Although it has been reported that mesenchymal stem cells isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells, the findings and their interpretation have been challenged. The main argument against this process is that the cells rapidly adopt neuron-like morphologies through retraction of the cytoplasm rather than active neurite extension. In this study, we examined the sequence of biological events during neural differentiation of human periodontal ligament-derived stem cells (hPDLSCs), human bone marrow-derived stem cells (hBMSCs) and human dental pulp-derived stem cells (hDPSCs) by time-lapse microscopy. We have demonstrated that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage and that they shrink dramatically and change their morphology to that of neuron-like cells through active neurite extension. Furthermore, we observed micronuclei movement and transient cell nuclei lobulation concurrent to in vitro neurogenesis from hBMSCs and hDPSCs. Our results demonstrate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step followed by differentiation to neural phenotypes, and therefore we definitively confirm that the rapid acquisition of the neural phenotype is via a differentiation trait. While asymmetric cell division is considered to be the mechanism by which the asymmetric inheritance of cellular components during mitosis defines the distinct fate of each daughter cell3.In the classical view of cell development, embryonic stem cells differentiate into progressively more precursor cells with an increasingly restricted lineage potential, until the final mature, specialised cell types are generated and functionally integrated into their respective tissues. The differentiated state of a cell was believed to be terminal and irreversible4. It has long been accepted that cells can change their identity, both in vitro and in vivo, a phenomenon known as cellular plasticity6. Cellular conversion falls into two major categories: dedifferentiation and transdifferentiation8. A definitive, well-accepted criterion for dedifferentiation and transdifferentiation has yet to be established9. In its simplest form, dedifferentiation is the conversion from one differentiated cell stage to a less differentiated stage. Transdifferentiation is the conversion of one differentiated cell type into another. It has been suggested that transdifferentiation may involve a dedifferentiation step before cells differentiate to a new mature phenotype, or it may occur directly in a process that bypasses such immature phenotypes10.However, there is increasing evidence that the rules of irreversibility and germ-layer restriction can be broken18. Although the transdifferentiation of human MSCs into neurons has aroused considerable interest, as it would have immense clinical potential in cell replacement therapy and regenerative medicine20, the findings and their interpretation have been challenged22. The main argument against this process is that the cells rapidly adopt neuron-like morphologies through retraction of the cytoplasm rather than by active neurite extensión24.Literature published over the past 20 years have shown that mesenchymal stem cells (MSCs) isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells25, we showed that human periodontal ligament-derived stem cells (hPDLSCs) can directly differentiate into neuron-like cells without passing through any mitotic stages. When hPDLSCs were exposed to a neural induction medium, we found that they rapidly underwent a dramatic change in shape and size; they initially adopted highly irregular forms before gradually contracting into round cells (neurogenesis). These round cells then slowly adopted a complex morphology and finally gave rise to a variety of neuron-like morphologies . Furthermore, we also reported nuclear remodelling concurrent to in vitro neurogenesis from hPDLSCs.In a recent studyThe present study is designed to confirm that human stem cells isolated from adult tissues in the early stages of exposure to a neural induction medium adopt a neuron-like morphology because of a differentiation trait rather than an artefact. We performed time-lapse phase-contrast microscopy to record the changes in the cell morphology of hPDLSCs, human bone marrow-derived stem cells (hBMSCs) and human dental pulp-derived stem cells (hDPSCs).We found that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage. All three types of stem cell shrunk dramatically and changed their morphology to that of neuron-like cells with active neurite extension. Additionally, we may have evidenced micronuclei movement and transient cell nuclei lobulation in parallel with in vitro neurogenesis from hBMSCs and hDPSCs. Our results clearly indicate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step before the cells differentiate to neural phenotypes. These findings definitively establish that the rapid acquisition of a neuron-like morphology during neural differentiation is due to a differentiation trait as opposed to an artefact.To conclusively affirm that hPDLSCs adopt a neuron-like morphology in the early stages of exposure to a neural induction medium is due to active neurite extension rather than retraction of the cytoplasm, we performed time-lapse phase-contrast microscopy and immunocytochemical analysis within the first 24 h of neural differentiation.Time-lapse imaging revealed that, after neural induction, the hPDLSCs underwent a rapid change in shape and size, first adopting highly irregular forms and then gradually contracting into round cells. Subsequently, we observed the growth of new neurites from the cell body of round cells Fig. , 2.FigurIn addition, cytoskeletal protein β-III tubulin and F-actin staining showed that hPDLSC-derived neuron-like cells developed distinct dendrite-like and axon-like domains Fig. b. Time-lhPDLSCs did not differentiate at the same time, so the cell culture simultaneously contained hPDLSCs at different stages of neurogenesis and neuronal polarisation. The results demonstrate that hPDLSCs can directly differentiate into neuron-like cells without passing through a mitotic stage, thus definitively confirming that the rapid acquisition of a neuron-like morphology during neural differentiation is by means of a differentiation trait rather than merely an artefact.27 and dental pulp31 have been described previously. Under proliferation conditions, hBMSCs and hDPSCs displayed a fibroblast-like morphology with actin microfilaments and β-III tubulin microtubules oriented parallel to the cell’s longitudinal axis . In addition, no unusual nuclear structures were observed as hPDLSC-derived neuron-like cells gradually acquired a more mature neuron-like morphology .In a recent study25. Chromatin-containing bodies arise from the main nuclei and start to move towards specific positions within the cell, temporarily forming lobed nuclei. Subsequently, these lobed nuclei connect together to form nucleoplasmic bridges and finally the internuclear bridges facilitate the merger into a single nucleus with an eccentric position within the cell.While we acknowledge that the definitive nuclear remodelling sequence, when hPDLSCs become near-spherical, can only be determined by detecting fluorescent labelled cell nuclei in time-lapse microscopy, our accumulated data suggest how these steps may occurTo determine whether nuclear remodelling also occurs when hBMSCs and hDPSCs take on a near-spherical shape, we differentiated hPDLSCs, hBMSCs and hDPSCs in the same neural induction medium Figs. , 7, 8.FiWithin 2 h of exposure to the neural induction medium we observed DNA-containing structures arise from the main nuclei of hPDLSCs Fig. a, hBMSCsWithin 24 h of exposure to the neural induction medium we observed lobed nuclei in hDPSCs Fig. d, lobed 33 and further support the idea that hBMSCs, hPDLSCs and hDPSCs follow similar morphological neural development sequences in vitro.The results indicate that nuclear remodelling also occurred during in vitro neurogenesis from hBMSCs and hDPSCs. hBMSCs and hDPSCs also exhibited unusual nuclear structures and extranuclear bodies in the cellular cytoplasm when they approach a near-spherical shape. These DNA-containing structures displayed a spherical or ovoid shape Fig. a and somIn this study, we have shown that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage. In fact, the spindle-shaped cells (differentiated cell stage) were reset and started their neuronal development as round spheres (less differentiated cell stage). It would, therefore, be interesting to examine whether cell rounding is a common feature of cellular plasticity.Our results indicatate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step (neurogenesis) before the cells differentiate to neural phenotypes .In the present work, we have demonstrated that hPDLSC-derived, hBMSC-derived and hDPSC-derived neuron-like cells produce neurites that gradually adopt a complex morphology, acquiring dendrite-like and axon-like identities. Therefore, our results definitively establish that the rapid acquisition of a neuron-like morphology during neural differentiation occurs though a differentiation trait and is not just an artefact.34.Although further research is required to confirm the successful differentiation of hPDLSCs, hBMSCs and hDPSCs into a neural lineage, and finally facilitate the production of autologous cells for cell replacement in a diseased central nervous system, our results provide additional evidence that cells derived from human adult tissues can be converted into neuronal cells without genetic manipulation25, we reported that nuclear remodelling occurred during in vitro neurogenesis from hPDLSCs. We observed that many hPDLSCs had an unusual nuclear structure and chromatin-containing bodies in the cellular cytoplasm when they took on a near-spherical shape. In the present study, we have shown that nuclear remodelling also occurred during in vitro neurogenesis from hBMSCs and hDPSCs. We observed that hBMSCs and hDPSCs exhibited unusual nuclear structures and chromatin-containing bodies in the cellular cytoplasm when they became near-spherical in shape. It is important to mention that the hPDLSCs, hBMSCs and hDPSCs had a very similar nuclear morphology during in vitro neurogenesis. These results confirm our previous findings33 and further support that hPDLSCs, hBMSCs and hDPSCs follow similar morphological neural development sequences in vitro.In a previous publication35), vertebrates, invertebrates, plants and protozoa37. However, the exact role of these extranuclear bodies and their possible correlation with unusual nuclear structures is not yet known38. Our results suggest these unusual nuclear structures and extranuclear bodies may be associated with nuclear movement within the cell. Although future analysis involving live-cell nucleus fluorescence labelling and time-lapse microscopy is necessary to determine whether chromatin-containing bodies move within the cell, and if there is any