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Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome–endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex–containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)–independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex–containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis. Cancer Res; 70(4); 1668–78

Supplementary Figure 4 from Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

Supplementary Figure 3 from Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

Supplementary Methods, Figure Legends 1-4, Tables 1-5, References from Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation

Розроблено математичну модель нагрівання сталевих заготовок у полуменевихпечах камерного типу. Задачу теплообміну вирішено за допомогою зонального методу зурахуванням наявності факела, руху нагрівального середовища, втрат теплоти через ого-родження печі, а також акумульовано кладкою. Досліджено вплив довжини факела, а та-кож імпульсного режиму опалювання печі на якісні показники нагрівання металу. Встанов-лено, що достатньо рівномірного нагрівання металу досягнуто за довжини факела, яка єзіставленою із шириною робочого об’єму камери, та під час реалізації імпульсного режимуопалювання печі у період витримки.

Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell–cell communication in cancer.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Acute intermittent porphyria (AIP), an autosomal dominant disorder due to the half-normal activity of hydroxymethylbilane synthase (HMBS), is characterized by acute neurovisceral attacks that are precipitated by factors that induce heme biosynthesis. Molecular diagnosis is the most sensitive and specific diagnostic test for AIP, and importantly, it permits the identification of asymptomatic family members for genetic counseling and avoidance of precipitating factors. Here, we report the identification of 40 novel HMBS mutations, including 11 missense, four nonsense, 16 small insertions or deletions, eight consensus splice site mutations, and a complex insertion-deletion mutation in unrelated individuals with AIP. Prokaryotic expression of the missense mutations demonstrated that all mutants had ≤5% of expressed wildtype activity, except for c.1039G>C (p.A347P), which had 51% residual HMBS activity but was markedly thermolabile. Of note, the mutation c.612G>T (p.Q204H) altered the last nucleotide of exon 10, which resulted in an alternative HMBS transcript with an in-frame nine base-pair deletion at the 3'-terminus of exon 10 (encoding protein Q204HΔ3). When expressed, Q204HΔ3 and an in-frame three base-pair deletion (c.639_641delTGC) had no detectable HMBS activity. Western blot analyses and mapping of the missense mutations on the human HMBS crystal structure revealed that mutations near the active site or at the dimerization interface resulted in stably expressed proteins, while most that altered surface residues resulted in unstable proteins, presumably due to improper protein folding. These studies identified novel pathogenic HMBS mutations and expanded the molecular heterogeneity of AIP.

Wnt5A signals through various receptors that confer versatile biological functions. Here, we used Wnt5A overexpressing human ovarian SKOV-3 and OVCAR-3 stable clones for assessing integrin expression, cell proliferation, migration, invasion, and the ability of multicellular aggregates (MCAs) formation. We found here, that Wnt5A regulates differently the expression of its receptors in the stable Wnt5A overexpressing clones. The expression levels of Frizzled (FZD)-2 and -5, were increased in different clones. However ROR-1, -2 expression levels were differently regulated in clones. Wnt5A overexpressing clones showed increased cell proliferation, migration, and clonogenicity. Moreover, Wnt5A overexpressing SKOV-3 clone showed increased MCAs formation ability. Cell invasion had been increased in OVCAR-3-derived clones, while this was decreased in SKOV-3-derived clone. Importantly, αv integrin expression levels were increased in all assessed clones, accompanied by increased cell attachment to fibronectin and focal adhesion kinase activity. Moreover, the treatment of clones with Box5 as a Wnt5A/FZD5 antagonist abrogates ITGAV increase, cell proliferation, migration, and their attachment to fibronectin. Accordingly, we observed significantly higher expression levels of ITGAV and ITGB3 in human high-grade serous ovarian cancer specimens and ITGAV correlated positively with Wnt5A in metastatic serous type ovarian cancer. In summary, we hypothesize here, that Wnt5A/FZD-5 signaling modulate αv integrin expression levels that could be associated with ovarian cancer cell proliferation, migration, and fibronectin attachment.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study we focus on the profibrotic properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNA seq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrated that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin alpha 6 beta 1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. (c) 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extracellular vesicles (EVs) contain various bioactive molecules such as DNA, RNA, and proteins, and play a key role in the regulation of cancer progression. Furthermore, cancer-associated EVs carry specific biomarkers and can be used in liquid biopsy for cancer detection. However, it is still technically challenging and time consuming to detect or isolate cancer-associated EVs from complex biofluids (e.g., blood). Here, a novel EV-capture strategy based on dip-pen nanolithography generated microarrays of supported lipid membranes is presented. These arrays carry specific antibodies recognizing EV- and cancer-specific surface biomarkers, enabling highly selective and efficient capture. Importantly, it is shown that the nucleic acid cargo of captured EVs is retained on the lipid array, providing the potential for downstream analysis. Finally, the feasibility of EV capture from patient sera is demonstrated. The demonstrated platform offers rapid capture, high specificity, and sensitivity, with only a small need in analyte volume and without additional purification steps. The platform is applied in context of cancer-associated EVs, but it can easily be adapted to other diagnostic EV targets by use of corresponding antibodies.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (?liquid biopsies?). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood. Non

