Double Imprinted Nanoparticles for Sequential Membrane-to-Nuclear Drug Delivery.

Efficient and site-specific delivery of therapeutics drugs remains a critical challenge in cancer treatment. Traditional drug nanocarriers such as antibody-drug conjugates are not generally accessible due to their high cost and can lead to serious side effects including life-threatening allergic reactions. Here, these problems are overcome via the engineering of supramolecular agents that are manufactured with an innovative double imprinting approach. The developed molecularly imprinted nanoparticles (nanoMIPs) are targeted toward a linear epitope of estrogen receptor alfa (ERα) and loaded with the chemotherapeutic drug doxorubicin. These nanoMIPs are cost-effective and rival the affinity of commercial antibodies for ERα. Upon specific binding of the materials to ERα, which is overexpressed in most breast cancers (BCs), nuclear drug delivery is achieved via receptor-mediated endocytosis. Consequentially, significantly enhanced cytotoxicity is elicited in BC cell lines overexpressing ERα, paving the way for precision treatment of BC. Proof-of-concept for the clinical use of the nanoMIPs is provided by evaluating their drug efficacy in sophisticated three-dimensional (3D) cancer models, which capture the complexity of the tumor microenvironment in vivo without requiring animal models. Thus, these findings highlight the potential of nanoMIPs as a promising class of novel drug compounds for use in cancer treatment.

Chemical mapping of xyloglucan distribution and cellulose crystallinity in cotton textiles reveals novel enzymatic targets to improve clothing longevity.

Pilling is a form of textile mechanical damage, forming fibrous bobbles on the surface of garments, resulting in premature disposal of clothing by consumers. However, our understanding on how the structural properties of the cellulosic matrix compliment the three-dimensional shape of cotton pills remains limited. This knowledge gap has hindered the development of effective 'pillase' technologies over the past 20 years due to challenges in balancing depilling efficacy with fabric integrity preservation. Therefore, the main focus here was characterising the role of cellulose and the hemicellulose components in cotton textiles to elucidate subtle differences between the chemistry of pills and fibre regions involved in structural integrity. State-of-the-art bioimaging using carbohydrate binding modules, monoclonal antibodies, and Leica SP8 and a Nikon A1R confocal microscopes, revealed the biophysical structure of cotton pills for the first time. Identifying regions of increased crystalline cellulose in the base of anchor fibres and weaker amorphous cellulose at dislocations in their centres, enhancing our understanding of current enzyme specificity. Surprisingly, pills contained a 7-fold increase in the concentration of xyloglucan compared to the main textile. Therefore, xyloglucan offers a previously undescribed target for overcoming this benefit-to-risk paradigm, suggesting a role for xyloglucanase enzymes in future pillase systems.

Functional and Molecular Analysis of Human Osteoarthritic Chondrocytes Treated with Bone Marrow-Derived MSC-EVs.

Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1ß on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.

XPR1: a regulator of cellular phosphate homeostasis rather than a Pi exporter.

Phosphate (Pi) is an essential nutrient, and its plasma levels are under tight hormonal control. Uphill transport of Pi into cells is mediated by the two Na-dependent Pi transporter families SLC34 and SLC20. The molecular identity of a potential Pi export pathway is controversial, though XPR1 has recently been suggested by Giovannini and coworkers to mediate Pi export. We expressed XPR1 in Xenopus oocytes to determine its functional characteristics. Xenopus isoforms of proteins were used to avoid species incompatibility. Protein tagging confirmed the localization of XPR1 at the plasma membrane. Efflux experiments, however, failed to detect translocation of Pi attributable to XPR1. We tested various counter ions and export medium compositions (pH, plasma) as well as potential protein co-factors that could stimulate the activity of XPR1, though without success. Expression of truncated XPR1 constructs and individual domains of XPR1 (SPX, transmembrane core, C-terminus) demonstrated downregulation of the uptake of Pi mediated by the C-terminal domain of XPR1. Tethering the C-terminus to the transmembrane core changed the kinetics of the inhibition and the presence of the SPX domain blunted the inhibitory effect. Our observations suggest a regulatory role of XPR1 in cellular Pi handling rather than a function as Pi exporter. Accordingly, XPR1 senses intracellular Pi levels via its SPX domain and downregulates cellular Pi uptake via the C-terminal domain. The molecular identity of a potential Pi export protein remains therefore elusive.

Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.

High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.

