Endosomal disentanglement of a transducible artificial transcription factor targeting endothelin receptor A.

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

Clearance of neurotoxic peptides and proteins by meningothelial cells.

Meningothelial cells (MECs) are the cellular component of the meninges that provide physical protection to the central nervous system (CNS). Their main function is the formation of a barrier enclosing the brain including the cerebrospinal fluid (CSF). Further, MECs are involved in maintaining CSF homeostasis by clearing CSF from bacteria and apoptotic cells. Furthermore, secretion of pro- and anti-inflammatory cytokines and chemokines involves MECs in immunological processes in the CNS. We demonstrated that meningothelial Ben-Men-1 cells ingest neurotoxic peptides amyloid-ß (Aß1-40) and protein α-synuclein up to about 10-fold more efficiently compared to neuronal-like SH-SY5Y cells. Aß1-40 and α-synuclein are mainly taken up via macropinocytosis. Caveolar endocytosis in addition contributes to α-synuclein ingestion. Upon uptake, both are trafficked towards lysosomal degradation. While production of reactive oxygen species (ROS) following exposure to Aß25-35 and α-synuclein was similar between Ben-Men-1 and SH-SY5Y cells, mitochondrial function in Ben-Men-1 was significantly more robust to Aß25-35 treatment compared to neuronal-like SHSY5Y cells. Similarly, Ben-Men-1 were significantly less susceptible to Aß25-35-induced cell death than neuronal-like cells. Furthermore, co-culture with Ben-Men-1 offered significant protection to neuronal-like cells against Aß25-35-induced apoptosis. This study reveals for the first time the function of MECs as scavengers of neurotoxic Aß and α-synuclein, thereby connecting these cells to neuroprotective processes and suggesting a new mechanism and pathway for clearing neurotoxic substances from the CSF.

The extracellular matrix composition of the optic nerve subarachnoid space.

The meninges not only surround the brain and the spinal cord but also the optic nerve. Meningeal-derived extracellular matrix (ECM) is a crucial component of the pial basement membrane, glia limitans and important for maintenance of optic nerve axon integrity, homeostasis and retinal ganglion cell health. To get closer insight into optic nerve meningeal-derived ECM composition, we performed proteomic analysis of the sheep optic nerve subarachnoid space (SAS). Candidate components were confirmed in cultures of primary human meningothelial cells (phMECs) and human optic nerve samples. Sheep optic nerve SAS samples were analysed by LC-MS, identified proteins were matched to their human orthologs and filtered using gene lists representing all major ECM components. To validate these findings digital droplet PCR (ddPCR) to evaluate mRNA expression of all candidate components identified was performed on cultures of phMECs. In addition, one protein per major ECM group was stained on human optic nerve sections and on phMEC cultures. Employing LC-MS, 1273 proteins were identified and subjected to bioinformatic analysis. Gene ontology analysis revealed six out of forty-four collagen types (1A1, 1A2, 3A1, 6A2, 6A3 and 14A1), three out of eleven laminin subunits (A4, B2, C1) and six out of twenty-seven hyaluronan binding proteins (CD44, versican (VCAN), C1q binding protein, neurocan (NCAN), brevican (BCAN) and hyalaluronan proteoglycan link protein 2 (HAPLN2)) were present in our cohort. DdPCR in phMEC cell culture confirmed presence of all candidate components except NCAN, BCAN and HAPLN2. Immunohistochemistry (IHC) staining on human optic nerve sections and immunofluorescence (IF) staining on in vitro cultured phMECs showed strong immunopositivity for collagen-typeI-α1 (COL1A1), lamininγ1 (LAMC1), and VCAN. Fibronectin (FN1) was exclusively present in cultures of phMECs. Using a combined bioinformatics and immunohistological approach, we describe the ECM composition of the optic nerve subarachnoid space. As this space plays an important role in maintaining optic nerve function, a better understanding of ECM composition in this delicate environment might be key to further pathophysiological insight into optic nerve degeneration and associated disorders.

Impact of aging on meningeal gene expression.