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10−4 pmol of siRNA, and 1 × 10−3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP-ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs. S.R. and J.C. contributed equally to this work as co-first authors. C.O. and I.N. contributed equally to this work as co-senior authors. The authors thank Celso Reis and Joy Burchell for having generously shared the anti-Mucin-1 antibody, Gabriela Almeida and Mafalda Santos for sharing the H&E images from MKN45 and MKN74 tumors in mice, Maria Lazaro for the technical support with imaging flow cytometry data, Tanja Gainey-Schleicher and Elena Grueso Navarro for the support with EV–cell association assay, and Deborah Lawrie-Blum for proofreading the manuscript. S.R., R.K., J.C., C.O., A.T., A.K., and I.N. were involved in the conception and design of the study; S.R., J.C., and P.O. were involved in the establishment and characterization of 3D microwell array culture of GC cells, characterization of EVs, small RNA sequencing experiments, data analysis and validation, and functional assays; D.S., M.V., N.W., and R.K. were involved in all proteomics studies including data analysis and validation, and the analysis of cell–EV association; S.R., J.C., P.O., C.O., D.S., A.K., J.-C.S., A.T., and I.N. analyzed the data; S.R., J.C., P.O., C.O., A.K., and I.N. wrote the manuscript; S.R., J.C., P.O., C.O., A.K., and I.N. critically reviewed the manuscript; and all authors gave final approval of the manuscript.

Background. Synovial sarcoma is an aggressive soft-tissue malignancy. This study examines the presence of the SYT-SSX fusion transcript in synovial sarcoma microvesicles as well as its potential role as a biomarker for synovial sarcoma.Patients and Methods. Microvesicle release of synovial sarcoma cells was examined by transmission electron microscopy. RNA-content was analyzed by qPCR, nested PCR, nested qPCR, and droplet digital PCR to compare their sensitivity for detection of the SYT-SSX fusion gene transcript. Whole blood RNA, RNA of mononuclear cells, and microvesicle RNA of synovial sarcoma patients were analyzed for the presence of the fusion gene transcripts.Results. Electron microscopic analysis revealed synovial sarcoma cells releasing membrane-enclosed microvesicles.In vitro, the SYT-SSX fusion gene transcript was detected in both synovial sarcoma cells and microvesicles. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA. In contrast, the fusion gene transcript was not detected in peripheral blood cells and microvesicles of synovial sarcoma patients.Conclusion. Synovial sarcoma cells release microvesicles harboring the SYT-SSX fusion transcript. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA; however, more sensitive assays are needed to detect cancer-specific microvesicles in the peripheral blood of cancer patients.