correlation between the formation of these extranuclear bodies and the unusual nuclear structures, a recent study has demonstrated that chromatin-containing bodies arise from the main nuclei, move within the cell and are ultimately loaded in exosomes39.These unusual nuclear structures and extranuclear bodies were described independently in mammals  bears great similarities to the nuclear morphology of neural stem cells (NSCs) located in the ventricular-subventricular zone of the anterolateral ventricle wall in the human foetal brain45. However, the current models of adult NSC maintenance and differentiation are still contentious because the approaches used to probe stem cell division and differentiation are inherently limited46.It has generally been believed that adult neurogenesis occurs progressively through sequential phases of proliferation and neuronal differentiation of adult NSCs55, which would confirm that adult neurogenesis occurs progressively through sequential phases of proliferation. What is more, several studies indicate that some observations interpreted as cell division could be false positives60. The findings mentioned above and the similarities between the nuclear morphology of hPDLSCs, hBMSCs and hDPSCs during in vitro neurogenesis compared to that of adult NSCs located in neurogenic niches and cultured hippocampal neurons suggest that adult NSCs may also convert into neurons without involving cell division. Future research is necessary to determine the likelihood of this conjecture.It is important to note that very few reports describing newborn neurons in the adult brain have found mitotic chromosomes61. Our results demonstrate that hPDLSCs, hBMSCs and hDPSCs retain plasticity. Therefore, these cells could also help increase our understanding of the mechanisms underlying changes in cell fate and shed light on whether differentiation, dedifferentiation and transdifferentiation occur in different contexts or are different terms for the same cellular process.As noted above, there is increasing evidence to suggest that adult cells can assume new fates without asymmetric cell division through dedifferentiation and transdifferentiation processes. These findings call into question the rules of irreversibility and germ-layer restriction as well as the classical definition of a stem cellThe authors declare that all protocols used to obtain and process all human samples were approved by the local ethics committees  according to Spanish and European legislation and conformed to the ethical guidelines of the Helsinki Declaration. Donors provided written informed consent before obtaining samples.To obtain hPDLSCs, human premolars were extracted and collected from three different healthy adult donors undergoing orthodontic therapy in Murcia dental hospital (Spain). Human periodontal ligament (hPDL) was scraped from the middle third region of the root surface. To obtain hPDSCs, human third molars were extracted and collected from three different healthy adult donors undergoing orthodontic therapy in Murcia dental hospital (Spain). Human dental pulp (hDP) was harvested from the teeth.62. After washing the extracted hPDL and hDP with Ca and Mg-free Hank’s balance salt solution (HBSS), hPDL and hDP was digested with type I collagenase (3 mg/ml) and dispase II (4 mg/ml) in alpha modification minimum essential medium eagle (α-MEM) for 1 h at 37 °C. The reaction was stopped by the addition of α-MEM. hPDL and hDP derived from different subjects were pooled together to obtain single cell suspensions by passing the cells through a 70 μm strainer . Cells were centrifuged, and the pellet was resuspended in serum-containing media , composed of DMEM low glucose medium supplemented with fetal bovine serum (10%), non-essential amino acid solution, penicillin–streptomycin (100 units/ml) and l-glutamine (2 mM). The cell suspension was plated into six-well multiwell plates  and incubated at 37 °C in 5% CO2. All studies were performed using hPDLSCs and hDPSCs expanded within culture passages 3–4.Dissociated cell cultures of hPDL and hDP tissues were prepared as previously described2+/Mg2+-free phosphate buffered saline (PBS) and seeded into 175-cm2 culture flasks  at a cell density 1.5 × 105 cells/cm2 in DMEM low glucose medium supplemented with fetal bovine serum (10%), GlutaMAX (1%) and penicillin/streptomycin (1%). After 3 days of culture at 37ºC and 5% CO2, non-attached cells were removed and fresh complete medium was added. Culture media were renewed every 2 days, and the isolated hBMSCs were passaged when cultures were 70–80% confluent. All studies were performed using hBMSCs expanded within culture passages 3–4.Bone marrow aspirates were obtained by percutaneous direct aspiration from the iliac crest of 5 healthy volunteers at University Hospital Virgen de la Arrixaca . Bone marrow was collected with 20 U/ml sodium heparin, followed by a Ficoll density gradient-based separation by centrifugation at 540 g for 20 min. After, mononuclear cell fraction was collected, washed twice with CaWe used μ-Dish 35 mm, high Grid- 500 (Ibidi) for live cell imaging. Numeric marks on the bottom of each dish allow users to identify the location of cells. Cells were plated onto collagen IV coated plastic or glass coverslips, and maintained in basal media or neural induction media.To induce neural differentiation, cells at passage 3–4 were allowed to adhere to the plates overnight. Media was removed the following day and the cells were cultured for 2 days in serum-free media  consisting in Dulbecco’s modified Eagle’s medium/F12  supplemented with N2-supplement, glucose (0.6%), HEPES (5 mM), human serum albumin (0.5%), heparin (0.0002%), non-essential amino acid solution and penicillin–streptomycin (100 units/ml). On day 3, cells were cultured in neural induction media, consisting in the neural basal media supplemented with retinoic acid (500 nM), dibutyryl cAMP (1 mM) and growth factors like BDNF , GDNF  and IGF-1 . We perform time-lapse phase-contrast microscopy within the first 24 h after neural induction media was added directly to the cells.62. Cells were plated onto collagen IV coated plastic or glass coverslips, and maintained in basal media or neural induction media. Cells were rinsed with PBS and fixed in freshly prepared paraformaldehyde (4%). Fixed cells were blocked for 2 h in PBS containing normal horse serum (10%) and Triton X-100 (0.25%) and incubated overnight at 4 °C with antibodies against β-III-tubulin  in PBS containing normal horse serum (1%) and Triton X-100 (0.25%). On the next day, cells were rinsed and incubated with the secondary antibody: Alexa Fluor® 594 . Cell nuclei were counterstained with DAPI . Alexa Fluor 488® phalloidin (Molecular Probes) was used to selectively stains F-actin. Data are representative of ten independent experiments per condition.A standard immunocytochemical protocol was used as previously described25. We used an inverted Leica DM IRB microscope equipped with a digital camera Leica DFC350FX (Nussloch) and confocal laser scanning microscope Leica TCS-SP8. Digitized images were analyzed using LASX Leica confocal software. We used Photoshop software to improve the visibility of fluorescence images without altering the underlying data.Analyses and photography of visible and fluorescent stained samples were carried out as previously describedSupplementary Figures.


M) and changes in intracellular [Ca2+] driven by voltage-gated Ca2+ channels (VGCCs). Arterial VM is in turn set by the dynamic interplay among ion channel species, the strongly inward rectifying K+ (Kir) channel being of special interest. Kir2 channels possess a unique biophysical signature in that they strongly rectify, display negative slope conductance, respond to elevated extracellular K+ and are blocked by micromolar Ba2+. While functional Kir2 channels are expressed in both smooth muscle and endothelium, they lack classic regulatory control, thus are often viewed as a simple background conductance. Recent literature has provided new insight, with two membrane lipids, phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, noted to (1) stabilize Kir2 channels in a preferred open or closed state, respectively, and (2) confer, in association with the cytoskeleton, caveolin-1 (Cav1) and syntrophin, hemodynamic sensitivity. It is these aspects of vascular Kir2 channels that will be the primary focus of this review.Cerebral arteries contain two primary and interacting cell types, smooth muscle (SMCs) and endothelial cells (ECs), which are each capable of sensing particular hemodynamic forces to set basal tone and brain perfusion. These biomechanical stimuli help confer tone within arterial networks upon which local neurovascular stimuli function. Tone development is intimately tied to arterial membrane potential (V This va cycling . The eleial (VM) . This abchannels .M values negative to EK, activation by extracellular K+, and rapid blockade by micromolar Ba2+ , with a primary focus on cerebral vascular tone and perfusion control.vs. fluid lipid components, which in turn depends primarily on the temperature, and the length/degree of unsaturation of phospholipid fatty acids.Every cell in a living organism contains a thick ∼4–10 nm hydrophobic layer which serves as a physical and selectively permeable barrier between two aqueous and heterogeneous environments: the cytoplasm and the outside of the cell. The plasma membrane represents a mosaic of multiple components—including lipids, embedded proteins, and carbohydrates—that are constantly subjected to spontaneous reorganization by rotational or lateral motion . InteresMore than 1,000 different species of lipids coexist within the plasma membrane, heterogeneously separated between the two leaflets and giving rise to complex lipid environments at the cell surface . These l2), which has emerged as a highly versatile player in cell signaling, constitutes only ∼1–3% of the phospholipids in eukaryotic cell membranes  domains—which bind specifically to membrane PtdIns—have more accurately estimated its effective concentration to range from 2 to 30 μM  in two successive phosphorylation steps by the enzymes PI4 and PI5 kinases (PIK4 and PIK5). Moreover, the efficient hydrolysis of PIP2 provides the source of two important secondary messengers in the cell, inositol trisphosphate (IP3) and diacylglycerol (DAG). Structurally, PIP2 contains an inositol head group with an average orientation of 45° with respect to the bilayer surface  is a central lipid in mammalian cells involved in multiple cellular processes at the vascular plasma membrane. It accounts for up to 50% of total membrane lipid molecules, as more than 90% of total cellular cholesterol is confined to the plasma membrane , altering their conformational states and regulating their activity  while passing small outward currents at more depolarized voltages , a fundamental process that ensures regional increases in blood flow in to maintain tissue function and