BACKGROUND: The three-layered meninges cover and protect the central nervous system and form the interface between cerebrospinal fluid and the brain. They are host to a lymphatic system essential for maintaining fluid dynamics inside the cerebrospinal fluid-filled subarachnoid space and across the brain parenchyma via their connection to glymphatic structures. Meningeal fibroblasts lining and traversing the subarachnoid space have direct impact on the composition of the cerebrospinal fluid through endocytotic uptake as well as extensive protein secretion. In addition, the meninges are an active site for immunological processes and act as gatekeeper for immune cells entering the brain. During aging in mice, lymphatic drainage from the brain is less efficient contributing to neurodegenerative processes. Aging also affects the immunological status of the meninges, with increasing numbers of T cells, changing B cell make-up, and altered macrophage complement. METHODS: We employed RNASeq to measure gene expression and to identify differentially expressed genes in meninges isolated from young and aged mice. Using Ingenuity pathway, GO term, and MeSH analyses, we identified regulatory pathways and cellular functions in meninges affected by aging. RESULTS: Aging had profound impact on meningeal gene expression. Pathways related to innate as well as adaptive immunity were affected. We found evidence for increasing numbers of T and B lymphocytes and altered activity profiles for macrophages and other myeloid cells. Furthermore, expression of pro-inflammatory cytokine and chemokine genes increased with aging. Similarly, the complement system seemed to be more active in meninges of aged mice. Altered expression of solute carrier genes pointed to age-dependent changes in cerebrospinal fluid composition. In addition, gene expression for secreted proteins showed age-dependent changes, in particular, genes related to extracellular matrix composition and organization were affected. CONCLUSIONS: Aging has profound effects on meningeal gene expression; thereby affecting the multifaceted functions meninges perform to maintain the homeostasis of the central nervous system. Thus, age-dependent neurodegenerative processes and cognitive decline are potentially in part driven by altered meningeal function.

Large-scale morphometry of the subarachnoid space of the optic nerve.

BACKGROUND: The meninges, formed by dura, arachnoid and pia mater, cover the central nervous system and provide important barrier functions. Located between arachnoid and pia mater, the cerebrospinal fluid (CSF)-filled subarachnoid space (SAS) features a variety of trabeculae, septae and pillars. Like the arachnoid and the pia mater, these structures are covered with leptomeningeal or meningothelial cells (MECs) that form a barrier between CSF and the parenchyma of the optic nerve (ON). MECs contribute to the CSF proteome through extensive protein secretion. In vitro, they were shown to phagocytose potentially toxic proteins, such as α-synuclein and amyloid beta, as well as apoptotic cell bodies. They therefore may contribute to CSF homeostasis in the SAS as a functional exchange surface. Determining the total area of the SAS covered by these cells that are in direct contact with CSF is thus important for estimating their potential contribution to CSF homeostasis. METHODS: Using synchrotron radiation-based micro-computed tomography (SRµCT), two 0.75 mm-thick sections of a human optic nerve were acquired at a resolution of 0.325 µm/pixel, producing images of multiple terabytes capturing the geometrical details of the CSF space. Special-purpose supercomputing techniques were employed to obtain a pixel-accurate morphometric description of the trabeculae and estimate internal volume and surface area of the ON SAS. RESULTS: In the bulbar segment, the ON SAS microstructure is shown to amplify the MECs surface area up to 4.85-fold compared to an "empty" ON SAS, while just occupying 35% of the volume. In the intraorbital segment, the microstructure occupies 35% of the volume and amplifies the ON SAS area 3.24-fold. CONCLUSIONS: We provided for the first time an estimation of the interface area between CSF and MECs. This area is of importance for estimating a potential contribution of MECs on CSF homeostasis.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

VCP/p97 cofactor UBXN1/SAKS1 regulates mitophagy by modulating MFN2 removal from mitochondria.

Initiation of PINK1- and PRKN-dependent mitophagy is a highly regulated process involving the activity of the AAA-ATPase VCP/p97, a cofactor-guided multifunctional protein central to handling ubiquitinated client proteins. Removal of ubiquitinated substrates such as the mitofusin MFN2 from the outer mitochondrial membrane by VCP is critical for PRKN accumulation on mitochondria, which drives mitophagy. Here we characterize the role of the UBA and UBX-domain containing VCP cofactor UBXN1/SAKS1 during mitophagy. Following mitochondrial depolarization and depending on PRKN, UBXN1 translocated alongside VCP to mitochondria. Prior to mitophagy, loss of UBXN1 led to mitochondrial fragmentation, diminished ATP production, and impaired ER-mitochondrial apposition. When mitophagy was induced in cells lacking UBXN1, mitochondrial translocation of VCP and PRKN was impaired, diminishing mitophagic flux. In addition, UBXN1 physically interacted with PRKN in a UBX-domain depending manner. Interestingly, ectopic expression of the pro-mitophagic VCP cofactor UBXN6/UBXD1 fully reversed impaired PRKN recruitment in UBXN1-/- cells. Mechanistically, UBXN1 acted downstream of PINK1 by facilitating MFN2 removal from mitochondria. In UBXN1-/- cells exposed to mitochondrial stress, MFN2 formed para-mitochondrial blobs likely representing blocked intermediates of the MFN2 removal process partly reversible by expression of UBXN6. Presence of these MFN2 blobs strongly correlated with impaired PRKN translocation to depolarized mitochondria. Our observations connect the VCP cofactor UBXN1 to the initiation and maintenance phase of PRKN-dependent mitophagy, and indicate that, upon mitochondrial stress induction, MFN2 removal from mitochondria occurs through a specialized process.

