👤 H Massa

Annotation of regulatory elements is essential for understanding mechanisms underlying gene regulation, particularly tissue-specific regulation in human and animals. Here, we characterize 274,682 enhancers and 25,975 promoters across 24 tissues from an adult female sheep using ChIP-seq, ATAC-seq, CAGE-seq, RRBS, WGBS, and RNA-seq. We identify seven neural development-related genes with over 10 enhancers in brain tissues, highlighting the role of tissue-specific regulation. Cis-regulatory enhancer-promoter combinations provide insights into tissue-specific enhancers, such as the cerebellum-specific enhancer (chr15: 57390520-57390685) regulating BDNF, which is expressed in both the cerebellum and cerebral cortex. Comparative analysis of enhancer-promoter combinations in human, mouse, pig, cattle, and sheep reveals ruminant-specific pathways, including pentose catabolism and long-chain fatty acid import regulation. A milk fat yield quantitative trait locus (QTL) identified within an enhancer interacts with the fat metabolism-related gene COMMD1, and a birth weight-associated QTL detected within a cerebellum-specific enhancer regulates XKR4. This study provides a robust framework for exploring cis-regulatory mechanisms and tissue-specific regulation, advancing the functional annotation of the sheep reference genome. Show less

The transient, heterogeneous nano-bio interface defined by the protein corona in biological environments dictates the biodistribution, immune recognition, metabolism, and clearance of nanomaterials. Far from being a drawback, this corona can be harnessed for targeted nanodrug delivery when its composition is predictably tuned or deliberately modulated. We hypothesized that preloading apolipoprotein E (ApoE), previously identified as a constituent of the corona of β-sheet-breaker peptide-functionalized gold nanoparticles (AuNPs), would enhance transport across the blood-brain barrier (BBB) and increase brain uptake. To test this, we synthesized AuNPs (approximately 12 nm) functionalized (AuNP-f) with CLPFFD or THRPPMWSPVWPCLPFFD peptides, both containing the β-sheet-breaker motif LPFFD, which recognizes β-amyloid aggregates implicated in Alzheimer's disease. After incubation with human plasma, hard-corona proteins were profiled by 2D IEF/SDS-PAGE and LC-MS/MS. Proteins were ranked based on their roles in nanoparticle trafficking and BBB transcytosis, and ApoE was selected for deliberate enrichment due to its recurrent presence. ApoE-decorated AuNP-f were evaluated in an in vitro BBB model and in vivo biodistribution assays using Sprague-Dawley rats. Brain accumulation was assessed ex vivo. Preloading ApoE onto AuNP-f significantly enhanced nanoparticle transport across the BBB in vitro and increased brain accumulation in rats. These results demonstrate that rational corona enrichment with ApoE improves BBB transit and brain accumulation without altering nanoparticle surface chemistry. Corona engineering thus offers a pragmatic route to brain-targeted nanodrug delivery and may be extended to other protein-receptor axes for organ-specific targeting. Show less

Immune checkpoint inhibitors (ICIs) are a key component of different stages of hepatocellular carcinoma (HCC) treatment, particularly in the first line of treatment. A lesson on the primary resistance which hampers their efficacy and activity was learned from the failure of the trials which tested them as first-line mono-therapies. Despite the combination of anti-PD(L)1 agents with anti-VEGF, anti CTLA4, or TKIs demonstrating relevant improvements in efficacy, the "doublets strategy" still shows room for improvement, due to a limited overall survival benefit and a high rate of progressive disease as best response. In this review, we discuss the results from the currently tested doublet strategies (i.e., atezolizumab+bevacizumab, durvalumab+tremelimumab with a mention to the newly presented ICIs/TKIs combinations), which highlight the need for therapeutic improvement. Furthermore, we examine the rationale and provide an overview of the ongoing trials testing the treatment intensification strategy with triplet drugs: anti-PD1+anti-CTLA4+anti-VEGF/TKIs and anti-PD1+anti-VEGF+alternative immunity targets. Lastly, we report on the alternative strategy to integrate ICIs into the new paradigm of immune therapeutics constituted by CAR-T and anti-cancer vaccines. This review provides up-to-date knowledge of ongoing clinical trials of the aforementioned strategies and critical insight into their mechanistic premises. Show less

