Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton’s tyrosine kinase

AbstractThe SH3 domain of Bruton’s tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and 15N NMR relaxation measurements are all consistent with a monomer–dimer equilibrium with a dissociation constant on the order of 60 μM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk

Missing-in-metastasis and IRSp53 deform PI(4,5)P-2-rich membranes by an inverse BAR domain-like mechanism

The actin cytoskeleton plays a fundamental role in various motile and morphogenetic processes involving membrane dynamics. We show that actin-binding proteins MIM (missing-in-metastasis) and IRSp53 directly bind PI(4,5)P-2-rich membranes and deform them into tubular structures. This activity resides in the N-terminal IRSp53/MIM domain (IMD) of these proteins, which is structurally related to membrane-tubulating BAR (Bin/amphiphysin/Rvs) domains. We found that because of a difference in the geometry of the PI(4,5)P-2-binding site, IMDs induce a membrane curvature opposite that of BAR domains and deform membranes by binding to the interior of the tubule. This explains why IMD proteins induce plasma membrane protrusions rather than invaginations. We also provide evidence that the membrane-deforming activity of IMDs, instead of the previously proposed F-actin - bundling or GTPase-binding activities, is critical for the induction of the filopodia/microspikes in cultured mammalian cells. Together, these data reveal that interplay between actin dynamics and a novel membrane-deformation activity promotes cell motility and morphogenesis

Different expression of BRAFV600E, ALK and PD-L1 in melanoma in children and adolescents : a nationwide retrospective study in Finland in 1990-2014

Background Pediatric melanoma may have a different biological background and more favorable prognosis compared with melanoma in adults. The aim of this study was to investigate melanoma in children and adolescents in the Finnish population in terms of incidence, clinical course, treatment, prognosis and BRAFV600E-, ALK- and PD-L1-positivity of the primary tumors. Materials and Methods Primary tumor samples and clinical records of all patients aged 0-19 years diagnosed with cutaneous melanoma in Finland in 1990-2014 were collected using the Finnish Cancer Registry database, Finnish hospitals and private pathology laboratories. BRAFV600E, ALK and PD-L1 were analyzed from 54 primary tumors and BRAFV600E from six metastasis samples. Results A total of 122 patients diagnosed with cutaneous melanoma were retrieved from the Cancer Registry database. The primary tumor samples of 73 patients were obtained for the review, and 56 cases were included in the study. The incidence of pediatric melanoma increased from 0.2 to 1.0/100 000 during the period 1990-2014. Spitzoid melanoma was the most common subtype (66%). The 10-year cancer-specific survival (CSS) was 88.7% in all patients. The 10-year-CSS did not differ in SLNB-positive or -negative groups. BRAFV600E was positive in 48%, ALK in 9% and PD-L1 in 2% of the tumors. BRAFV600E mutation was associated with 83% of melanoma deaths. Conclusions Young melanoma patients had more favorable prognosis and a different staining profile for BRAFV600E, ALK, and PD-L1 in primary tumor than reported in adults. SLNB status was not an indicator for survival. BRAFV600E-positive patients have worse prognosis and could benefit from surveillance and treatment similarly to adults.Peer reviewe

Internalization of novel non-viral vector TAT-streptavidin into human cells

BACKGROUND: The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT(47–57)-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. RESULTS: By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs. CONCLUSION: This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells

GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth

Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including he-patocellular carcinoma (HCC), as well as in viral in-fections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh -7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accu-mulation of sphingolipids, such as ceramides, hex-osylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lyso-phosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi struc-ture and distribution were observed both by electron microscopy imaging and immunofluorescence mi-croscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid ho-meostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demon-strate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.Peer reviewe

Expanded national database collection and data coverage in the FINDbase worldwide database for clinically relevant genomic variation allele frequencies

FINDbase (http://www.findbase.org) is a comprehensive data repository that records the prevalence of clinically relevant genomic variants in various populations worldwide, such as pathogenic variants leading mostly to monogenic disorders and pharmacogenomics biomarkers. The database also records the incidence of rare genetic diseases in various populations, all in well-distinct data modules. Here, we report extensive data content updates in all data modules, with direct implications to clinical pharmacogenomics. Also, we report significant new developments in FINDbase, namely (i) the release of a new version of the ETHNOS software that catalyzes development curation of national/ethnic genetic databases, (ii) the migration of all FINDbase data content into 90 distinct national/ethnic mutation databases, all built around Microsoft’s PivotViewer (http://www.getpivot.com) software (iii) new data visualization tools and (iv) the interrelation of FINDbase with DruGeVar database with direct implications in clinical pharmacogenomics. The above mentioned updates further enhance the impact of FINDbase, as a key resource for Genomic Medicine applications

Increased incidence of melanoma in children and adolescents in Finland in 1990-2014 : nationwide re-evaluation of histopathological characteristics