health , different from that of homomeric Kir2.1 or Kir2.2 channels   Ba2+ exclusively blocks the inward rectification at voltages more hyperpolarized than EK and, (2) tissues expressing Kir channels exclusively exhibit [K+]-induced vasodilation sensitive to Ba2+  . The selnic tone , 60. Acc surface  and crem surface .2 and cholesterol, which stimulates and suppresses the Kir2 channel activity, respectively. Specifically, PIP2 is required at certain levels in the plasma membrane to stabilize Kir2 channels in a preferred open (active) state, and transient depletion of PIP2 by PLC activation in vivo suppresses these functions  amino acid residues  of Kir2 channels to directly modulate their activity. In particular, the majority of these basic amino acid residues are positioned at the interface between the transmembrane and the cytosolic domains of the channel allowing PIP2 to interact with both regions (2 (2 for Kir channel regulation seems to be the number of phosphates in the polar head as other PtdIns species (PtdIns(4)P and/or PtdIns) exhibited diminished activity or failed to stimulate Kir2 channels  and facilitating channel conductance  the cells with diverse pharmacological drugs that modify membrane PIP2 concentrations. Consistent with PIP2 facilitating Kir channel activity, depletion of this signaling lipid by dropping the intracellular ATP concentration to zero, blocking PIK, or adding neomycin—an aminoglycoside that impairs the electrostatic interaction between PIP2 and the Kir channel—to the patch pipette induced an evident steady decrease of the Kir current. This whole-cell Kir current rundown was further prevented by incorporating the water-soluble short-chain analog of PIP2  to the intracellular solution, a finding that poises membrane PIP2 as an intrinsic regulator of endothelial Kir2 channels by promoting channel stabilization in a preferred open (active) state. Surprisingly, Kir2 channels residing in SMCs were insensitive to these pharmacological manipulations over time, suggesting that membrane PIP2 is a minor modulator of this channel pool (vs. SMCs) in cerebral resistance arteries which are distinctly regulated by their neighboring lipid microenvironment.In the brain circulation, our recent study  revealednel pool . These f2 has been reported as a requisite molecule to maintain EC Kir2.1 channels in a preferred open state. This property makes it essential for the generation of retrograde electrical signals from capillaries which dilate upstream arterioles and contribute to the fundamental process by which CBF increases to satisfy the metabolic demands of active neurons   and Ca2+ signaling (TRPV4) —the major cause of stroke and dementia  arteries give rise to penetrating and parenchymal arterioles, and eventually to a capillary network containing hundreds of capillaries which vastly extend the arteriolar territory of perfusion. Capillaries, the narrowest blood vessels in the body, are tubes made up of a single layer of ECs which act as a neural activity-sensing network being thus much more than simple conduits for blood . At the peremia) . Since P (TRPV4) . In thisdementia .2, membrane cholesterol strongly suppresses the activity of Kir channels by placing the channel in a preferred closed (silent or inactivated) state  sharing a common regulatory hub that controls the Kir2.1 channel state (open vs. silent). Interestingly, this attribute is absent in Kir2.2 channels as they lack the aforementioned overlap in PIP2- and cholesterol-binding residues (Contrarily to PIPd) state . This nod) state . The mold) state . Paradoxresidues . While cresidues . These fresidues . In thisresidues . Upon faresidues   that contribute to peripheral vascular resistance. The vascular wall of these blood vessels displays structural and intrinsic  responses to ever-dynamic forces to eventually regulate vascular tone and match blood flow to tissue metabolic demands. This ability is accomplished by the plasma membranes of two of the principal constituents of the vascular wall—smooth muscle and endothelium—which are constantly exposed to mechanical stimuli, mainly originated by a pulsatile blood flow—stretch (tensile stress) and/or shear stress . In thisThe vascular smooth muscle layer of a resistance artery (or of downstream penetrating arterioles) possesses an inherent ability (independently of the endothelium and nerves) to contract—and reduce the luminal diameter—in response to an abrupt increase of transmural pressure  . In the 2+ (M) and initiating the myogenic response  shear stress is opposed by tension and deformation in the endothelium and initiates a complex signaling cascade—involving mechanosensory complexes containing multiple surface molecules including ion channels and integrins—that in short timescales causes vessel dilation to preserve the functionality of the vasculature . ECs are2+-activated K+ channels (SK) , and SK   have beeels (SK) . In the , and SK , 61, 108, and SK , 115 or , and SK . Notablysal tone .Mechanosensitive ion channels represent a firmly established set of biological molecules which possess an extraordinary capacity to sense and process external mechanical stimuli by initiating an electrical or chemical signal, eventually integrated into a coherent cellular response . Among t2+ influx and membrane depolarization. However, the precise identification of the ion channel species and scaffolding protein intermediates involved in this process remain undefined  with a critical role in the maintenance of cellular integrity , whereasndefined . In thisndefined .DMD gene encoding Dystrophin causes Duchenne muscular dystrophy, a rare but distinctive disease characterized by progressive muscle weakness and wasting primarily in males. The severe pathological effects of this rare genetic disease have stimulated a solid focus of research exploring the function of dystrophin and associated proteins  sub-membrane cytoskeleton protein that interacts with syntrophin and various protein elements to constitute the DAPC in vascular smooth muscle . Loss ofproteins , 133. Dyproteins . The C-tproteins , 135. Coproteins . Consequproteins . In the proteins .Syntrophins  are 58 kDa membrane-associated adaptor proteins involved in DAPC formation and the recruitment and regulation of surrounding signaling proteins—including ion channels—into a regulatory macromolecular assembly . The syn– currents are suppressed in Cav1-deficient cell lines but restored by transient expression of Cav1  . Caveola of Cav1 .2+ channels as they reside in caveolae and their activity is tightly regulated by the actin cytoskeleton . Disruption of caveolae-forming proteins similarly prevented suppression of smooth muscle Kir2 currents during cell-swelling. Moreover, immunofluorescence approaches revealed the expression of both scaffolding proteins, syntrophin and Cav1, in vascular smooth muscle and a proximity ligation assay highlighted the intimate structural association with Kir2.2 subunits (within 40 nm of one another), suggesting their participation in channel mechanosensing. These findings provided compelling evidence that Kir2 channel mechanosensitivity involves unique interactions with the cytoskeleton which are likely driven by scaffolding proteins.myogenic tone) of brain resistance arteries. Specifically, Kir2 currents recorded from native SMCs were potently suppressed during hypoosmotic challenge (swelling), a stimulus known to activate mechanically sensitive inward currents, leading to myogenic-like responses in the resistance vasculature (2+-induced constrictions (employed as an index of Kir activity) were observed when the arteries were exposed to low intravascular pressures, where the channel is most active. We further demonstrated that the lipid microenvironment surrounding Kir2 can impact the ability of the channel to respond to the pressure stimuli. In this framework, membrane cholesterol depletion prevented pressure-induced Kir2 inhibition in vitro and significantly augmented Ba2+-induced vasoconstriction in response to high intraluminal pressures. Together, these provocative data suggest that cholesterol-smooth muscle Kir interactions are crucial for hemodynamic sensing of pressure in the cerebral vasculature  by increasing the open probability of K+ channels—a response entirely blocked by micromolar concentrations of extracellular Ba2+ (a specific blocker of Kir2 channels) or Cs+ and/or completely abolished in mesenteric ECs freshly isolated from genetically modified mice lacking the Kir2.1 gene (heterozygous deletion of Kir2.1)   , 158. Moepletion . These ipulation . Paradoxpulation . These fon (–/–) , in whicon (–/–) . In lineon (–/–) , however’s group  and prel’s group  revealed’s group .2 and cholesterol depending on their cellular location (endothelium vs. smooth muscle); being these specific lipid-Kir2 channel interactions crucial to confer shear stress- and pressure sensitivity to the endothelial and smooth muscle Kir channel pools, respectively. These two cellular Kir2 populations have been proposed to actively interact to drive an integrated vascular Kir2 conductance designed to ultimately govern VM and basal tone in the brain circulation  and shear stress (flow). These vascular cell types express functional Kir2 channels, and each cellular population is distinctly modulated by key lipid signaling molecules which can sense diverse hemodynamic forces. Particularly, Kir2 channels are uniquely modulated by membrane PIPculation  22). Th. Th2 andculation . It is a2 to cholesterol) would suppress endothelial Kir2 activity, impair shear stress-induced activation, and ultimately impact brain blood flow. This future area of research is intended to analyze the possible role of endothelial dysfunction as an early predictor of atherosclerosis. Undoubtedly, the translation of these observations to humans will be the basis for the development of accurate therapeutic avenues to treat patients at risk of early cardiovascular disease (These mechanistic insights may provide important translational implications in the framework of dyslipidemia, a lipid disorder characterized by elevated plasma LDL-cholesterol levels. Given the endothelium anatomical location, ECs would be constantly exposed to higher levels of LDL-cholesterol, which in turn may impact lipid-Kir interactions and cause Kir2 channel stabilization to a preferred closed state. Therefore, under dyslipidemia a regulatory switch (PIP disease .MS and JF wrote, designed the figures, and edited the manuscript. DGW edited the manuscript. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