Inhalation of particulate matter (PM) from residential biomass combustion is epidemiologically associated with cardiovascular and pulmonary diseases. This study investigates PM0.4-1 emissions from combustion of commercial Miscanthus straw (MS), softwood chips (SWC) and beech wood chips (BWC) in a domestic-scale boiler (40 kW). The PM0.4-1 emitted during combustion of the MS, SWC and BWC were characterized by ICP-MS/OES, XRD, SEM, TEM, and DLS. Cytotoxicity and genotoxicity in human alveolar epithelial A549 and human bronchial epithelial BEAS-2B cells were assessed by the WST-1 assay and the DNA-Alkaline Unwinding Assay (DAUA). PM0.4-1 uptake/translocation in cells was investigated with a new method developed using a confocal reflection microscope. SWC and BWC had a inherently higher residual water content than MS. The PM0.4-1 emitted during combustion of SWC and BWC exhibited higher levels of Polycyclic Aromatic Hydrocarbons (PAHs), a greater variety of mineral species and a higher heavy metal content than PM0.4-1 from MS combustion. Exposure to PM0.4-1 from combustion of SWC and BWC induced cytotoxic and genotoxic effects in human alveolar and bronchial cells, whereby the strongest effect was observed for BWC and was comparable to that caused by diesel PM (SRM 2 975), In contrast, PM0.4-1 from MS combustion did not induce cellular responses in the studied lung cells. A high PAH content in PM emissions seems to be a reliable chemical marker of both combustion efficiency and particle toxicity. Residual biomass water content strongly affects particulate emissions and their toxic potential. Therefore, to minimize the harmful effects of fine PM on health, improvement of combustion efficiency (aiming to reduce the presence of incomplete combustion products bound to PM) and application of fly ash capture technology, as well as use of novel biomass fuels like Miscanthus straw is recommended.

Porphyria Cutanea Tarda (PCT) is a cutaneous porphyria that results from the hepatic inhibition of the heme biosynthetic enzyme uroporphyrinogen decarboxylase (UROD), and can occur either in the absence or presence of an inherited heterozygous UROD mutation (PCT subtypes 1 and 2, respectively). A heterozygous UROD mutation causes half-normal levels of UROD activity systemically, which is a susceptibility factor but is not sufficient alone to cause type 2 PCT. In both Types 1 and 2 PCT, the cutaneous manifestations are precipitated by additional factors that lead to generation of an inhibitor that more profoundly reduces hepatic UROD activity. PCT is an iron-related disorder, and many of its known susceptibility factors, which include infections (e.g. hepatitis C virus, HIV), high alcohol consumption, smoking, estrogens, and genetic traits (e.g. hemochromatosis mutations) can increase hepatic iron accumulation. Hepatoerythropoietic Porphyria (HEP) is a rare autosomal recessive disease that results from homozygosity or compound heterozygosity for UROD mutations and often causes infantile or childhood onset of both erythropoietic and cutaneous manifestations. During the 11-year period from 01/01/2007 through 12/31/2017, the Mount Sinai Porphyrias Diagnostic Laboratory provided molecular diagnostic testing for 387 unrelated patients with PCT and four unrelated patients with HEP. Of the 387 unrelated individuals tested for Type 2 PCT, 79 (20%) were heterozygous for UROD mutations. Among 26 family members of mutation-positive PCT patients, eight (31%) had the respective family mutation. Additionally, of the four unrelated HEP patients referred for UROD mutation analyses, all had homozygosity or compound heterozygosity for UROD mutations, and all eight asymptomatic family members were heterozygotes for UROD mutations. Of the UROD mutations identified, 19 were novel, including nine missense, two nonsense, one consensus splice-site, and seven insertions and deletions. These results expand the molecular heterogeneity of PCT and HEP by adding a total of 19 novel UROD mutations. Moreover, the results document the usefulness of molecular testing to confirm a genetic susceptibility trait in Type 2 PCT, confirm a diagnosis in HEP, and identify heterozygous family members.

