FF is positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of the FF is predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This new and essential information will inform future mechanistic studies on fibrosis progression.