F9) gene leading to deficient or defective coagulation F9. Our study aimed at identifying the causative mutations within a sample of HB Egyptian patients. The present study comprised clinical data of eleven HB patients descending from six unrelated families and a seventh family including a carrier mother with a history of deceased HB sibling. Sequencing of F9 gene was performed.Hemophilia B (HB)  is a rare X-linked recessive disorder characterized by spontaneous or prolonged hemorrhages caused by mutations in Factor 9  and NM_000133.3:c.1305T>G, (p.Cys435Trp), and two nonsense mutations NM_000133.3:c.880C>T, (p.Arg294*) and NM_000133.3:c.1150C>T, (p.Arg384*), identified mutations spanned exons 6 and 8 of which a total of three mutations are located in hotspot exon 8 of F9 gene in HB Egyptian patients. Consistent genotype/phenotypic severity correlation could be concluded, helping proper genetic counseling and prenatal decision taking.Reviewing the literature, this is the first molecular analysis of  Hemophilia-B  is the second most common form of hemophilia after hemophilia-A accounting for 15-20% of hemophilia affection. HB incidence is estimated as one in 30,000 live male births worldwide with rare reports of affected females [F9, HB patients are clinically classified as severe (<1%), moderate (1-5%), and mild (5-40%). Severely affected males suffer from frequent spontaneous bleeding mainly in joints and muscles while mild or moderate cases experience excessive bleeding only upon trauma [Based on the plasma levels of n trauma .F9 gene. The F9 gene (OMIM 300746) located on the Xq27.1 is 34 kb in length encompassing 8 exons encoding 2.8 kb mRNA. Full length F9 gene contains 451 amino acid residues and includes a signal peptide (1–28 residues), a propertied (29–46) domain Gla enriched with glutamate (47–92), two EGF-like domain EGF1 (93–129) and EGF2 (130–171), a linker (172–191), an activation peptide (192–226), and SP domain of serine protease (227–451) [Hemophilia-B is caused by a wide variety of heterogeneous and infrequent mutations in OMIM 30076 locatedF9 gene mutations is responsible for the phenotypic severity and bleeding tendency. A wide spectrum of F9 gene mutations causing HB has been identified in all regions of the gene. The most common mutation type is point mutations which account for about 73%, followed by deletion mutations 16.3%, and, in minor proportion; insertions, duplications, small indels, and large rearrangements. More than 3940 unique mutations have been reported so far in HGMD [F9 gene can be detected by direct DNA sequencing; however, for large gross deletions, the multiple ligation—dependent probe amplification (MLPA) assay could be employed [Underlying This study aims at the identification of pathogenic mutations verified by gene screening and describing the clinical outcomes from variants identified in a sample of Egyptian HB patients referred to the Hereditary Blood Disorders clinic, (NRC) Egypt, and develop a first step in building knowledge about the molecular basis of this disease in Egyptian patients ultimately helping to provide proper genetic counseling.The Institutional Review Board NRC according to the World Medical Association Declaration of Helsinki approved the current research. Eleven HB male patients descending from six unrelated pedigrees and a seventh family including a carrier mother with a history of deceased HB patient were recruited from the Hereditary Blood Disorders clinic, NRC. Patients’ ages ranged from 1 to 20 years old and their ages at disease onset ranged from 22 days-3 years. They were all descending from non-consanguineous families. Patients were included based on the presence of a history of bleeding and laboratory findings including prolonged activated partial thromboplastin time (aPTT), normal thromboplastin time (PT), and deficient F9. Patients were subjected to detailed medical history recording including three-generation pedigree analyses, demographic data, initial complaint, age at onset, history of present illness, and disease progression, coupled with thorough clinical evaluation with emphasis on hemarthrosis. Patients were then phenotypically classified according to bleeding episodes and the level of F9 activity into one mild (>5%) and two moderate (1–5%) HB patients with history of severe bleeding following trauma, and eight severe (<1%) HB patients presenting with history of spontaneous joints and/or muscles bleeding episodes. Written informed consents were obtained from all participants and/or legal guardians. Peripheral blood samples (3 ml) were collected in EDTA tubes from all recruited patients and their available family members for genomic DNA extraction.Genomic DNA was extracted from 2 ml peripheral blood of affected HB patients and their available family members using “DNA Mini kit Quiagen” following the manufacturer’s instructions and quantification of the isolated DNA was performed by spectrometry in a “Nanodrop 2000.”F9 gene were amplified by polymerase chain reaction (PCR) of primers designed by ExonPrimer SOFTWARE. The coding regions and exon/intron boundaries of approximately 50 bp sequence were investigated to recognize any splice site variants as well. The PCR products were purified using Exo-SAP PCR purification kit  and both directions were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit and analyzed on the ABI Prism 3500 Genetic Analyzer (Applied Biosystems) according to manufacturer’s instructions.Eight exons of F9 gene was compared with reference genomic and cDNA sequence of the gene nucleotide numbering based on the Genebank accession numbers IX (F9), RefSeqGen LRG 556 on chromosome X, NM_000133.3 and NP_000124.1., with c.1 denoting the first position of the translation start codon obtained from National Center for Biotechnology Information (NCBI) database. Mutation nomenclature was given according to Genetic Variations approved by the Human Genome Variation Society (HGVS) (http://www.hgvs.org). Results were examined using Ensembl genome browser (https://www.ensembl.org/index.html).The sequence data of F9 gene databases .To ascertain whether a variant was novel or not, all of the mutations in this study were compared to the international F9 activity, eight HB cases were phenotypically classified as severe, two as moderate, and one mild. Family 7 presented with a history of previous death of HB sibling with severe phenotype.In this study, eleven HB male patients descending from six unrelated pedigrees were investigated. Patients’ ages ranged from 1 to 20 years old and their ages at disease onset ranged from 22 days-3 years. They were all descending from non-consanguineous families residing in different governorates in Egypt. According to F9 gene identified biallelic pathogenic mutations in all our HB patients  which is located in exon eight Reviewing the literature, this is the first molecular HB study in Egypt. The pathogenic variants for HB were successfully identified in all studied patients; mutations were homozygous in patients and heterozygous in carrier mothers as per the X-linked recessive inheritance mode of the disease.F9 gene in eleven clinically diagnosed Egyptian hemophilia-B patients and carrier mothers revealed four point mutations including two missense and two nonsense mutations that were correlating with phenotypic severity within the studied patients. Up to November 2018, the F9 gene mutation database (EAHD Coagulation Factor Variant Databases) has recorded a total of 1094 mutations in 3713 HB patients [F9 gene (exon 8).Mutational screening of patients ; point mpatients , 8–17. IOur patients presented with the classical phenotypic characteristics of the HB disease including severely affected males suffering from frequent spontaneous bleeding mainly in joints and muscles while boys with mild or moderate HB disease excessive bleeding only upon trauma.We identified one missense mutation  located in exon 6 in two HB patients (patients 1 and 2) who clinically presented with moderate phenotype. However, severe and moderate forms of the disease resulting from this mutation have been reported earlier in different populations like China, Turkey, and France –20; thisThe missense mutation c.676C>G where cytosine was substituted to guanine at nucleotide 676 results in the replacement of positively polar arginine with uncharged polar amino acid glycine at codon 226 in activation peptide domain causing an alteration in its structure and decreased protein function, since arginine residues are known to be involved in the formation of salt bridges and hydrogen bonds [Another identified missense mutation  was detected in one patient (patient 3) who presented with a mild phenotype. This mutation was previously reported in two Tunisian patients presenting with both severe and mild forms of the disease . AccordiInterestingly, there are seventy patients’ data available in the Factor IX Variant database reporting the detected nonsense mutation  in exon 8, leading mostly to moderate and severe forms of the disease in patients from different ethnic backgrounds. In the current study, the severe form of the disease was observed in three studied patients  and their affected families members where factor IX activity was <1% , 23–26.F9 gene which was previously reported in Colombian, Brazilian, and Argentinian populations with moderate and severe forms of the disease [The seventh family (7) was seeking pre-conception counseling for a history of a deceased HB child. DNA from the carrier mother was sequenced and the causative mutation was characterized as a nonsense mutation  in exon 8 of the  disease , 27, 28.The two nonsense mutations  were detected in eight patients descending from four unrelated HB families  who presented with the severe phenotype, were previously described in patients bearing moderate and severe forms of the disease , 23–26. F9 gene (exon 8), where CpG dinucleotides are considered to be mutation hot spots that are the highly susceptible site for mutation within the F9 gene [In addition, these two mutations  present in the serine protease domain; the greater part of which (codons 280–451) is coded by the largest exon in the  F9 gene –29.F9 gene.Currently, the multiplex ligation dependent probe amplification (MLPA) method is used to detect large deletion or duplication mutations in F9 gene, however, it cannot detect point mutations that are considered as the major type of causative mutations in HB , and ourF9 gene in our cohort of eleven Egyptian HB patients descending from 6 unrelated pedigrees and one carrier female from a seventh family. A total of four different mutations were identified, which further confirms the high degree of heterogeneity in the mutations within F9 gene. Mutation detection allows proper genetic counseling, carrier detection of female relatives, and prenatal genetic services.We could successfully define the mutational spectrum of Further studies with larger patients’ numbers are highly recommended to build an Egyptian database and confirm the genotype–phenotype correlation within our ethnic background. In absence of definitive curative treatment of hemophilia B disease, which results in most instances in patients’ mortality and/or disability, this database could help in trials for modern therapeutic possibilities where the molecular background has to be defined such as the evolving gene-editing technology.


