Luminal Space Research Articles

Finite Element Analysis of Glucose Diffusivity in Cellulose Nanofibril Peripheral Nerve Conduits.

Peripheral neuropathy arising from physical trauma is estimated to afflict 20 million people in the United States alone. In one common surgical intervention, neural conduits are placed over the nerve stumps to bridge the gap and create a microenvironment conducive to regeneration. It has been proposed that a biocompatible material such as cellulose nanofiber may serve as a viable conduit material, providing a non-inflammatory and mechanically stable system. Preliminary studies have shown that cellulose nanofiber conduits successfully aid neural regeneration and further, that the dimensions of the conduit relative to the nerve gap have an impact on efficacy in murine models. It has been hypothesized that the reliance of regeneration upon the physical dimensions of the conduit may be related to modified modes of diffusion and/or distances of key cellular nutrients and waste metabolites to/from the injury site. The present work investigates the concentration profile of glucose within the conduit via finite element analysis as a function of the physical dimensions of the conduit. It was determined that the magnitude of glucose diffusion was greater through the conduit walls than through the luminal space between the nerve and the inner wall of the conduit, and that as such radial diffusion is dominant over axial diffusion.

Clinical Assessment and Lesion-Specific Management of Orbital Vascular Malformations.

The systematic classification of vascular disease as proposed and refined by the International Society for the Study of Vascular Anomalies (ISSVA) divides vascular pathology first into tumors and malformations. Malformations are described as simple and complex, where simple malformations contain a single vascular system and complex malformations comprised of multiple vascular systems. Arteriovenous malformations are considered in terms of inflow characteristics which are primarily responsible for the key management challenges. Management utilizing endovascular embolization and/or surgical resection is often employed; however, recurrence can occur, particularly in diffuse cases. There may be an increasing role for systemic antiangiogenic therapy in such cases. Lymphaticovenous malformations are divided into the principle components on the lymphatic and venous sides for clarity of discussion. Lymphatic malformations are described morphologically as macrocystic and microcystic, and physiologically in terms of the processes responsible for growth. In both cases, surgical options are challenging and local therapeutics intended to close large luminal spaces in the case of macrocystic and to slow biological signaling for growth in microcystic. Venous malformations are described physiologically in terms of flow and distensibility, as volume plays a critical role in the limited space of the orbital cavity. Combined embolic-surgical approaches can be effective for management. More complicated, combined lesions can be managed by dividing the lesion into principal components and treating each appropriately.

On dark matter and dark energy

The dark matter hypothesis was created to explain the reason for the preservation of stellar clusters from dispersion. The weak point of this hypothesis is the great age of space, which is 13.8 billion years. Based on experimental data, it is shown that the age of space does not exceed 10 thousand years. In this case, the hypothesis of dark matter is not needed, since stellar clusters cannot scatter in such short cosmic time. The dark energy hypothesis was created to explain the reason for the accelerated expansion of space. The basis for this phenomenon is a large amount of spectral redshift of distant luminous space objects. It is shown that this value is mainly determined by the significant absorption of light energy of distant space objects by a huge amount of intergalactic gas, and not by the movement of these objects. In this case, the hypothesis of dark energy is not needed, and space should not rapidly expand and scatter in space.

Abstract PO-234: Relationship between increased concentrations of circulating chemokines and population differences in tumor biology