Background Changes in the incidence of melanoma in children and adolescents have been reported in Europe and in the USA in the recent decades. Aims The aim of this study was to examine the incidence of paediatric and adolescent melanomas in Finland in 1990-2014, and the associated clinical and histopathological characteristics to reveal temporal trends, such as changes in diagnostic sensitivity of Spitzoid melanomas. Methods Information on 122 patients diagnosed with cutaneous melanoma at 0-19 years of age in Finland in 1990-2014 were retrieved from the Finnish Cancer Registry. 73 primary melanoma archival samples were re-evaluated by two dermatopathologists to allow comparability over time. Results A 5.6% annual increase was observed in the incidence of melanoma among children and adolescents during the study period. Fifty-six tumours were confirmed as malignant melanomas in the re-evaluation. After correction for tumour misclassification in the Cancer Registry, the age-adjusted annual incidence was estimated to have increased from 1.4/1 000 000 in 1990-1994 to 5.8/1 000 000 in 2010-2014. The change in incidence was most prominent among adolescents and in Spitzoid melanoma subtype. Melanomas diagnosed 1990-2002 and 2003-2014 did not differ in terms of their clinicopathological characteristics or prognosis (hazard ratio for melanoma-related death 1.53, 95% CI 0.30 to 7.88). Spitzoid melanomas were diagnosed at a younger age, were of higher stage and had higher Clark level than other melanomas, yet the hazard ratio for death was 0.52 (95% CI 0.10 to 2.58) for Spitzoid versus other melanomas. Conclusions The incidence of cutaneous melanoma has clearly increased among the young in Finland, especially among adolescents. No evidence for overdiagnosis of Spitzoid melanomas as the underlying cause of the increased incidence was observed. Key message A nationwide retrospective re-evaluation of the cutaneous melanomas recorded in the Finnish Cancer Registry among patients aged 0-19 years in Finland in 1990-2014 revealed an approximately 4-fold increase in the incidence. The increase in the incidence was most prominent among adolescents and in the Spitzoid melanoma subtype. Our results contrast those reported in other countries, where the incidence of melanoma among adolescents has declined.Peer reviewe

Follow-Up of Cancer Patients Receiving Anti-PD-(L)1 Therapy Using an Electronic Patient-Reported Outcomes Tool (KISS): Prospective Feasibility Cohort Study

Background: Immune checkpoint inhibitors (ICIs) have become a standard of care for various tumor types. Their unique spectrum of side effects demands continuous and long-lasting assessment of symptoms. Electronic patient-reported outcome (ePRO) follow-up has been shown to improve survival and quality of life of cancer patients treated with chemotherapy.Objective: This study aimed to investigate whether ePRO follow-up of cancer patients treated with ICIs is feasible. The study analyzed (1) the variety of patient reported symptoms, (2) etiology of alerts, (3) symptom correlations, and (4) patient compliance.Methods: In this prospective, one-arm, multi-institutional study, we recruited adult cancer patients whose advanced cancer was treated with anti-programmed cell death protein 1 (PD)-ligand (L)1 agents in outpatient settings. The ePRO tool consisted of a weekly questionnaire evaluating the presence of typical side effects, with an algorithm assessing the severity of the symptom according to National Cancer Institute Common Terminology Criteria for Adverse Events and an urgency algorithm sending alerts to the care team. A patient experience survey was conducted monthly. The patients were followed up to 6 months or until disease progression.Results: A total of 889 symptom questionnaires was completed by 37 patients (lung cancer, n=15; melanoma, n=9; genitourinary cancer, n=9; head and neck cancer, n=4). Patients showed good adherence to ePRO follow-up. The most common grade 1 symptoms were fatigue (28%) and itching (13%), grade 2 symptoms were loss of appetite (12%) and nausea (12%), and grade 3-4 symptoms were cough (6%) and loss of appetite (4%). The most common reasons for alerts were loss of appetite and shortness of breath. In the treatment benefit analysis, positive correlations were seen between clinical benefit and itching as well as progressive disease and chest pain.Conclusions: According to the results, ePRO follow-up of cancer patients receiving ICIs is feasible. ePROs capture a wide range of symptoms. Some symptoms correlate to treatment benefit, suggesting that individual prediction models could be generated

Cutaneous lupus erythematosus after treatment with paclitaxel and bevacizumab for metastatic breast cancer: a case report

Abstract Introduction The monoclonal anti-vascular endothelial growth factor antibody bevacizumab is increasingly used in the treatment of several malignant tumors. The usual side effects of this drug are hypertension and proteinuria. Paclitaxel is widely used in the treatment of breast cancer and head and neck carcinomas. Neither of these two drugs typically causes skin disorders. Paclitaxel-related cutaneous lupus erythematosus has been described before, but in earlier cases patients had a history of autoimmune disease. Case presentation We report a case of a 65-year-old Caucasian woman who presented with cutaneous lupus erythematosus after receiving paclitaxel-bevacizumab combination treatment as first-line therapy for metastatic breast cancer. Her cutaneous symptoms and increased serum anti-SSA and anti-SSB antibodies disappeared shortly after the discontinuation of therapy. Conclusion We conclude that cutaneous lupus erythematosus can also be seen in patients without earlier anamnesis of autoimmune disorders and that, furthermore, bevacizumab might cause atypical cutaneous side effects.</p

Distribution of immunodeficiency fact files with XML – from Web to WAP

BACKGROUND: Although biomedical information is growing rapidly, it is difficult to find and retrieve validated data especially for rare hereditary diseases. There is an increased need for services capable of integrating and validating information as well as proving it in a logically organized structure. A XML-based language enables creation of open source databases for storage, maintenance and delivery for different platforms. METHODS: Here we present a new data model called fact file and an XML-based specification Inherited Disease Markup Language (IDML), that were developed to facilitate disease information integration, storage and exchange. The data model was applied to primary immunodeficiencies, but it can be used for any hereditary disease. Fact files integrate biomedical, genetic and clinical information related to hereditary diseases. RESULTS: IDML and fact files were used to build a comprehensive Web and WAP accessible knowledge base ImmunoDeficiency Resource (IDR) available at . A fact file is a user oriented user interface, which serves as a starting point to explore information on hereditary diseases. CONCLUSION: The IDML enables the seamless integration and presentation of genetic and disease information resources in the Internet. IDML can be used to build information services for all kinds of inherited diseases. The open source specification and related programs are available at