In contrast, food allergy sensitive Il4raF709 mice housed under standard temperature conditions translocated the luminal antigens in the SI across the epithelium via secretory antigen passages (SAPs). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg allergens at standard temperature predisposed Il4raF709 mice to develop an anaphylactic reaction. Housing Il4raF709 mice at Tn altered systemic type 2 cytokine, IL-4, and the landscape of SI antigen passage patterning (villus and crypt involvement). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg antigen under Tn conditions led to the robust induction of egg-specific IgE and development of food-induced mast cell activation and hypovolemic shock. Similarly, Tn housing of WT BALB/c mice altered the cellular patterning of SI antigen passage (GAPs to SAPs). Activation of SI antigen passages and the oral challenge of WT BALB/c mice with egg antigen led to systemic reactivity to egg and mast cell activation. Together these data demonstrate that Tn housing alters antigen passage cellular patterning and landscape, and concurrent oral exposure of egg antigens and SAP activation is sufficient to induce oral antigen sensitization.Food allergy is an emerging epidemic, and the underlying mechanisms are not well defined partly due to the lack of robust adjuvant free experimental models of dietary antigen sensitization. As housing mice at thermoneutrality (Tn) - the temperature of metabolic homeostasis (26–30°C) – has been shown to improve modeling various human diseases involved in inflammation, we tested the impact of Tn housing on an experimental model of food sensitization. Here we demonstrate that WT BALB/c mice housed under standard temperature  conditions translocated the luminal antigens in the small intestine (SI) across the epithelium  The forRecent investigations demonstrate that housing temperature robustly influences mice physiology . VivariuOne of the limitations of studying human immunological disease processes in mice is that mice do not necessarily respond to immunological challenges similarly to humans. For example, mice are resistant to developing human disease conditions such as obesity , atherosF709IL4ra mice at Ts conditions compared to WT BALB/c mice. Activation of antigen passage formation predisposed F709IL4ra mice to oral food sensitization at Ts conditions. Tn housing of F709IL4ra mice altered the antigen passage landscape and increased penetrance of food sensitization and reactivity. Tn housing of WT BALB/c mice altered the SI antigen passage cellular patterning (GAP to SAP) and landscape promoting passage formation from the villus to the crypt region. Activation of antigen passages and oral exposure of Tn conditioned housed F709IL4ra and WT BALB/c mice to egg antigen significantly exacerbated allergen-specific IgE and IgG1 responses and led to hypovolemic shock that coincided with the activation of mucosal mast cells following systemic and oral allergen challenge.Herein, we show that antigen passage cellular patterning was altered in Il4raF709 mice  were maintained and bred under standard housing conditions . Mice were transferred to the thermoneutral housing conditions  after weaning and acclimated for at least two weeks prior to experimentation. We have previously demonstrated that two weeks is sufficient to decrease serum stress hormones in WT mice (2) within the facility was set at 30°C (86°F), with a 12-hour light/dark cycle. The room is equipped with a laminar flow workstation and a Rodent Cage system to maintain the mice in the room at all times. Mouse cages are maintained in a Rodent Cage system that delivers HEPA-filtered ventilated air and is equipped with an automated water supply to individual cages providing air and water at ambient temperature. The room temperature is monitored daily to ensure the temperature is maintained between 27.7-31.1 °C (82-88 °F). Six to 10-week old mice were used for all the experiments described in the study. All animals were maintained and used as approved by the Animal Care and Use Committee at CCHMC and the University of Michigan.BALB/c WT and  WT mice . The Tn Food allergen, egg white powder from Jay Robb Enterprises  was stored at -20°C until the time of use. Reagents used are as follows: Imaging antigen, lysine fixable dextran tetramethylrhodamine in 10,000 MW (Invitrogen), CCh, and paraformaldehyde (Sigma-Aldrich). Antibodies used are as follows: wheat germ agglutinin (WGA) conjugated to Alexa 488 (Invitrogen), rat anti-mouse IgE (BD Bioscience), anti rat IgG conjugated to biotin (Vector lab), avidin conjugated to HRP (BD Bioscience), or anti-mouse IgG1 (Abcam) conjugated to HRP.F709Il4ra mice were orally exposed to egg 23 mg in 400 μl water twice a week for two weeks. The mice were fasted for 5 hours and treated subcutaneously with either saline or CCh  15 minutes before oral food allergen exposure. The serum was harvested on day 14. On day 16, mice were challenged intravenously with 100 μg of egg allergen, and body temperature and hemoconcentration were measured as previously described  in 400 μl water every 2 weeks after 4 weeks of oral allergen exposure, and serum was harvested following each oral allergen challenge. WT BALB/c mice were exposed to egg 23 mg in 400 μl water twice a week for four weeks. Each oral allergen exposure was performed as described earlier. The serum was harvested on day 28. On day 30, mice were challenged intravenously with 100 μg of egg allergen, and blood was harvested, hemoconcentration were measured as previously described (escribed . For oraescribed .Mononuclear cells in the lamina propria (LP) from the small intestine (SI) were isolated as previously described . The isoThe intestinal epithelium isolated from the mononuclear cell preparation of the lamina propria were used to extract RNA as previously described . 1 μg ofin vitro cytokine capture assay (IVCCA) for IL-4 was performed as previously described  as described by the manufacture. Allergen specific IgE and IgG1 were captured with allergens coating of the plate, and the levels were detected with rat anti-mouse IgE along with biotinylated anti rat IgG or biotinylated IgG1, detection antibodies in the presence of TMB substrate solution (B.D. Bioscience). The escribed . Serum cHarvested tissues were fixed and processed as previously described . ImmunofStools were collected from mice housed at the standard temperature. The mice were transferred to the thermoneutral temperature to collect stools after the acclimation period. DNA was isolated from the stools, as previously described . DNA libStudent t-test or one-way ANOVA was performed to determine statistical significance using GraphPad Prism 8 unless otherwise noted. P<0.05 is considered statistical significance unless otherwise noted.Il4raF709 and not WT BALB/C mice induce food sensitization due to the effects of heightened IL-4rα signaling on the immune compartment . In contrast, we observed WGA+ and WGA- antigen passages in the saline-treated Il4raF709 mice, indicating the presence of SAPs  or CCh treatment to induce antigen passage formation (Il4raF709 mice lead to a shock response (decrease in core body temperature) in 55% of the mice compared to 0% of the saline treated Il4raF709 mice , ILC2 (Lin- CD4- CD8- c-kit- IL17RB+), or the regulatory T (Tregs) (Lin- CD3+ CD4+ GITR+ OX40+) cells were not significantly different between Ts- and Tn-housed Il4raF709 mice (Il4raF709 mice (data not shown and Il25 mRNA levels were similar between Ts and Tn housed Il4raF709 mice suggesting that housing temperature has very little impact on pro type 2 immune phenotype at a steady state (data not shown). Taken together, the data suggest that Tn housing did not alter the gastrointestinal immune compartment in Il4raF709 mice.We have previously reported that Tn housing decreases serum stress hormones in WT C57BL/6 mice . To conf709 mice , 32, we Il4raF709 mice housed at Ts and Tn conditions. Analysis of 16 S rRNA gene content high-throughput sequencing of V4 amplicons from fecal samples revealed no significant separation between the microbiome of Ts and Tn housed Il4raF709 mice  and OTU0008 (Bacteroides) were similarly represented between Ts and Tn housed Il4raF709 mice supporting the principal component analysis  in the villus of the SI epithelium  at The animal study was reviewed and approved by University of Michigan and Cincinnati Children’s Medical Center.TN, J-BL, VG, AY, and ST performed experiments. TN and SH analyzed the data, and wrote the manuscript. CB and GH processed stool samples and analyzed the microbiome. SD, RN, and GH assisted in study design and discussion. SH supervised and acquired funding. All authors contributed to the article and approved the submitted version.Funding was provided by National Institutes of Health grants; AI138177 (to SH), and AI112626R01(to SH), AI138348 (to GH); DK099222 (to SD); the Mary H. Weiser Food Allergy Center (MHWFAC) , and the Nina and Jerry D. Luptak Endowment of the MHWFAC (to GH) and the Askwith Endowment of the MHWFAC (to SH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pathway analysis of genes associated with breast-specific veQTL revealed an enrichment of four genes  involved in the C21-steroidal biosynthesis pathway that converts cholesterol to breast-related hormones (e.g. oestrogen). Each of these four genes were significantly more variable in individuals homozygous for rs11075995 (A/A) breast cancer risk allele located in the FTO gene, which encodes an RNA demethylase. The A/A allele was also found associated with reduced expression of FTO, suggesting an epi-transcriptomic mechanism may underlie the dysregulation of genes involved in hormonal biosynthesis leading to an increased risk of breast cancer. These findings provide evidence that genetic variants govern high levels of expression variance in breast tissue, thus building a more comprehensive insight into the underlying biology of breast cancer risk loci.Genome wide association studies (GWAS) have identified more than 180 variants associated with breast cancer risk, however the underlying functional mechanisms and biological pathways which confer disease susceptibility remain largely unknown. As gene expression traits are under genetic regulation we hypothesise that differences in gene expression variability may identify causal breast cancer susceptibility genes. We performed variable expression quantitative trait loci (veQTL) analysis using tissue-specific expression data from the Genotype-Tissue Expression (GTEx) Common Fund Project. veQTL analysis identified 70 associations ( Risk variants are often located in non-coding regions making it difficult to determine pathogenic pathways. Approximately 700 potential gene targets of breast cancer risk variants have been identified using analytical methods that employ genomic data from chromatin interactions, enhancer–promoter correlations, transcription binding, topologically associated domains and gene expression3.Genome wide association studies (GWAS) in breast cancer have identified more than 180 common risk variants4. For