MicroRNAs (miRNAs) are class of small RNA molecules with major impact on gene regulation. We analyzed the potential of miRNAs secreted from pre-implantation embryos into the embryonic culture media as biomarkers to predict successful pregnancy. Using microarray analysis, we profiled the miRNome of the 56 spent culture media (SCM) after embryos transfer and found a total of 621 miRNAs in the SCM. On average, we detected 163 miRNAs in SCM of samples with failed pregnancies, but only 149 SCM miRNAs of embryos leading to pregnancies. MiR-634 predicted an embryo transfer leading to a positive pregnancy with an accuracy of 71% and a sensitivity of 85%. Among the 621 miRNAs, 102 (16.4%) showed a differential expression between positive and negative outcome of pregnancy with miR-29c-3p as the most significantly differentially expressed miRNA. The number of extracellular vehicles was lower in SCM with positive outcomes (3.8 × 109/mL EVs), as compared to a negative outcome (7.35 × 109/mL EVs) possibly explaining the reduced number of miRNAs in the SCM associated with failed pregnancies. The analysis of the miRNome in the SCM of couples undergoing fertility treatment lays the ground towards development of biomarkers to predict successful pregnancy and towards understanding the role of embryonic miRNAs found in the SCM.

Cholesteryl ester storage disease (CESD) and Wolman disease are autosomal recessive later-onset and severe infantile disorders, respectively, which result from the deficient activity of lysosomal acid lipase (LAL). LAL is encoded by LIPA (10q23.31) and the most common mutation associated with CESD is an exon 8 splice junction mutation (c.894G>A; E8SJM), which expresses only ∼3%-5% of normally spliced LAL. However, the frequency of c.894G>A is unknown in most populations. To estimate the prevalence of CESD in different populations, the frequencies of the c.894G>A mutation were determined in 10,000 LIPA alleles from healthy African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals from the greater New York metropolitan area and 6,578 LIPA alleles from African-American, Caucasian, and Hispanic subjects enrolled in the Dallas Heart Study. The combined c.894G>A allele frequencies from the two cohorts ranged from 0.0005 (Asian) to 0.0017 (Caucasian and Hispanic), which translated to carrier frequencies of 1 in 1,000 to ∼1 in 300, respectively. No African-American heterozygotes were detected. Additionally, by surveying the available literature, c.894G>A was estimated to account for 60% (95% confidence interval [CI]: 51%-69%) of reported mutations among multiethnic CESD patients. Using this estimate, the predicted prevalence of CESD in the Caucasian and Hispanic populations is ∼0.8 per 100,000 (∼1 in 130,000; 95% CI: ∼1 in 90,000 to 1 in 170,000). Conclusion: These data indicate that CESD may be underdiagnosed in the general Caucasian and Hispanic populations, which is important since clinical trials of enzyme replacement therapy for LAL deficiency are currently being developed. Moreover, future studies on CESD prevalence in African and Asian populations may require full-gene LIPA sequencing to determine heterozygote frequencies, since c.894G>A is not common in these racial groups. (HEPATOLOGY 2013;53:958–965)

X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670-1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.

Tetraspanins comprise a large family of integral membrane proteins involved in the regulation of cell adhesion, migration and fusion. In humans it consists of 33 members divided in four subfamilies. Here, we examined the spatial and temporal gene expression of four related tetraspanins during the embryonic development of Xenopus laevis by quantitative real-time PCR and in situ hybridization: Tspan3 (encoded by the gene Tm4sf8 gene) Tspan4 (encoded by the gene Tm4sf7), Tspan5 (encoded by the gene Tm4sf9) and Tspan7 (encoded by the gene Tm4sf2). These genes appeared first in the vertebrates during the evolution and are conserved across different species. In humans, they were associated with several diseases such as sclerosis, mental retardation and cancer; however their physiological role remained unclear. This work provides a comprehensive comparative analysis of the expression of these tetraspanins during the development of X. laevis. The more closely related tetraspanins Tspan3, Tspan4 and Tspan7 exhibited very similar spatial expression patterns, albeit differing in their temporal occurrence. The corresponding transcripts were found in the dorsal animal ectoderm at blastula stage. At early tailbud stages (stage 26) the genes were expressed in the migrating cranial neural crest located in the somites, developing eye, brain, and in otic vesicles. In contrast, Tspan5 appeared first at later stages of development and was detected prominently in the notochord. These data support close relatedness of Tspan3, Tspan4 and Tspan7. The expression of these tetraspanins in the cells with a high migratory potential, e.g. neural crest cells, suggests their role in the regulation of migration processes, characteristic for tetraspanin family members, during development. Similarity of the expression profiles might indicate at least partial functional redundancy, which is in concordance with earlier findings of tissue-limited or absent phenotypes in the knock-down studies of tetraspanins family members performed.