Abstract Genetic ancestry is a significant determinant of multiple aspects of tumor biology, immunity, and inflammatory responses. In lung cancer, in addition to increased incidence in African American (AA) men compared with other racial and ethnic groups in the US, differences in tumor biology between AAs and European Americans (EAs) have been identified. Natural selection is a strong driver of gene selection leading to population differences that can be linked to geographic and genetic ancestry. A classic example involves the Duffy antigen receptor for chemokines, also known as DARC. Individuals with African ancestry are more likely to lack DARC expression on their red blood cells (also known as Duffy null), as this confers resistance to malaria. Loss of erythrocyte expression of DARC is associated with increased circulating chemokine levels in the CXCR and CCR families as DARC serves as a dummy chemokine reservoir. Additionally, Duffy null individuals have lower circulating neutrophils, known as benign ethnic neutropenia. However, loss of DARC expression is exclusive to erythrocytes in Duffy null individuals. As venous endothelial cells and tumor cells also express DARC, we questioned whether the increased circulating bioavailability of DARC ligands in AAs led to increased DARC- dependent signaling in the tumor microenvironment (TME) of AA lung cancer patients. DARC expression on tumor cells is associated with increased aggressiveness and invasion, and endothelial cells have been shown to transcytose DARC ligands into the luminal space. As DARC ligands are associated with angiogenesis and granulocyte recruitment, we hypothesized that tumors of AAs would present with increased blood vessel invasion and granulocyte recruitment to the TME. Using the Somalogic multiplex proteomic assay, we observed significantly elevated levels of the ELR+ CXCR chemokines, with the exception of IL-8 (CXCL8), in serum samples from AA healthy control and stage 1 lung adenocarcinoma patients compared with EAs (samples from the NCI-UMD Case-Control Study), We did not find any evidence of increased angiogenesis as measured by CD31 immunohistochemical staining in AA patients (n= 39) compared with EA patients (n= 107). Immune cell composition did not differ between AAs and EAs as measured by CIBERSORT analysis in both healthy controls and patients between AAs and EAs. Therefore, while AAs have significantly higher levels of circulating DARC ligands compared with EAs, they do not appear to contribute to increased DARC-dependent signaling in normal and tumor lung tissue microenvironment in AAs. Citation Format: Sheryse Taylor, Julian Candia, Adriana Zingone, John Tsang, Brid Ryan. Relationship between increased concentrations of circulating chemokines and population differences in tumor biology [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-234.

Intestinal Epithelium-Derived Luminally Released Extracellular Vesicles in Sepsis Exhibit the Ability to Suppress TNF-a and IL-17A Expression in Mucosal Inflammation.

Sepsis is a systemic inflammatory disorder induced by a dysregulated immune response to infection resulting in dysfunction of multiple critical organs, including the intestines. Previous studies have reported contrasting results regarding the abilities of exosomes circulating in the blood of sepsis mice and patients to either promote or suppress inflammation. Little is known about how the gut epithelial cell-derived exosomes released in the intestinal luminal space during sepsis affect mucosal inflammation. To study this question, we isolated extracellular vesicles (EVs) from intestinal lavage of septic mice. The EVs expressed typical exosomal (CD63 and CD9) and epithelial (EpCAM) markers, which were further increased by sepsis. Moreover, septic-EV injection into inflamed gut induced a significant reduction in the messaging of pro-inflammatory cytokines TNF-α and IL-17A. MicroRNA (miRNA) profiling and reverse transcription and quantitative polymerase chain reaction (RT-qPCR) revealed a sepsis-induced exosomal increase in multiple miRNAs, which putatively target TNF-α and IL-17A. These results imply that intestinal epithelial cell (IEC)-derived luminal EVs carry miRNAs that mitigate pro-inflammatory responses. Taken together, our study proposes a novel mechanism by which IEC EVs released during sepsis transfer regulatory miRNAs to cells, possibly contributing to the amelioration of gut inflammation.

Transurethral Anastomosis after Transurethral Radical Prostatectomy: A Phantom Study on Intraluminal Suturing With Concentric Tube Robots.

Current surgical approaches to radical prostatectomy are associated with high rates of erectile dysfunction and incontinence. These complications occur secondary to the disruption of surrounding healthy tissue, which is required to expose the prostate. The urethra offers the least invasive access to the prostate, and feasibility has been demonstrated of enucleating the prostate with an endoscope using Holmium laser, which can itself be aimed by concentric tube robots. However, the transurethral approach to radical prostatectomy has thus far been limited by the lack of a suitable means to perform an anastomosis of the urethra to the bladder after prostate removal. Only a few intraluminal anastomotic devices currently exist, and none are small enough to pass through the urethra. In this paper we describe a new way to perform an anastomosis in the small luminal space of the urethra, harnessing the dexterity and customizability of concentric tube manipulators. We demonstrate a successful initial proof-of-concept anastomosis in an anthropomorphic phantom of the urethra and bladder.

