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Retinal pigment epithelium is a monolayer of cells located beneath photoreceptors of the retina maintaining their functionality. Malfunction of RPE leads to retinal degenerative diseases, such as age-related macular degeneration and Stargardt disease. Ca2+ is a ubiquitous ion that takes part in regulation of vital cellular processes. The knowledge of Ca2+ dynamics is essential for understanding RPE physiology. This is especially important for functionality assessment of cells intended for transplantation and for drug testing. The aim of this thesis was to study spontaneous and mechanically induced Ca2+ activity in human RPE and to assess the effect of cellular maturation and wounding on the [Ca2+]i dynamics. For this, various methods, such as fluorescent Ca2+ imaging, immunofluorescence staining, PCR, and mathematical modeling were applied. In addition, novel methods were developed to analyze large amounts of Ca2+ imaging data. ARPE-19 and human embryonic stem cell-derived RPE cells (hESC-RPE) were used as RPE cell models. In this thesis, it was shown that both ARPE-19 and hESC-RPE exhibit intercellular Ca2+ waves upon mechanical stimulation. With live-cell Ca2+ imaging and mathematical modeling, it was demonstrated that in ARPE-19 cells, the mechanically induced Ca2+ waves propagate intracellularly through gap junctions and extracellularly involving diffusion of a paracrine factor. By applying in-house image analysis tools for the experimental fluorescence time-series, it was found that in hESC-RPE cells, spontaneous [Ca2+]i transients and the ability to propagate intercellular Ca2+ waves upon mechanical stimulation strongly depend on the maturation status of the cells. Finally, it was demonstrated that wounding affects spontaneous Ca2+ activity close to the wound edges, and cells within the healed areas resemble Ca2+ dynamics of immature hESC-RPE. To conclude, this thesis has provided important insights into human RPE Ca2+ dynamics, as well as into the events of single cell mechanical stimulation and large scale monolayer wounding. In addition, it was demonstrated that maturation drastically affects RPE Ca2+ dynamics. This knowledge and the developed image analysis algorithms contribute to understanding RPE physiology and can facilitate establishment of novel tools for assessment of RPE functionality prior to transplantation and in drug testing assays.
BACKGROUND: Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls) is an international consortium of 62 centres in 20 countries evaluating the use of targeted PCa screening in men with BRCA1/2 mutations. OBJECTIVE: To report the first year's screening results for all men at enrollment in the study. DESIGN, SETTING AND PARTICIPANTS: We recruited men aged 40-69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrollment, and those men with PSA >3 ng/ml were offered prostate biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. RESULTS AND LIMITATIONS: We recruited 2481 men (791 BRCA1 carriers, 531 BRCA1 controls; 731 BRCA2 carriers, 428 BRCA2 controls). A total of 199 men (8%) presented with PSA >3.0 ng/ml, 162 biopsies were performed, and 59 PCas were diagnosed (18 BRCA1 carriers, 10 BRCA1 controls; 24 BRCA2 carriers, 7 BRCA2 controls); 66% of the tumours were classified as intermediate- or high-risk disease. The positive predictive value (PPV) for biopsy using a PSA threshold of 3.0 ng/ml in BRCA2 mutation carriers was 48%-double the PPV reported in population screening studies. A significant difference in detecting intermediate- or high-risk disease was observed in BRCA2 carriers. Ninety-five percent of the men were white, thus the results cannot be generalised to all ethnic groups. CONCLUSIONS: The IMPACT screening network will be useful for targeted PCa screening studies in men with germline genetic risk variants as they are discovered. These preliminary results support the use of targeted PSA screening based on BRCA genotype and show that this screening yields a high proportion of aggressive disease. PATIENT SUMMARY: In this report, we demonstrate that germline genetic markers can be used to identify men at higher risk of prostate cancer. Targeting screening at these men resulted in the identification of tumours that were more likely to require treatment