collaboration au projet H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD); International audience; Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and.Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

In recent years there has been tremendous interest in both the basic biology and applications of extracellular vesicles (EVs) in translational cancer research. This includes a better understanding of their biogenesis and mechanisms of selective cargo packaging, their precise roles in horizontal communication, and their application as non-invasive biomarkers. The rapid advances in next-generation omics technologies are the driving forces for these discoveries. In this review, the authors focus on recent results of EV research in ovarian cancer. A deeper understanding of ovarian cancer-derived EVs, the types of cargo molecules and their biological roles in cancer growth, metastases and drug resistance, could have significant impact on the discovery of novel biomarkers and innovative therapeutics. Insights into the role of EVs in immune regulation could lead to novel approaches built on EV-based immunotherapy.

Hereditary coproporphyria (HCP) is an autosomal dominant acute hepatic porphyria due to the half-normal activity of the heme biosynthetic enzyme, coproporphyrinogen oxidase (CPOX). The enzyme catalyzes the step-wise oxidative decarboxylation of the heme precursor, coproporphyrinogen III, to protoporphyrinogen IX via a tricarboxylic intermediate, harderoporphyrinogen. In autosomal dominant HCP, the deficient enzymatic activity results primarily in the accumulation of coproporphyrin III. To date, only a few homozygous HCP patients have been described, most having Harderoporphyria, a rare variant due to specific CPOX mutations that alter enzyme residues D400-K404, most patients described to date having at least one K404E allele. Here, we describe a Turkish male infant, the product of a consanguineous union, who presented with the Harderoporphyria phenotype including neonatal hyperbilirubinemia, hemolytic anemia, hepatosplenomegaly, and skin lesions when exposed to UV light. He was homoallelic for the CPOX missense mutation, c.980A > G (p.H327R), and had massively increased urinary uroporphyrins I and III (9,250 and 2,910 mu M, respectively) and coproporphyrins I and III (895 and 19,400 mu M, respectively). The patient expired at 5 months of age from an apparent acute neurologic porphyric attack. Structural studies predicted that p.H327R interacts with residue W399 in the CPOX active site, thereby accounting for the Harderoporphyria phenotype.

Intracellular signaling governed by serine/threonine kinases comprises the molecular interface between cell surface receptors and the nuclear transcriptional machinery. The protein kinase C (PKC) family members are involved in the control of many signaling processes directing cell proliferation, motility, and survival. Here, we examined a role of different PKC isoenzymes in protein phosphatase 2A (PP2A) and HRSL3 tumor suppressor–dependent cell death induction in the ovarian carcinoma cell line OVCAR-3. Phosphorylation and activity of PKC isoenzymes were measured in response to PP2A or phosphoinositide 3-kinase inhibition or HRSL3 overexpression. These experiments indicated a regulation of PKCθ, ϵ, ζ, and ι through PP2A and/or HRSL3, but not of PKCα and β. Using isoform-specific peptide inhibitors and overexpression approaches, we verified a contribution to PP2A- and HRLS3-dependent apoptosis only for PKCζ, suggesting a proapoptotic function of this kinase. We observed a significant proportion of human ovarian carcinomas expressing high levels of PKCζ, which correlated with poor prognosis. Primary ovarian carcinoma cells isolated from patients also responded to okadaic acid treatment with increased phosphorylation of PKCζ and apoptosis induction. Thus, our data indicate a contribution of PKCζ in survival control in ovarian carcinoma cells and suggest that upregulation or activation of tyrosine kinase receptors in this tumor might impinge onto apoptosis control through the negative regulation of the atypical PKCζ. Mol Cancer Res; 8(6); 919–34. ©2010 AACR.