example, identification of expression quantitative trait loci (eQTL) has been a key approach for investigating tissue-specific effects of breast cancer risk variants under the hypothesis that non-breast tissue may be involved in breast cancer risk5. Gene expression patterns are often explored assuming genetic control of mean expression level, however the variability of gene expression is also genetically controlled9. Just as differences in expression means have been associated with genotype so too differences in expression variability can be associated with genotype.Gene expression traits are under genetic regulation and the heritability of differences in genotypes have been extensively described10, yeast7 and complex multicellular organisms13. Furthermore, gene expression variability had been shown to be important in early human development14, schizophrenia15 and cancer subtypes13. The effects of genetic variation on gene expression variability has been recently described in human derived lymphoblastoid cell lines from HapMap individuals8 and in the TwinsUK cohort17.Gene expression variability has been described in a wide range of organisms including prokaryotes19. However, the mechanisms underlying breast cancer risk for the majority of variants remains to be uncovered. Here, we demonstrate variable expression quantitative trait loci (veQTL) as a method for testing the association of variants with gene expression variability. We performed veQTL analysis on 181 variants that have been previously associated with breast cancer risk and identified 60 new candidate genes and pathways associated with 27 breast cancer risk variants.Breast cancer risk variants associated with eQTL, based on mean gene expression, have been investigated in both breast tissue , and non-breast tissueGenotype and expression data were acquired through the database of Genotypes and Phenotypes (dbGaP) and the Genotype-Tissue Expression (GTEx) Common Fund Project (release version phs000424.v7.p2.) under the project title “Identification of variable expression quantitative trait loci that are associated with cancer risk”. Datasets from breast, ovarian, lung and kidney tissue used in this study were obtained through the dbGaP approval number 17463.Genotype data from 635 individuals acquired through GTEx were converted to chromosome-specific matrices, where the genotypes were numbered by the minor allele count. For tissue specific analysis, only genotypes from individuals with tissue expression data in a given tissue  were retained. Genotypes were filtered so that only bi-allelic genotypes of at least 10 subjects with two or more genotypes  were retained.https://www.ensembl.org/) gene ids were split into tissue-specific datasets. For each dataset, only transcripts with RPKM > 0.1 in at least 10 samples were retained. Subjects with multiple tissue-specific samples were collapsed by calculating the average RPKM values. Linear regression models were used to correct expression data for age and sex as covariates.Normalised Reads Per Kilobase of transcript, per Million mapped reads (RPKM) counts for 56,203 unique Ensembl  was used to calculate Brown–Forsythe test-statistics , Ni is the number of samples in group i, Zi is the mean of the absolute deviation from the medians for group i and Z.. is the mean of the absolute deviations from all samples from their respective group medians. The resulting W statistics follows the F-distribution with degrees of freedom df1 = k – 1 and df2 = N – k20.Tissue specific veQTL were mapped for breast cancer risk variants that passed the filtering criteria  gene expression variability associated with a breast cancer risk variants, (ii) the significant veQTL association was only observed in breast tissue and (iii) the gene was only associated with a change in expression variability (i.e. veQTL) and not change in mean expression (i.e. eQTL).We limited proposed breast cancer susceptibility genes to those that had: (i) significant , lung (n = 387), kidney (n = 41) and ovary (n = 123) were used. A major proportion of breast cancer risk variants are predicted to alter expression of cancer susceptibility gene(s) in breast tissue. To identify veQTL that specifically increase risk in breast tissue, even if the genes in the veQTL are ubiquitously expressed in multiple tissues, we only considered veQTL that were uniquely identified in breast tissue (i.e. breast-specific veQTL). These assumptions, would however miss breast cancer susceptibility genes whose expression variability is tolerated in other tissue but not breast.RNA-sequencing and genotype data were split into tissue-specific datasets and filtered to remove low frequency genotypes and genes with low expression. After pre-processing 33,059, 29,522, 25,026 and 35,137 transcripts were retained for the breast, ovary, kidney and lung, respectively.Large genome-wide association studies (GWAS) have identified variants associated with breast cancer risk or subtype specific breast risk. In total we identified 181 breast cancer risk variants in the literature (Supplementary Table p < 5 × 10–8) veQTL interactions with breast cancer risk variants in the breast (70), ovary (9) and lung (109)  in the breast, lung and ovary, respectively. A greater proportion of eQTL were observed in cis compared to veQTL, with approximately 5% of veQTL and 13% eQTL acting in cis  were generated and genomic factors estimated for each tissue Fig. a. No subp values distributions were similar between veQTL and eQTL analyses  all of which were associated with the risk variant rs11075995. By comparison, the 88 veQTL that were significant in lung tissue were not significantly enriched for any pathway using pathway analysis (data not shown).The majority of breast cancer variants have no known associations with other traits, however 25 variants have previously been associated with a phenotype other than breast cancer risk  of rs11075995, which is associated with ER negative breast cancer risk, was found to be associated with increased variability in expression of four genes by veQTL analysis  associated with rs11075995 are all involved in the conversion of cholesterol to hormones via the C21 steroidal biosynthesis pathway 26. Variants in this locus are associated with increased body mass index (BMI), the mechanism of action has been linked to expression changes of the neighbouring gene IRX3 in the human brain and in particular the hypothalamus28. Furthermore, there is conflicting evidence of rs11075995 association with breast cancer risk. Recent studies identified a loss of breast cancer risk association after adjusting for BMI29. However, Garcia-Closas and colleagues tested the association with ER negative breast cancer risk after adjusting for BMI and observed no change30. We therefore explored the effects of the rs11075995 on the expression of both FTO and IRX3 in breast tissue. Neither FTO nor IRX3 had significant breast eQTL or veQTL associations with rs11075995. However, FTO (p = 0.05), and not IRX3 (p = 0.29), had decreased expression in the homozygous minor allele individuals in breast tissue  veQTL unique to breast tissue were considered. This approach identified 60 candidate genes that were associated with 27 variants. The majority of significant veQTL were class I and displayed a homozygous recessive like phenotype  all of which were variable in association with the risk allele of rs11075995. Furthermore, rs11075995 produced the strongest signal for variable expression for all four candidate genes and was the most likely casual variant  and are secreted into the circulating system to act on distant tissues (e.g. breast). The activation of local hormone biosynthesis, associated with the risk allele of rs11075995, through the metabolism of cholesterol to pregnenlone may lead to greater exposure and/or hormone imbalance in breast tissues, which may drive tumourigenesis. Local steroidogenesis and ultimately production of androgens has been observed in androgen independent advance prostate cancers32. In prostate cancer, the local production of androgens may explain the development of hormonal treatment resistance in late-stage prostate cancers.Breast cancer development has been associated with exposure to steroid hormoneswww.gwascentral.org) identified significant associations of rs11075995 with overall and ER negative breast cancer risk and with body mass index  expression in breast tissue associated with individuals homozygous for the rs11075995 risk allele. FTO is involved in demethylation of RNA adenosine (m6A). Methylated adenosine are post-transcriptional modifications which signals RNAs for processing, including degradation and splicing34. The four genes associated with rs11075995 all have the m6A target site (GGACU). RNA variability may occur due to dysregulation of these pathways (mRNA degradation and splicing) in response to decreased FTO expression.The variant rs11075995 is located in intron 2 of the FTO have been strongly associated with obesity and changes in BMI35, however these variants act on the expression of the neighbouring gene IRX3 in the hypothalamus region of the brain28. Iroquois homeobox protein 3 (IRX3) is a highly conserved transcription factor typically expressed during neural development36. The role of IRX3 in obesity is yet to be fully elucidated with conflicting reports of body mass associated to deficient Irx3. Smemo et al., described a 30% increase in body weight of Irx3-deficient mice28. While in contrast the partial depletion of Irx3 through a lentiviral system resulted in mice with greater body mass37.Variants in intron 1 and 2 of IRX3 and FTO are highly expressed in the hypothalamus, a region of the brain important to hormonal regulation37. It is unknown whether risk variants, for either BMI or breast cancer, directly disrupt the regulation of hormonal control in the hypothalamus. Furthermore, it is unclear what effect IRX3 expression would have on breast cancer risk and whether any effect would be independent of the risk attributed to obesity alone. A better understanding of the downstream transcriptional targets of IRX3 may identify pro-tumourgeneic pathways.Intriguingly, both FTO locus may be associated with tissue-specific hormonal control and subsequently different pathologies. Consequently, we would expect differences in the regulation of C21 hormones in breast tissue for the different rs11075995 genotypes. Furthermore, candidate genes identified through veQTL analysis require functional validation. A major challenge with assessment of intra-sample gene expression variability is the limitation of single-point ‘grind and bind’ approaches. However, approaches such as RNA hybridisation in situ and single cell RNA-sequencing do provide the ability to detect expression variability. It is of further importance to derive the mechanism of variability which may be driven by interaction of genotypes with exposures or epistasis.Our results are based on data from largely white, European derived ancestry (GTEx is 85% white), hence extrapolation to more diverse ancestry is a limitation of this study. However, these results are consistent with the hypothesis that different variants in the In summary, breast cancer risk variants are associated with variable expression of candidate breast cancer susceptibility genes. These included genes involved in hormonal biosynthetic pathways that are associated with a single variant (rs11075995). To our knowledge, this is the first time gene expression variability has been used to identify candidate cancer susceptibility genes.Supplementary Information