Epigenetic therapy for breast cancer.

Both genetic and epigenetic alterations can control the progression of cancer. Genetic alterations are impossible to reverse, while epigenetic alterations are reversible. This advantage suggests that epigenetic modifications should be preferred in therapy applications. DNA methyltransferases and histone deacetylases have become the primary targets for studies in epigenetic therapy. Some DNA methylation inhibitors and histone deacetylation inhibitors are approved by the US Food and Drug Administration as anti-cancer drugs. Therefore, the uses of epigenetic targets are believed to have great potential as a lasting favorable approach in treating breast cancer.

Correlation of telomere length shortening with promoter methylation profile of p16/Rb and p53/p21 pathways in breast cancer.

Unregulated cell growth, a major hallmark of cancer, is coupled with telomere shortening. Measurement of telomere length could provide important information on cell replication and proliferation state in cancer tissues. Telomere shortening and its potential correlation with downregulation of cell-cycle regulatory elements were studied by the examination of relative telomere length and methylation status of the TP53, P21 and P16 promoters in tissues from breast cancer patients. Telomere length was measured in 104 samples (52 tumors and paired adjacent normal breast tissues) by quantitative PCR. Methylation profile of selected genes was analyzed in all samples using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results demonstrated a significant shortening of tumor telomere regions compared with paired adjacent normal tissues (P<0.001). Similarly, telomere lengths were significantly shorter in advanced stage cases and in those with higher histological grades (P<0.05). Telomere shortening in cancer tissues was correlated with a different level of hypermethylation in the TP53, P21 and P16 promoters (r=-0.33, P=0.001; r=-0.70, P<0.0001 and r=-0.71, P<0.0001, respectively). The results suggested that inactivation of p16/Rb and/or p53/p21 pathways by hypermethylation may be linked to critical telomere shortening, leading to genome instability and ultimately to malignant transformation. Thus, telomere shortening and promoter hypermethylation of related genes both might serve as breast cancer biomarkers.

Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors.

BACKGROUND: With the aim to simplify cancer management, cancer research lately dedicated itself more and more to discover and develop non-invasive biomarkers. In this connection, circulating cell-free DNA (ccf DNA) seems to be a promising candidate. Altered levels of ccf nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have been found in several cancer types and might have a diagnostic value. METHODS: Using multiplex real-time PCR we investigated the levels of ccf nDNA and mtDNA in plasma samples from patients with malignant and benign breast tumors, and from healthy controls. To evaluate the applicability of plasma ccf nDNA and mtDNA as a biomarker for distinguishing between the three study-groups we performed ROC (Receiver Operating Characteristic) curve analysis. We also compared the levels of both species in the cancer group with clinicopathological parameters. RESULTS: While the levels of ccf nDNA in the cancer group were significantly higher in comparison with the benign tumor group (P < 0.001) and the healthy control group (P < 0.001), the level of ccf mtDNA was found to be significantly lower in the two tumor-groups (benign: P < 0.001; malignant: P = 0.022). The level of ccf nDNA was also associated with tumor-size (<2 cm vs. >2 cm<5 cm; 2250 vs. 6658; Mann-Whitney-U-Test: P = 0.034). Using ROC curve analysis, we were able to distinguish between the breast cancer cases and the healthy controls using ccf nDNA as marker (cut-off: 1866 GE/ml; sensitivity: 81%; specificity: 69%; P < 0.001) and between the tumor group and the healthy controls using ccf mtDNA as marker (cut-off: 463282 GE/ml; sensitivity: 53%; specificity: 87%; P < 0.001). CONCLUSION: Our data suggests that nuclear and mitochondrial ccf DNA have potential as biomarkers in breast tumor management. However, ccf nDNA shows greater promise regarding sensitivity and specificity.

Decreased mitochondrial DNA content in blood samples of patients with stage I breast cancer.

BACKGROUND: Alterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters. METHODS: Peripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein. RESULTS: The content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed. CONCLUSION: Early detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer.

Current understanding of mitochondrial DNA in breast cancer.