The large number of complete mitochondrial DNA (mtDNA) sequences available for metazoan species makes it a good system for studying genome diversity, although little is known about the mechanisms that promote and/or are correlated with the evolution of this organellar genome. By investigating the molecular evolutionary history of the catalytic and accessory subunits of the mtDNA polymerase, pol γ, we sought to develop mechanistic insight into its function that might impact genome structure by exploring the relationships between DNA replication and animal mitochondrial genome diversity. We identified three evolutionary patterns among metazoan pol γs. First, a trend toward stabilization of both sequence and structure occurred in vertebrates, with both subunits evolving distinctly from those of other animal groups, and acquiring at least four novel structural elements, the most important of which is the HLH-3β (helix-loop-helix, 3 β-sheets) domain that allows the accessory subunit to homodimerize. Second, both subunits of arthropods and tunicates have become shorter and evolved approximately twice as rapidly as their vertebrate homologs. And third, nematodes have lost the gene for the accessory subunit, which was accompanied by the loss of its interacting domain in the catalytic subunit of pol γ, and they show the highest rate of molecular evolution among all animal taxa. These findings correlate well with the mtDNA genomic features of each group described above, and with their modes of DNA replication, although a substantive amount of biochemical work is needed to draw conclusive links regarding the latter. Describing the parallels between evolution of pol γ and metazoan mtDNA architecture may also help in understanding the processes that lead to mitochondrial dysfunction and to human disease-related phenotypes.
Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10−4 to 102 ng/mL)
BACKGROUND: Many men with elevated prostate-specific antigen (PSA) levels in serum do not have aggressive prostate cancer and undergo unnecessary biopsy. Retrospective studies using cryopreserved serum suggest that four kallikrein markers can predict biopsy outcome. METHODS: Free, intact and total PSA, and kallikrein-related peptidase 2 were measured in cryopreserved blood from 6129 men with elevated PSA (≥3.0ng/mL) participating in the prospective, randomized trial Prostate Testing for Cancer and Treatment. Marker levels from 4765 men providing anticoagulated plasma were incorporated into statistical models to predict any-grade and high-grade (Gleason score ≥7) prostate cancer at 10-core biopsy. The models were corrected for optimism by 10-fold cross validation and independently validated using markers measured in serum from 1364 men. All statistical tests were two-sided. RESULTS: The four kallikreins enhanced prostate cancer detection compared with PSA and age alone. Area under the curve (AUC) for the four kallikreins was 0.719 (95% confidence interval [CI] = 0.704 to 0.734) vs 0.634 (95% CI = 0.617 to 0.651, P < .001) for PSA and age alone for any-grade cancer, and 0.820 (95% CI = 0.802 to 0.838) vs 0.738 (95% CI = 0.716 to 0.761) for high-grade cancer. Using a 6% risk of high-grade cancer as an illustrative cutoff, for 1000 biopsied men with PSA levels of 3.0ng/mL or higher, the model would reduce the need for biopsy in 428 men, detect 119 high-grade cancers, and delay diagnosis of 14 of 133 high-grade cancers. Models exhibited excellent discrimination on independent validation among men with only serum samples available for analysis. CONCLUSIONS: A statistical model based on kallikrein markers was validated in a large prospective study and reduces unnecessary biopsies while delaying diagnosis of high-grade cancers in few men.
Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values.