The hypothalamic paraventricular nucleus (PVN) is a key target of the melanocortin system, which orchestrates behavioral and metabolic responses depending on energy availability. The mechanistic target of rapamycin complex 1 (mTORC1) and the endocannabinoid type 1 receptor (CB1R) pathways are two key signaling systems involved in the regulation of energy balance whose activity closely depends upon energy availability. Here we tested the hypothesis that modulation of mTORC1 and CB1R signaling regulates excitatory glutamatergic inputs onto the PVN. Patch-clamp recordings in C57BL/6J mice, in mice lacking the mTORC1 component Rptor or CB1R in pro-opio-melanocortin (POMC) neurons, combined with pharmacology targeting mTORC1, the melanocortin receptor type 4 (MC4R), or the endocannabinoid system under chow or a hypercaloric diet. Acute pharmacological inhibition of mTORC1 in C57BL/6J mice decreased glutamatergic inputs onto the PVN via a mechanism requiring modulation of MC4R, endocannabinoid 2-AG mobilization by PVN parvocellular neurons, and retrograde activation of presynaptic CB1R. Further electrophysiology studies using mice lacking mTORC1 activity or CB1R in POMC neurons indicated that the observed effects involved mTORC1 and CB1R-dependent regulation of glutamate release from POMC neurons. Finally, energy surfeit caused by hypercaloric high-fat diet feeding, rapidly and time-dependently altered the glutamatergic inputs onto parvocellular neurons and the ability of mTORC1 and CB1R signaling to modulate such excitatory activity. These findings pinpoint the relationship between mTORC1 and endocannabinoid-CB1R signaling in the regulation of the POMC-mediated glutamatergic inputs onto PVN parvocellular neurons and its rapid alteration in conditions favoring the development of obesity. Show less

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. Show less

Williams syndrome (WS) is a contiguous gene deletion disorder caused by haploinsufficiency of genes at 7q11.23. We have shown that hemizygosity of elastin is responsible for one feature of WS, supravalvular aortic stenosis (SVAS). We have also implicated LIM-kinase 1 hemizygosity as a contributing factor to impaired visual-spatial constructive cognition in WS. However, the common WS deletion region has not been completely characterized, and genes for additional features of WS, including mental retardation, infantile hypercalcemia, and unique personality profile, are yet to be discovered. Here, we present a physical map encompassing 1.5 Mb DNA that is commonly deleted in individuals with WS. Fluorescence in situ hybridization analysis of 200 WS individuals shows that WS individuals have the consistent deletion interval. In addition, we identify three novel genes from the common deletion region: WS-betaTRP, WS-bHLH, and BCL7B. WS-betaTRP has four putative beta-transducin (WD40) repeats, and WS-bHLH is a novel basic helix-loop-helix leucine zipper (bHLHZip) gene. BCL7B belongs to a novel family of highly conserved genes. We describe the expression profile and genomic structure for each of these genes. Hemizygous deletion of one or more of these genes may contribute to developmental defects in WS. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by multiple bony outgrowths from the juxtaepiphyseal region of long bones. In a small proportion of cases, these exostoses progress to malignant chondrosarcomas. Genetic linkage of this disorder has been described to three independent loci on chromosomes 8q24.1 (EXT1), 11p11-13 (EXT2), and 19p (EXT-3). The EXT1 and EXT2 genes were isolated recently and show extensive sequence homology to each other. These genes are deleted in exostoses-derived tumors, supporting the hypothesis that they encode tumor suppressors. We have identified a third gene that shows striking sequence similarity to both EXT1 and EXT2 at the nucleotide and amino acid sequence levels, and have derived its entire coding sequence. Although the mRNA transcribed from this gene is similar in size to that from EXT1 and EXT2, its pattern of expression is quite different. We have localized this gene by fluorescence in situ hybridization to metaphase chromosomes and by whole genome radiation hybrid mapping to chromosome 1p36.1 between DIS458 and DIS511, region that frequently shows loss of heterozygosity in a variety of tumor types. This gene, EXTL (for EXT-like), is therefore a new member of the EXT gene family and is a potential candidate for several disease phenotypes. Show less