BAT optimization based Retinal artery vein classification

The investigation of artery vein changes over time is considered to be the significant diagnosis process of retinal diseases like diabetic retinopathy. The diagnosis includes the characteristics analysis of artery vein vessels, changes in its tortuosity level and artery vein ratio; hence, it is important to classify the artery and vein in a better way. Computer-aided diagnosis requires the automated classification of retinal artery and vein for diagnosing the progression of diseases. In this paper, a supervised classification with Bat algorithm is proposed to discriminate the artery and vein vessels in the retinal fundus images. A novel feature vector space, including both additive colour space as well as luminous chromaticity model colour space, is constructed. BAT algorithm is applied to select the feature group which improve the classification accuracy and also to reduce the dimensionality of feature space. The proposed method is developed and analyzed using the publicly available databases DRIVE, IOSTAR and STARE.

Formation and function of bacterial organelles.

Advances in imaging technologies have revealed that many bacteria possess organelles with a proteomically defined lumen and a macromolecular boundary. Some are bound by a lipid bilayer (such as thylakoids, magnetosomes and anammoxosomes), whereas others are defined bya lipid monolayer (such as lipid bodies), a proteinaceous coat (such as carboxysomes) or have a phase-defined boundary (such as nucleolus-like compartments). These diverse organelles have various metabolic and physiological functions, facilitating adaptation to different environments and driving the evolution of cellular complexity. This Review highlights that, despite the diversity of reported organelles, some unifying concepts underlie their formation, structure and function. Bacteria have fundamental mechanisms of organelle formation, through which conserved processes can form distinct organelles in different species depending on the proteins recruited to the luminal space and the boundary of the organelle. These complex subcellular compartments provide evolutionary advantages as well as enabling metabolic specialization, biogeochemical processes and biotechnological advances. Growing evidence suggests that the presence of organelles is the rule, rather than the exception, in bacterial cells.

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion.

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host-bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.

Three-Dimensional Explant Platform for Studies on Choroid Plexus Epithelium.

The choroid plexus (CP) plays a major role in controlling the entry of substances and immune cells into the brain as it forms the blood-cerebrospinal fluid barrier (BCSFB) in the brain ventricles. Dysregulated immune cell trafficking through the epithelial cell (EC) layer of CP is central for the pathogenesis of infectious diseases in the brain and many neurodegenerative disorders. In vitro studies elucidating the function of the CP have so far been limited to the monolayer culture of CP ECs. To mimic immune cell migration across the CP barrier, a three-dimensional model would be advantageous. Here, we present an in vitro platform for studies of the immune cell trafficking based on CP explants/organoids. The explants were generated from fragments of mouse CPs in Matrigel, where the cells formed luminal spaces and could be maintained in culture for at least 8 weeks. We demonstrate expression of the major CP markers in the explants, including transthyretin and aquaporin 1 as well as ZO1 and ICAM-1, indicating a capacity for secretion of cerebrospinal fluid (CSF) and presence of tight junctions. CP explants displayed CP-like cell polarization and formed an intact EC barrier. We also show that the expression of transthyretin, transferrin, occludin and other genes associated with various functions of CP was maintained in the explants at similar levels as in native CP. By using dendritic cells and neutrophils, we show that the migration activity of immune cells and their interactions with CP epithelium can be monitored by microscopy. Thereby, the three-dimensional CP explant model can be used to study the cellular and molecular mechanisms mediating immune cell migration through CP epithelium and other functions of choroid EC. We propose this platform can potentially be used in the search for therapeutic targets and intervention strategies to improve control of (drug) substances and (immune) cell entry into the central nervous system.

A review of the manufacturing process and infection rate of 3D-printed models and guides sterilized by hydrogen peroxide plasma and utilized intra-operatively

3D printing in the context of medical application can allow for visualization of patient-specific anatomy to facilitate surgical planning and execution. Intra-operative usage of models and guides allows for real time feedback but ensuring sterility is essential to prevent infection. The additive manufacturing process restricts options for sterilisation owing to temperature sensitivity of thermoplastics utilised for fabrication. Here, we review one of the largest single cohorts of 3D models and guides constructed from Acrylonitrile butadiene styrene (ABS) and utilized intra-operatively, following terminal sterilization with hydrogen peroxide plasma. We describe our work flow from initial software rendering to printing, sterilization, and on-table application with the objective of demonstrating that our process is safe and can be implemented elsewhere. Overall, 7% (8/114 patients) of patients developed a surgical site infection, which was not elevated in comparison to related studies utilizing traditional surgical methods. Prolonged operation time with an associated increase in surgical complexity was identified to be a risk factor for infection. Low temperature plasma-based sterilization depends upon sufficient permeation and contact with surfaces which are a particular challenge when our 3D-printouts contain diffusion-restricted luminal spaces as well as hollows. Application of printouts as guides for power tools may further expose these regions to sterile bodily tissues and result in generation of debris. With each printout being a bespoke medical device, it is important that the multidisciplinary team involved in production and application understand potential pitfalls to ensuring sterility as to minimize infection risk.