BACKGROUND: The effect of prostate-specific antigen (PSA) screening on prostate cancer mortality remains debated, despite evidence from randomized trials. We investigated the association between prostate cancer incidence, reflecting uptake of PSA testing, and prostate cancer mortality. METHODS: The study population consisted of all men aged 50 to 74 years residing in eight counties in Sweden with an early increase in prostate cancer incidence and six counties with a late increase during two time periods. Incidence of metastatic prostate cancer was investigated in the period from 2000 to 2009, and prostate cancer-specific mortality and excess mortality were investigated in the period from 1990 to 1999 and the period from 2000 to 2009 by calculating rate ratios for high- vs low-incidence counties and rate ratios for the period from 2000 to 2009 vs the period from 1990 to 1999 within these two groups. All statistical tests were two-sided. RESULTS: There were 4528134 person-years at risk, 1577 deaths from prostate cancer, and 1210 excess deaths in men with prostate cancer in high-incidence counties and 2471373 person-years at risk, 985 prostate cancer deaths, and 878 excess deaths in low-incidence counties in the period from 2000 to 2009. Rate ratios in counties with high vs low incidence adjusted for time period were 0.81 (95% confidence interval [CI] = 0.73 to 0.90) for prostate cancer- specific mortality and 0.74 (95% CI = 0.64 to 0.86) for excess mortality, and the rate ratio of metastatic prostate cancer was 0.85 (95% CI = 0.79 to 0.92). CONCLUSIONS: The lower prostate cancer mortality in high-incidence counties reflecting a high PSA uptake suggests that more-intense as compared with less-intense opportunistic PSA screening reduces prostate cancer mortality.
BACKGROUND: Odontogenic tumors such as ameloblastic fibro-odontoma (AFO) are rare conditions in children and are often asymptomatic. AFOs are found by routine clinical and radiological examination or when they cause obvious intra- or extra-oral swelling. MATERIALS AND METHODS: A case of an AFO in a 7-year-old girl is described, and 107 cases from the literature and this report are analyzed. RESULTS: The total of 108 cases revealed the average age at presentation of AFO to be 6.3 years in boys and 9.6 years in girls. There was a slight male predilection and AFO lesions most often occurred in the posterior mandible. AFO was almost always associated with an unerupted tooth or teeth. CONCLUSIONS: While the recurrence rate of AFO was found to be 5.5%, long-term postoperative clinical and radiological follow-up is advised to ensure no future signs of aggressive recurrence.
Background The rapid advancement of high-throughput tools for quantitative measurement of proteins has demonstrated the potential for the identification of proteins associated with cancer. However, the quantitative results on cancer tissue specimens are usually confounded by tissue heterogeneity, e.g. regions with cancer usually have significantly higher epithelium content yet lower stromal content. Objective It is therefore necessary to develop a tool to facilitate the interpretation of the results of protein measurements in tissue specimens. Methods Epithelial cell adhesion molecule (EpCAM) and cathepsin L (CTSL) are two epithelial proteins whose expressions in normal and tumorous prostate tissues were confirmed by measuring staining intensity with immunohistochemical staining (IHC). The expressions of these proteins were measured by ELISA in protein extracts from OCT embedded frozen prostate tissues. To eliminate the influence of tissue heterogeneity on epithelial protein quantification measured by ELISA, a color-based segmentation method was developed in-house for estimation of epithelium content using H&E histology slides from the same prostate tissues and the estimated epithelium percentage was used to normalize the ELISA results. The epithelium contents of the same slides were also estimated by a pathologist and used to normalize the ELISA results. The computer based results were compared with the pathologist’s reading. Results We found that both EpCAM and CTSL levels, measured by ELISA assays itself, were greatly affected by epithelium content in the tissue specimens. Without adjusting for epithelium percentage, both EpCAM and CTSL levels appeared significantly higher in tumor tissues than normal tissues with a p value less than 0.001. However, after normalization by the epithelium percentage, ELISA measurements of both EpCAM and CTSL were in agreement with IHC staining results, showing a significant increase only in EpCAM with no difference in CTSL expression in cancer tissues. These results were obtained with normalization by both the computer estimated and pathologist estimated epithelium percentage. Conclusions Our results show that estimation of tissue epithelium percentage using our color-based segmentation method correlates well with pathologists' estimation of tissue epithelium percentages. The epithelium contents estimated by color-based segmentation may be useful in immuno-based analysis or clinical proteomic analysis of tumor proteins. The codes used for epithelium estimation as well as the micrographs with estimated epithelium content are available online.
The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. Here we have studied this response in detail, and found that Toll signaling in several different tissues can activate a cellular immune defense, and that this response does not require Toll signaling in the blood cells themselves. Like in the humoral immune response, we show that Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen.