The recent surge in mitochondrial research has been driven by the identification of mitochondria-associated diseases and the role of mitochondria in apoptosis and aging. Mitochondrial DNA (mtDNA) has been proposed to be involved in carcinogenesis because of its high susceptibility to mutations and limited repair mechanisms in comparison to nuclear DNA. As mtDNA lacks introns, it has been suggested that most mutations will occur in coding sequences. The subsequent accumulation of mutations may lead to tumor formation. By virtue of their clonal nature, high copy number and high frequent mutations may provide a powerful molecular biomarker for the detection of cancer. It has been suggested that the extent of mtDNA mutations might be useful in the prognosis of cancer outcome and/or the response to certain therapies. In this review article, we aim to provide a brief summary of our current understanding of mitochondrial genetics and biology, review the mtDNA alterations reported in breast cancer, and offer some perspectives as to the emergence of mtDNA mutations, including their functional consequences in cancer development, diagnostic criteria, and therapeutic implications.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR.

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

A perfusion bioreactor-based 3D model of the subarachnoid space based on a meningeal tissue construct.

BACKGROUND: Altered flow of cerebrospinal fluid (CSF) within the subarachnoid space (SAS) is connected to brain, but also optic nerve degenerative diseases. To overcome the lack of suitable in vitro models that faithfully recapitulate the intricate three-dimensional architecture, complex cellular interactions, and fluid dynamics within the SAS, we have developed a perfusion bioreactor-based 3D in vitro model using primary human meningothelial cells (MECs) to generate meningeal tissue constructs. We ultimately employed this model to evaluate the impact of impaired CSF flow as evidenced during optic nerve compartment syndrome on the transcriptomic landscape of MECs. METHODS: Primary human meningothelial cells (phMECs) were seeded and cultured on collagen scaffolds in a perfusion bioreactor to generate engineered meningeal tissue constructs. Engineered constructs were compared to human SAS and assessed for specific cell-cell interaction markers as well as for extracellular matrix proteins found in human meninges. Using the established model, meningeal tissue constructs were exposed to physiological and pathophysiological flow conditions simulating the impaired CSF flow associated with optic nerve compartment syndrome and RNA sequencing was performed. RESULTS: Engineered constructs displayed similar microarchitecture compared to human SAS with regards to pore size, geometry as well as interconnectivity. They stained positively for specific cell-cell interaction markers indicative of a functional meningeal tissue, as well as extracellular matrix proteins found in human meninges. Analysis by RNA sequencing revealed altered expression of genes associated with extracellular matrix remodeling, endo-lysosomal processing, and mitochondrial energy metabolism under pathophysiological flow conditions. CONCLUSIONS: Alterations of these biological processes may not only interfere with critical MEC functions impacting CSF and hence optic nerve homeostasis, but may likely alter SAS structure, thereby further impeding cerebrospinal fluid flow. Future studies based on the established 3D model will lead to new insights into the role of MECs in the pathogenesis of optic nerve but also brain degenerative diseases.

Juvenile xanthogranuloma involving concurrent iris and skin: Clinical, pathological and molecular pathological evaluations.

PURPOSE: To report a case of juvenile xanthogranuloma involving the iris and skin that clincally was diagnosed with an obvious cutaneous lesion. OBSERVATIONS: A four month-old girl with hyphema and increased intraocular pressure of the left eye persisting for 2 weeks. A suspicious yellow-brown mass with nodular surface and traversed by irregular vascularization was noted on the inferior iris surface. Ultrasound biomicroscopy (UBM; 35 MHz) of the mass revealed multiple nodular irregular hyperreflective lesions in the peripheral iris. Using a biopsy of an obvious cutaneous abdominal skin lesion a diagnosis was made based on histopathological analyses. The biopsy showed dense dermal infiltrate consisting of foamy histiocytes. Additional stains revealed CD68 positivity and CD1a and S100 negativity. This mass revealed histopathologic features identical to juvenile xanthogranuloma and was concurrent with the iris lesion. Next-generation sequencing using Ion AmpliSeqTM Cancer Hotspot Panel revealed a missense mutation of FGFR3 (p.F386L). CONCLUSION AND IMPORTANCE: The diagnosis of a xanthogranuloma of the iris with hyphema can be made easier in patients with obvious cutaneous lesions as described in our case. The significance of FGFR3 mutation in association with JXG is unknown and should be further investigated.

UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy.