Pluripotent stem cell-derived cholangiocytes and cholangiocyte organoids.

The development of protocols for pluripotent stem cell (PSC) differentiation into cholangiocytes and cholangiocyte organoids in three-dimensional structures represent a huge advance in both research and medical fields because of the limited access to primary human cholangiocytes and the potential bias induced by animal models used to study cholangiopathies in vivo. PSC-derived cholangiocyte organoids consisting of either cysts with luminal space or branching tubular structures are composed of cells with apico-basal polarity that can fulfill cholangiocyte functions like the transport of bile salts. Several protocols of PSC differentiation have already been published but we added to the detailed protocol we describe here some notes or advice to facilitate its handling by new users. We also propose detailed protocols to carry out some of the characterization analyses using immunofluorescence to study the expression of specific markers and a functionality test to visualize bile acid transport using cholyl-lysyl-fluorescein (CLF).

Adjustment in Luminance and Color Spaces to Generate Visible Contrasts in People with Abnormal Trichromatic Color Blindness

The perception of color allows visibility in individuals but is something relative to the subject and the light of the surrounding environment. Such perception is given by two types of cells in the eyes, cones and gallons. Deficiency in the capture of light in some of these cells causes colors to be perceived in different ways, from the absence of a color to the confusion between shades. In this article we present a method of adjustment in luminance and color spaces to generate visible contrasts in people with abnormal trichromatic color blindness.

Drug Screen Identifies Leflunomide for Treatment of Inflammatory Bowel Disease Caused by TTC7A Deficiency

Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient anddevelop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease. We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport. TTC7A-KO HAP1cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function. In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.

Histological differences in cancer cells, stroma, and luminal spaces strongly correlate with in vivo MRI-detectability of prostate cancer

The current study aimed to investigate the plausible histopathological factors that affect the detectability of prostate cancers on multiparametric magnetic resonance imaging (MP-MRI). This retrospective study included 59 consecutive patients who had undergone MP-MRI and subsequent radical prostatectomy. The cases were standardized according to the tumor size ranging from 10 to 20 mm on the final pathological diagnosis. Histopathological review and semi-automated imaging analysis were performed to evaluate the relative area fractions of the histological components, including cancer cells, stroma, and luminal spaces. Among the 59 prostatectomy specimens, no case showed two or more foci of cancer that matched the size criteria. Of the 59 lesions, 35 were MRI-detectable [Prostate Imaging Reporting and Data System (PIRADS) score of 3 or greater] and 24 were MRI-undetectable (PIRADS score of 2 or less). No significant differences were observed in Gleason Grade Group, percentage of Gleason pattern 4, and predominant subtype of Gleason pattern 4 between MRI-detectable and MRI-undetectable cancers. On the other hand, significantly higher mean area fraction of cancer cells (60.9% vs. 42.7%, P < 0.0001) and lower mean area fractions of stroma (33.8% vs. 45.1%, P = 0.00089) and luminal spaces (5.2% vs. 12.2%, P < 0.0001) were observed in MRI-detectable cancers than in MRI-undetectable cancers. In a multivariable analysis performed upon exclusion of area fraction of stroma due to its multicollinearity with that of cancer cells, area fractions of cancer cells (P = 0.0031) and luminal space (P = 0.0035) demonstrated strong positive and negative correlation with MRI-detectability, respectively. Changes in cancer cells, stroma, and luminal spaces, rather than conventional histological parameters, could be considered one of the best predictors to clinical, in vivo MRI-detectability of prostate cancer.

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Pathophysiologic Findings in the Human Endolymphatic Sac in Endolymphatic Hydrops: Functional and Molecular Evidence.