Impact of retrotransposon protein L1 ORF1p expression on oncogenic pathways in hepatocellular carcinoma: the role of cytoplasmic PIN1 upregulation.

BACKGROUND: Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood. METHODS: We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems. RESULTS: We observed positive associations between L1 and activated TGFß-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFß-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFß-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p. DISCUSSION: Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

The Neuroprotective Effects of mGlu1 Receptor Antagonists Are Mediated by an Enhancement of GABAergic Synaptic Transmission via a Presynaptic CB1 Receptor Mechanism.

In this study, we investigated the cross-talk between mGlu1 and CB1 receptors in modulating GABA hippocampal output in whole-cell voltage clamp recordings in rat hippocampal acute slices, in organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD) and in gerbils subjected to global ischemia. CB1 receptor expression was studied using immunohistochemistry and the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by LC-MS/MS. Our results show that mGlu1 receptor antagonists enhance sIPSCs in CA1 pyramidal cells and the basal and ischemic hippocampal release of GABA in vivo in a manner that is mediated by CB1 receptor activation. In hippocampal slices exposed to OGD and in ischemic gerbils, mGlu1 receptor antagonists protected CA1 pyramidal cells against post-ischemic injury and this effect was reduced by CB1 receptor activation. OGD induced a transient increase in the hippocampal content of AEA and this effect is prevented by mGlu1 receptor antagonist. Finally, OGD induced a late disruption of CB1 receptors in the CA1 region and the effect was prevented when CA1 pyramidal cells were protected by mGlu1 antagonists. Altogether, these results suggest a cooperative interaction between mGlu1 receptors and the endocannabinoid system in the mechanisms that lead to post-ischemic neuronal death.

High-resolution simulations of chromatin folding at genomic rearrangements in malignant B cells provide mechanistic insights into proto-oncogene deregulation.

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to 3D genome structure remains poorly understood. Here, we used the highly predictive heteromorphic polymer (HiP-HoP) model to predict chromatin conformations at the proto-oncogene CCND1 in healthy and malignant B cells. After confirming that the model gives good predictions of Hi-C data for the nonmalignant human B cell-derived cell line GM12878, we generated predictions for two cancer cell lines, U266 and Z-138. These possess genome rearrangements involving CCND1 and the immunoglobulin heavy locus (IGH), which we mapped using targeted genome sequencing. Our simulations showed that a rearrangement in U266 cells where a single IGH super-enhancer is inserted next to CCND1 leaves the local topologically associated domain (TAD) structure intact. We also observed extensive changes in enhancer-promoter interactions within the TAD, suggesting that it is the downstream chromatin remodeling which gives rise to the oncogene activation, rather than the presence of the inserted super-enhancer DNA sequence per se. Simulations of the IGH-CCND1 reciprocal translocation in Z-138 cells revealed that an oncogenic fusion TAD is created, encompassing CCND1 and the IGH super-enhancers. We predicted how the structure and expression of CCND1 changes in these different cell lines, validating this using qPCR and fluorescence in situ hybridization microscopy. Our work demonstrates the power of polymer simulations to predict differences in chromatin interactions and gene expression for different translocation breakpoints.

Mitochondrial complex I subunit deficiency promotes pancreatic α-cell proliferation.

OBJECTIVE: There is strong evidence that mitochondrial DNA mutations and mitochondrial dysfunction play a role in diabetes pathogenesis. The homozygous knock-in mtDNA mutator mouse is a model of premature aging due to the accumulation of mitochondrial DNA mutations. We used this mouse model to investigate the relationship between mitochondrial subunit expression and pancreatic islet cell composition. METHODS: Quadruple immunofluorescence was used to quantify mitochondrial subunit expression (complex I and IV) and cell composition in pancreatic islets from mitochondrial DNA mutator mice (PolgAmut/mut) and control C57BL/6 mice at 12 and 44 weeks of age. RESULTS: Mitochondrial complex I subunit expression was decreased in islets from 12 week PolgAmut/mut mice. This complex I deficiency persisted with age and was associated with decreased insulin staining intensity at 44 weeks. Complex I deficiency was greater in α-cells compared with ß-cells in islets from 44 week PolgAmut/mut mice. Islet cell composition was normal in 12 week PolgAmut/mut mice, but the ß: α cell ratio was decreased in islets from 44 week PolgAmut/mut mice. This was due to an increase in α-cell number linked to an increase in α-cell proliferation. CONCLUSION: Complex I deficiency promotes α-cell proliferation and alters islet cell composition.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Mitochondrial Translation Occurs Preferentially in the Peri-Nuclear Mitochondrial Network of Cultured Human Cells.