Prisoners are recognised as a high-risk population and prisons as high-risk locations for the transmission of hepatitis c virus (HCV) infection. Injecting drug use (IDU) is the main driver of HCV infection in prisoners and harm reduction services are often suboptimal in prison settings. HCV prevalence and incident data in prisoners is incomplete which impacts the public health opportunity that incarceration provides in identifying, treating and preventing HCV infection. The aim of this study is to identify new HCV infection and associated risk factors in an Irish male prison.We conducted a follow up (18-month) cohort study on prisoners who had previously tested negative, self-cleared or had been successfully treated for HCV infection. We conducted the study in a male medium security prison located in Dublin Ireland (Mountjoy Prison) using HCV serology, a review of medical records and a researcher-administered questionnaire.n = 77), had self-cleared (n = 9) or achieved sustained virological response (n = 12). Small numbers of prisoners continued to engage in risk-behaviour including, IDU both in the prison (n = 2) and the community (n = 3), sharing syringes (n = 1) and drug taking paraphernalia (n = 6) and receiving non-sterile tattoos (n = 3).99 prisoners with a mean age of 33.2 yrs. participated in the study and 82(82.8%) completed a research-administered questionnaire. Over half (51%) had a history of drug use from a young age (14.8 yrs.), 49.9% a history of heroin use and 39% a history of IDU. The prevalence of HIV and hepatitis B virus core antibody was 3% and HCV antibody was 22.2%. No new HCV infections were identified in those who had never been infected (Despite the high numbers of Irish prisoners with a history of IDU and HCV infection, new HCV infection is low or non-existent in this population. Small numbers of prisoners continue to engage in risk behaviour and larger studies are required to further understand HCV transmission in this cohort in an Irish and international context. Hepatitis C (HCV) infection is a major public health concern and a leading cause of liver-related morbidity and mortality worldwide . InjectiEpidemiological data on HCV infection in prisoners is lacking in many countries . From avIreland has one of the lowest incarceration rates in Europe with approximately 4000 people incarcerated across 14 locations in the Irish Prison Service (IPS) daily .Studies on Irish prisoners report high rates of opioid use (50%), IDU (43%) and HCV infection (13–37%) –8. RecenThe study site is one of three locations where in-reach hepatology services, through specialist nurses, are provided in the IPS. HCV direct-acting antivirals (DAAs) have been available in Ireland since 2014, with initial availability restricted on clinical need. DAAs (including 8-week and pan-genotypic regimens) can now be prescribed to all HCV-infected people needing treatment including prisoners.This study is a follow up study from a previously published 2017 cross sectional study that estimated the prevalence of untreated chronic HCV infection and associated risk factors in a male Irish prisoners .n = 422) reported a HIV and hepatitis B virus (HBV) core antibody prevalence of 4.0 and 3.0% respectively and a HCV antibody prevalence of 22.8% among the general prison population, increasing to 79.7% among prisoners with history of IDU [The 2017 study  . Peer-suSimilar to other jurisdiction, Ireland struggles to increase rates of HCV screening and treatment in prisoners , 15, 16.Enablers identified include; access to health care, opt-out screening at committal, peer support, stability of prison life and in-reach hepatology services and fibroscanning .The aims of this study are to estimate the HCV incident infection among Irish male prisoners and to describe levels of risk behaviour among prisoner who are aware of not having HCV infection or having a documented sustained virological response (SVR) post-treatment. This study will add to the existing literature on the epidemiology of HCV infection in prisoners including incident infection and risks. Findings will also evaluate the effectiveness of harm reduction services in Irish prisons and may inform future development of these services internationally.This study reports on the final part of a larger European HCV seek and treatment study (HepCare Europe project) and was a follow up to a previously reported prevalence study completed in 2017 , 19. EthThe original study included 422 participants of which 403 had HCV serology completed. This study found that 311 prisoners were HCV antibody negative and 92 (22.8%) were HCV antibody positive. A review of medical records found of those who were HCV antibody positive 23 (25%) had spontaneously cleared, 12 (13%) had a sustained virological response (SVR) and 53 (57%) had chronic infection.n = 77), had achieved spontaneous clearance (n = 9) or had an SVR post-treatment (n = 13) were incarcerated at the time of this updated study. All 99 prisoners agreed to participate in this follow up cohort study and 82 agreed to complete a research-administered questionnaire which answers are presented in Table A review of prison and medical records found that 99 of the original study participants who were either; HCV antibody negative  [Data on study variables were collected from two sources; prisoners’ electronic medical records and a researcher-administered questionnaire. All prisoners routinely complete a nurse committal interview on the day of incarceration which is recorded in the prisoners’ medical records in the Prisoner Healthcare Management System (PHMS). From the medical records review, we collected the following variables: age, country of origin, incarceration history, pre-incarceration accommodation, drug and alcohol use and treatment, previous HCV-related risk behaviour  history of tattooing and the sharing of toothbrushes and razors while incarcerated).The risk questionnaire was developed and piloted by the research team in conjunction with national experts in the area of HCV infection and prisoner groups. The questionnaire covered the 18 month time period since the previous study and included questions on: incarceration history, history of current HCV-related risk behaviour both in the community and in prison  and engagement with harm reduction services (MAT and NSPs).Questionnaires were completed prior to screening, but priority was given to completing screening over questionnaires, particularly in areas of the prison that housed enhanced-security prisoners. All study participants were given a patient information sheet and asked to sign a consent form. No inducements were offered.me HCV assay. HCV genotyping was conducted on samples with detectable HCV RNA using the Abbott RealTime HCV Genotype II assay.All participants were offered repeat serological screening for HIV, HBV, HCV antibody and reflex HCV RNA testing. First-line serological screening for hepatitis B virus (HBV), HCV and human immunodeficiency virus (HIV) was carried out using chemiluminescent microparticle immunoassays on the Architect i4000sr automated platform . Confirmatory testing of reactive samples was carried out using alternative assays. HIV serological diagnosis was based on the Architect HIV Ag/Ab Combo  and confirmatory testing for reactive samples using the VIDAS HIV DUO Ultra  and HIV INNO-LIA HIV I/II Score  assays. The Abbott Architect assays were used for HBV-associated markers and to characterise the HBV infection status. Serological screening for HCV included the anti-HCV test (Abbott Architect), a third-generation immunoassay and the Abbott Architect HCV Ag assay. All anti-HCV reactive samples negative for HCV antigen were further investigated to confirm the presence of anti-HCV antibodies using the anti-HCV VIDAS (BioMérieux) and INNO-LIA HCV Score (Innogenetics) assays. When HCV was confirmed serologically, molecular detection of HCV RNA was performed using the Abbott RealTiAll data were anonymised and coded, double-entered and checked. Statistical analysis was performed using the Statistical Package for Social Sciences . Data were assessed for normality and where necessary, data were log-transformed for normalisation purposes. Data in tables are primarily expressed as means with standard deviation (SD) or numbers with percentages.A total of 99 prisoners with a mean age of 32.2 years (yrs.) consented to participate in the study. 94% reported Ireland as their country of origin and 21% were homeless prior to incarceration. This cohort were first incarcerated in their late teens (mean age = 18.4 yrs.), had experienced multiple incarcerations (mean = 6.4) and had spent the majority of their young adult lives in prison (mean = 9.9 yrs.). Just over half (51%) of participants had a history of drug use, 43.9% a history of heroin use and 39% a history of IDU. The mean age of first drug use was 14.8 yrs., first heroin use was 18.8 yrs., and first IDU was 20.6 yrs.In terms of historical risk factors for HCV acquisition, 42.7% gave a history of sharing drug taking equipment , 17.3% of sharing syringes in the community, 22.5% of having had a prison tattoo and 23.8% a non-sterile community tattoo. Small numbers reported sharing a razor or toothbrush in a prison setting (2.5 and 1.3% respectively). A total of 42.1% reported having a history of methadone treatment, and the mean length of time on treatment was 6.3 years (Table n = 99)) of the original study cohort were available to follow up  had been released and re-incarcerated. Small numbers of patients engaged in recent IDU while incarcerated (n = 2) and on release into the community (n = 3). Only one patient reported sharing syringes in the community post incarceration. Sharing drug taking paraphernalia both in the community (n = 2) and in the prison (n = 4) was a little more common than needle sharing. 