The endolymphatic sac (ES) is a cystic structure situated on the posterior fossa dura and is connected to the luminal space of the vestibular organ through the endolymphatic duct, which branches into the utricular and saccular ducts. Unlike the cochlea and vestibule, the ES does not contain sensory epithelium in its luminal space, and a single layer of epithelial cells line the luminal surface area. The ES in the inner ear is thought to play a role in the regulation of inner ear homeostasis, fluid volume, and immune reaction. If these functions of the ES are disrupted, dysfunction of the inner ear may develop. The most well-known pathology arising from dysfunction of the ES is endolymphatic hydrops, characterized by an enlarged endolymphatic space due to the accumulation of excessive endolymphatic fluid. Although, molecular identities and functional evidence for the roles were identified in animal studies, basic studies of the human ES are relatively uncommon compared with those using animal tissues, because of limited opportunity to harvest the human ES. In this study, molecular and functional evidence for the role of the human ES in the development of endolymphatic hydrops are reviewed. Although evidence is insufficient, studies using the human ES have mostly produced findings similar to those of animal studies. This review may provide a basis for planning further studies to investigate the pathophysiology of disorders with the finding of endolymphatic hydrops.

NME4 regulates TLR3 activation and type I IFN production through autophagy-dependent TRAF6 degradation

Abstract Nucleoside diphosphate kinase (NDPK)/Nm23 family proteins play multiple roles in energy metabolism, proliferation, differentiation, inflammatory responses and tumour invasion. Among these proteins, only NDPK-D (NME4) is located in the mitochondrial luminal space and the inner mitochondrial membrane. NME4 shows high affinity for cardiolipin (CL) and facilitates its redistribution to the outer mitochondrial membrane (OMM), which leads to mitophagy and inflammatory regulation. Here, we demonstrate that NME4 can regulate the type I interferon pathway by modulating the TLR3-TRAF6-IFN axis. NME4 deficiency promotes the degradation of TRAF6 and impairs the phosphorylation of p65 and p38. Overexpression of NME4 significantly upregulates the interferon production induced by a TLR3 ligand. However, when the NEM4 signal peptide for mitochondrial localization is deleted, the regulation of IFN production is abolished. We demonstrate that NME4 deficiency-mediated suppression of interferon production is rescued when ATG5 is knocked down following TLR3 stimulation, suggesting that NME4 is able to regulate interferon production through the autophagy pathway. Furthermore, influenza A virus-infected NME4 knockout mice showed higher mortality, which was associated with reduced interferon production compared to that in wild-type mice. Taken together, our results indicate that NME4 plays an important role in regulating TLR3-mediated interferon production.

ATP release into ADPKD cysts via pannexin-1/P2X7 channels decreases ENaC activity

Genetic predisposition is necessary for polycystic kidney disease (PKD) initiation, although there are other, incompletely identified downstream processes that are required for cyst growth. Their characterization may provide a unique opportunity for clinical interventions. One of the poorly studied phenomena in PKD is high ATP content in cysts. Unfortunately, neither origins of uncontrolled ATP release, nor consequences of abnormal purinergic signaling in relation to epithelial transport are well explored in the polycystic kidney. We tested the distribution of pannexin-1 (Panx1) and P2X7, two proteins potentially involved in ATP release, in the kidneys of the Pkd1RC/RC mice, a model of autosomal dominant PKD (ADPKD). Abundances of both proteins were abnormally increased in the cyst lining cells compared to non-dilated collecting ducts. To establish if pannexin-1 contributes to ATP release in the collecting ducts (CD), we measured luminal accumulation of ATP in M1 cell renal CD monolayers, and found that treatment with probenecid, a Panx1 blocker, prevents ATP release. Single channel patch clamp analysis of polarized M1 cells revealed that apical stimulation of P2X receptors with αβ-MeATP acutely reduces ENaC activity. We conclude that in ADPKD progression, an abnormal hyperexpression of both PANX1 and P2RX7 occurs in the cyst lining epithelial cells. High abundance of both proteins is not typical for non-dilated CDs but, when it happens in cysts, pannexin1/P2X7 cooperation elevates ATP release into the luminal space. High ATP level is a pathogenic factor facilitating cystogenesis by reducing ENaC-mediated reabsorption from the lumen.

PH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor—referred to as pH-Lemon—optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.