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in the distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

High-resolution imaging reveals compartmentalization of mitochondrial protein synthesis in cultured human cells.

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.

Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines.

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.

Distinctive Features of Orbital Adipose Tissue (OAT) in Graves' Orbitopathy.

Depot specific expansion of orbital-adipose-tissue (OAT) in Graves' Orbitopathy (GO) is associated with lipid metabolism signaling defects. We hypothesize that the unique adipocyte biology of OAT facilitates its expansion in GO. A comprehensive comparison of OAT and white-adipose-tissue (WAT) was performed by light/electron-microscopy, lipidomic and transcriptional analysis using ex vivo WAT, healthy OAT (OAT-H) and OAT from GO (OAT-GO). OAT-H/OAT-GO have a single lipid-vacuole and low mitochondrial number. Lower lipolytic activity and smaller adipocytes of OAT-H/OAT-GO, accompanied by similar essential linoleic fatty acid (FA) and (low) FA synthesis to WAT, revealed a hyperplastic OAT expansion through external FA-uptake via abundant SLC27A6 (FA-transporter) expression. Mitochondrial dysfunction of OAT in GO was apparent, as evidenced by the increased mRNA expression of uncoupling protein 1 (UCP1) and mitofusin-2 (MFN2) in OAT-GO compared to OAT-H. Transcriptional profiles of OAT-H revealed high expression of Iroquois homeobox-family (IRX-3&5), and low expression in HOX-family/TBX5 (essential for WAT/BAT (brown-adipose-tissue)/BRITE (BRown-in-whITE) development). We demonstrated unique features of OAT not presented in either WAT or BAT/BRITE. This study reveals that the pathologically enhanced FA-uptake driven hyperplastic expansion of OAT in GO is associated with a depot specific mechanism (the SLC27A6 FA-transporter) and mitochondrial dysfunction. We uncovered that OAT functions as a distinctive fat depot, providing novel insights into adipocyte biology and the pathological development of OAT expansion in GO.

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.

Differential mechanisms of tolerance induced by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning.

We investigated the molecular events triggered by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post-synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole-cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA-induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG-lipase inhibitor URB602, that increases arachidonoylglycerol (2-AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2-AG is responsible for the DHPG-induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1-AMPA receptors, DHPG by producing the endocannabinoid 2-AG.

In Situ Analysis Reveals That CFTR Is Expressed in Only a Small Minority of ß-Cells in Normal Adult Human Pancreas.

CONTEXT: Although diabetes affects 40% to 50% of adults with cystic fibrosis, remarkably little is known regarding the underlying mechanisms leading to impaired pancreatic ß-cell insulin secretion. Efforts toward improving the functional ß-cell deficit in cystic fibrosis-related diabetes (CFRD) have been hampered by an incomplete understanding of whether ß-cell function is intrinsically regulated by cystic fibrosis transmembrane conductance regulator (CFTR). Definitively excluding meaningful CFTR expression in human ß-cells in situ would contribute significantly to the understanding of CFRD pathogenesis. OBJECTIVE: To determine CFTR messenger ribonucleic acid (mRNA) and protein expression within ß-cells in situ in the unmanipulated human pancreas of donors without any known pancreatic pathology. DESIGN: In situ hybridization for CFTR mRNA expression in parallel with insulin immunohistochemical staining and immunofluorescence co-localization of CFTR with insulin and the ductal marker, Keratin-7 (KRT7), were undertaken in pancreatic tissue blocks from 10 normal adult, nonobese deceased organ donors over a wide age range (23-71 years) with quantitative image analysis. RESULTS: CFTR mRNA was detectable in a mean 0.45% (range 0.17%-0.83%) of insulin-positive cells. CFTR protein expression was co-localized with KRT7. One hundred percent of insulin-positive cells were immunonegative for CFTR. CONCLUSIONS: For the first time, in situ CFTR mRNA expression in the unmanipulated pancreas has been shown to be present in only a very small minority (<1%) of normal adult ß-cells. These data signal a need to move away from studying endocrine-intrinsic mechanisms and focus on elucidation of exocrine-endocrine interactions in human cystic fibrosis.

An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.