20% (n = 16) of participants were on MMT at the time of the study , had self-cleared (n = 9) or achieved SVR (n = 12). There was one treatment failure (previously identified) , but there is considerable uncertainty around this estimate among those who continue to inject . A numben = 13) of this study cohort had experienced prison release and re-incarceration between studies. Small numbers reported risk-behaviour which is encouraging. This finding may reflect the age of the cohort (mean age = 32.5 yrs.) and their existing knowledge of their HCV status. However the small numbers did not allow for comparison between those who remained incarcerated and those who had experienced release and re-incarceration. It also did not allow for comparisons between those never infected and those who had self-cleared or achieved SVR (previously infected). As already described most prisoners serve short sentences and many experience multiple cycles of release and re-incarceration. Community release can be particularly problematic and is associated with elevated risk of death and overdose [A small number  who were available for follow up and who completed the questionnaire (n = 82). The small numbers prevented any meaningful statistical comparison between those who remained incarcerated and those who had experienced released and re-incarcertaion and between those never infected and previously infected. A strength of the study is the use of serum samples rather than historical blood-borne virus screening data from chart records. The use of current serology is unusual in these types of studies and with high levels of both sensitivity and specificity for this method offers increased validity to this study over those using saliva samples. A further strength is that all eligible participants agreed to follow up serology.Incident studies in prison populations are rarely conducted. They are often difficult to design and implement. In this context the findings from this study are important since they add to what is a very small pool of data on incident HCV infection in prisoners. The findings are limited by the small numbers  known to be not infected or successfully treated for HCV. Small numbers engaged in HCV-related risk behaviour while incarcerated and on release. A larger multi-site study is required to fully understand the rates and associated risks of HCV infection in Irish prisoners, but this preliminary study suggests that existing measures including MAT availability are protective in preventing HCV infection in this group.This study reported no new HCV infections over an 18 month period in a cohort of prisoners (


Increasing evidence has demonstrated that lncRNAs are critical regulators in diverse biological processes, but the function of lncRNA in metabolic regulation remains largely unexplored. In this study, we evaluated the association between lncRNA and metabolic pathways and identified metabolism-related lncRNAs. Gastric cancer can be mainly subdivided into 2 clusters based on these metabolism-related lncRNA regulators. Comparative analysis shows that these subtypes are found to be highly consistent with previously identified subtypes based on other omics data. Functional enrichment analysis shows that they are enriched in distinct biological processes. Mutation analysis shows that ABCA13 is a protective factor in subtype C1 but a risk factor in C2. Analysis of chemotherapeutic and immunotherapeutic sensitivity shows that these subtypes tend to display distinct sensitivity to the same chemical drugs. In conclusion, these findings demonstrated the significance of lncRNA in metabolic regulation. These metabolism-related lncRNA regulators can improve our understanding of the underlying mechanism of lncRNAs and advance the research of immunotherapies in the clinical management of gastric cancer. Long non-coding RNAs (lncRNA), defined as transcripts over 200 nucleotides, do not have protein-coding potential3. They participate in the regulation of gene expression at different levels4. In recent years, a growing number of researches have focused on the identification of the function of lncRNA. LncRNA is found to be significantly associated with diverse biological processes, such as cell differentiation, proliferation, and apoptosis7. Moreover, human immunity is reported to be regulated by lncRNA8. But most of the lncRNAs remain largely unexplored and the function of these lncRNAs is still unknown. Therefore, it’s of great significance to identify the function of lncRNA.The human genome contains about 20,000 protein-coding genes, whereas most of the genome is transcribed into non-coding RNA9. LncRNA MT1JP is reported to function as a ceRNA in regulating FBXW7 through competitively binding to miR-92a-3p in gastric cancer10. Additionally, lncRNA is found to be significantly associated with immune pathways of primary human cancer11. But most of these analyses concentrated on a single lncRNA, functional characterization of lncRNA is still inadequate. It’s necessary to investigate the lncRNA that has not previously been studied.Growing researches have demonstrated the association between lncRNA and human cancer. LncRNA MEG3 is found to inhibit proliferation and metastasis of gastric cancer via p53 signaling pathway12. Cancer metabolism research revealed that metabolic profiles of individual tumors are highly heterogenous13 and metabolic expression subtypes of human cancers are significantly associated with patient survival14. But all of these studies concentrated on protein-coding genes. The regulation of lncRNA on metabolic pathways is unknown. Thus, it’s of great interest to study the underlying interactions between lncRNA and metabolic pathways. Deeply understanding the function of these metabolism-related lncRNAs can provide insights into the molecular mechanism of lncRNA.Currently, an increasing number of studies demonstrated the significance of metabolism in human cancer. Initiation and progression of colorectal cancer are found to be regulated by metabolic pathwaysIn this work, we aim to identify the interactions between metabolism and lncRNA and analyze the clinical relevance of these metabolism-related lncRNAs. After evaluating the association between lncRNA and metabolic pathways, we identified 1539 metabolism-related lncRNAs. Gastric cancer can be mainly classified into 2 subtypes based on these lncRNAs. Functional enrichment analysis shows that they are enriched in distinct biological processes. Genomic characterization shows that ABCA13 mutation is a protective factor in C1 but a risk factor in C2. Analysis of chemotherapeutic and immunotherapeutic sensitivity shows that different subtypes tend to display distinct sensitivity to the same chemical drugs, suggesting the clinical significance of these metabolism-related lncRNAs.11. Among these upregulated lncRNAs, 1138 (72.5%) lncRNAs are associated with at least one metabolic pathway and identified as metabolism-related lncRNAs 20 yielded 6 clusters of gastric cancer (N1–N6). Comparative analysis between these subtypes shows that the molecular subtypes derived from these two approaches achieve high consistency. Cluster N2 is highly enriched in cluster C2  enrichment analysis shows that these differentially expressed genes are enriched in various metabolic processes including hormone metabolic process, retinoid metabolic process, and diterpenoid metabolic process Fig. d. In add25 was used to analyze the functional difference between different subtypes. We collected 50 hallmark gene sets representing well-defined biological states or processes to check whether they are enriched in different pathways. We found that interferon-gamma response, interferon-alpha response, and inflammatory response are enriched in subtype C1. Pancreas beta cells and bile acid metabolism are enriched in subtype C2. The top 5 enriched pathways are presented in Fig. In addition, an independent method called gene set enrichment analysis (GSEA)22. To check whether there is a difference in mutational frequency between gastric cancer subtypes, We calculated TMB for each sample. Comparative analysis shows that there is no obvious difference in TMB between subtypes C1 and C2  is found to affect the immunotherapeutic sensitivity of cancer C2 Fig. a. In add C2 Fig. b.Figure 26. These specific genomic events are more or less likely to be co-selected during the initiation and progression of cancer. To study the mutational dependency of different subtypes, we used the SELECT algorithm to identify mutual exclusivity and co-occurrence events of genomic alterations27. Genomic analysis of subtype C1 shows that KMT2D mutation is co-occurred with ARID1A, PCLO, OBSCN, AFHX4, PLEC, RYR1, SACS, SYNE2, and RNF213 mutation  to analyze the chemotherapeutic sensitivity of gastric cancer. We constructed a prediction model based on the Genomics of Drug Sensitivity in Cancer (GDSC) dataset and predicted drug sensitivity IC50) for each sample of these four chemical drugs. We found that subtype C1 was more sensitive to gemcitabine than C2 . For the transcriptome data, we downloaded raw count data and Fragments Per Kilobase Million (FPKM) data. FPKM is then transformed to Transcripts Per Kilobase Million (TPM) for the representation of expression. LncRNA and mRNA are annotated using GENCODE tool. The information of previously identified molecular subtypes of gastric cancer is downloaded using R package TCGAbiolinks34. A total of 38 metabolic pathways are collected in this work. For analysis of GSEA between different subtypes, a total of 50 hallmark gene sets representing well-defined biological processes and states are also downloaded from MSigDB. For the analysis of immunotherapeutic sensitivity of gastric cancer, advanced melanoma treated with immune checkpoint blockade (GSE91061)35, metastatic urothelial cancer treated with an anti-PD-L1 agent (imvigor210)36, and metastatic melanoma treated with MAGE-A3 antigen (GSE35640)37 are collected in our analysis.For the identification of metabolism-related lncRNA, we collected metabolic pathways from The Molecular Signatures Database (MSigDB)P value < 0.05 and absolute fold change > 2. Differential expression analysis of lncRNA is conducted using R package edgeR38. The association between these differentially expressed lncRNAs and metabolic pathways is then evaluated using a previously proposed tool ImmLnc11. It calculates an association score for each pair of lncRNA and metabolic pathway. After setting a threshold for the association, the pairs surpassing the threshold are identified as significantly associated.To identify metabolism-related lncRNA, metabolic pathways are collected from MSigDB. LncRNA and mRNA expression profiles are prepared to analyze the association between lncRNA and metabolic pathways. Metabolism-related lncRNAs are identified based on a two-step framework. We first identified differentially expressed lncRNAs between gastric cancer and the adjacent normal samples based on pvalue and absolute fold change. A lncRNA is identified as differentially expressed if BH-adjusted Metabolism-related lncRNAs are used to identify molecular subtypes of gastric cancer. We choose the top 100 variable lncRNAs based on standard deviation as features  for each sample is evaluated using this prediction model for a specific chemical drug. We calculated the sensitivity of gastric cancer for a total of 138 chemical drugs. These analyses are conducted using R package pRRopheticFor the analysis of immunotherapeutic sensitivity of gastric cancer, submap is used to compare gene expression profiles between different subtypes. We compared our lncRNA subtypes with melanoma patients who received immunotherapies to check whether a specific subtype is significantly associated with the responder group.Supplementary Figure S1.Supplementary Figure S2.Supplementary Tables.