Clearance of damaged mitochondria through mitophagy is critical for maintaining mitochondrial fidelity and the prevention of neurodegeneration. Here, we report on the UBX domain-containing, p97/VCP cofactor UBXD1/UBXN6/UBXDC2 and its role in mitophagy. Recognizing depolarized mitochondria via its C-terminal UBX domain, UBXD1 translocates to mitochondria in a Parkin-dependent manner. During Parkin-independent mitophagy, UBXD1 shows no mitochondrial translocation. Once translocated, UBXD1 recruits p97 to mitochondria via a bipartite binding motif consisting of its N-terminal VIM and PUB domains. Recruitment of p97 by UBXD1 only depends on the presence of UBXD1 on mitochondria without the need for further mitochondrial signals. Following translocation of UBXD1 to CCCP-depolarized mitochondria and p97 recruitment, formation of LC3-positive autolysosomes is strongly enhanced and autophagic degradation of mitochondria is significantly accelerated. Diminished levels of UBXD1 negatively impact mitophagic flux in Parkin-expressing cells after CCCP treatment. Thus, our data supports a model, whereby the p97 cofactor UBXD1 promotes Parkin-dependent mitophagy by specifically recognizing damaged mitochondria undergoing autophagic clearance.

Cell-Cell Interaction Proteins (Gap Junctions, Tight Junctions, and Desmosomes) and Water Transporter Aquaporin 4 in Meningothelial Cells of the Human Optic Nerve.

PURPOSE: Meningothelial cells (MECs) play a central role in the maintenance of cerebrospinal fluid (CSF) homeostasis and in physiological and pathophysiological processes within the subarachnoid space (SAS) linking them to optic nerve (ON) pathologies. Still, not much is known about their structural properties that might enable MECs to perform specific functions within the ON microenvironment. METHODS: For closer characterization of the structural properties of the human MEC layer in the arachnoid, we performed immunohistological analyses to evaluate the presence of cell-cell interaction markers, namely, markers for tight junctions (JAM1, Occludin, and Claudin 5), gap junctions (Connexin 26 and 43), and desmosomes (Desmoplakin) as well as for water channel marker aquaporin 4 (AQP4) in retrobulbar, midorbital, and intracanalicular human ON sections. RESULTS: MECs displayed immunopositivity for markers of tight junctions (JAM1, Occludin, and Claudin 5) and gap junctions (Connexin 26 and 43) as well as for AQP4 water channels. However, no immunopositivity was found for Desmoplakin. CONCLUSION: MECs are connected via tight junctions and gap junctions, and they possess AQP4 water channels. The presence of these proteins emphasizes the important function of MECs within the ON microenvironment as part of the meningeal barrier. Beyond this barrier function, the expression of these proteins by MECs supports a broader role of these cells in signal transduction and CSF clearance pathways within the ON microenvironment.

Cell-free DNA in the circulation as a potential cancer biomarker.

In the course of the search for new biomarkers, circulating cell-free DNA (ccf-DNA) has become a popular target of interest. An elevated level of ccf-DNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since ccf-DNA in cancer patients often bears similar genetic and epigenetic features to the related tumor DNA, there is evidence that some of the ccf-DNA originates from tumoral tissue. This, and the fact that ccf-DNA can easily be isolated from the circulation and other body fluids of patients, makes it a promising candidate as a non-invasive biomarker of cancer. Yet ccf-DNA-based cancer tests have not come to fruitful clinical applications. This review evaluates the potential of ccf-DNA alterations as a biomarker for cancer management by addressing the question of how large the gap between ccf-DNA and the ideal cancer biomarker is.

Assessing the value of CAN-gene mutations using MALDI-TOF MS.

PURPOSE: To identify cancer-linked genes, Sjöblom et al. and Wood et al. performed a genome-wide mutation screening in human breast and colorectal cancers. 140 CAN-genes were found in breast cancer, which in turn contained overall 334 mutations. These mutations could prove useful for diagnostic and therapeutic purposes. METHODS: We used a MALDI-TOF MS 40-plex assay for testing 40 loci within 21 high-ranking breast cancer CAN-genes. To confirm mutations, we performed single-plex assays and sequencing. RESULTS: In general, the mutation rate of the analyzed loci in our sample cohort was very low. No mutation from the 40 loci analyzed could be found in the 6 cell lines. In tissue samples, a single breast cancer tissue sample showed heterozygosity at locus c.5834G>A within the ZFYVE26 gene (Zinc finger FYVE domain-containing gene 26). CONCLUSIONS: Sjöblom et al./Wood et al. already showed that the vast majority of CAN-genes are mutated at very low frequency. Due to the fact that we only found one mutation in our cohort, we therefore assume that at the selected loci, mutations might be low-frequency events and therefore, more rarely detectable. However, further evaluation of the CAN-gene mutations in larger cohorts should be the aim of further studies.