Perinteiset luuvaurioiden hoitomenetelmät ovat usein riittämättömiä kriittisen koon luuvaurioiden hoidoissa. Luun kudosteknologia pystyisi tarjoamaan tähän toimivia ja tehokkaita ratkaisuja hyödyntäen kehon omaa parantumiskykyä. Luun kudosteknologiassa käytettäviltä materiaaleilta vaaditaan mekaanista kestävyyttä johtuen luukudoksen tärkeästä roolista kehon tukirankana. Laktidipohjaiset polymeerit ovat yleisesti käytettyjä polymeereja luun kudosteknologiassa, sillä ne ovat mekaanisesti kestäviä, niiden hajoamista kehossa on helppo hallita ja ne on todettu erittäin kudosyhteensopiviksi polymeereiksi. Laktidipohjaiset polymeerit eivät kuitenkaan ole bioaktiivisia ja lisäksi niiden hydrofobisuus haittaa solujen ja kudoksen kiinnittymistä materiaalin pintaan. Polypyrroli(PPy)-pinnoitus on potentiaalinen menetelmä edellä mainittujen ongelmien välttämiseksi, koska sillä on edulliset pintaominaisuudet solujen kiinnittymiselle ja lisäksi siihen voidaan sisällyttää erilaisia bioaktiivisia molekyylejä. PPy on myös redox-aktiivinen, minkä vuoksi sen käyttö sähkövirtaa välittävänä biomateriaalina avaa mielenkiintoisia mahdollisuuksia osteogeenisten solujen stimuloinnissa. PPy:n on todettu olevan potentiaalinen substraatti osteogeenisille soluille, kuten mesenkymaalisille kantasoluille. Mesenkymaalisiin kantasoluihin lukeutuvat rasvan kantasolut ovat yksi potentiaalisimmista solutyypeistä niiden runsaslukuisuuden ja helpon saatavuuden ansiosta. Niitä ei ole kuitenkaan vielä tutkittu PPy:n ja sähköstimulaation yhdistelmässä. Tässä työssä arvioitiin PPy:n mahdollisuudet ihmisen rasvan kantasolujen substraattina luun kudosteknologiassa in vitro ja sekä luun uudismuodostumista edistävänä implantin pinnoitemateriaalina in vivo. Tutkimuksen ensimmäisessä osassa sähkökemiallisesti valmistettu PPy seostettiin kondroitiinisulfaatilla tai hyaluronihapolla ja tutkittiin sen soveltuvuutta pinnoitemateriaaliksi ihmisen rasvan kantasoluille. Tutkimuksen toisessa osassa PPy valmistettiin kemiallisesti kondroitiinisulfaatin läsnäollessa ja tutkittiin sen ominaisuuksia sähköäjohtavana pinnoitteena polylaktidista (PLA) valmistetuissa kuiturakenteissa. Kummassakin in vitro-osatyössä sähköstimulaatiota syötettiin PPy substraattien läpi. Soluvastetta tutkittiin solujen morfologian, elinkyvyn, proliferaation ja luuerilaistumisen kannalta. Tutkimuksen in vivo-osassa luun korjaukseen tarkoitetut kemiallisesti, kondoitiinisulfaatin läsnäollessa pinnoitetut biohajoavat poly(laktidi-co-glykolidi)- β-trikalsiumfosfaatti (PLGA-β-TCP) komposiittiruuvit implantoitiin kanin luukudokseen ja niiden kudosyhteensopivuutta ja vaikutusta uudisluun muodostukseen tutkittiin. Kondroitiinisulfaattiseostettujen PPy-pinnoitteiden (PPy-CS) päällä olevat solut osoittautuivat morfologialtaan hyviksi ja levittäytyivät tasaisesti pitkin pintaa. Sen sijaan hyaluronihapposeostettujen PPy-pinnoitteiden (PPy-HA) päällä olevat solut olivat aggregoituneet ja osittain irrottautuneet pinnasta. Tämä johtui todennäköisimmin PPy-HA-pinnoitteen merkittävästi suuremmasta pintakarheudesta. Solun ulkoisen tilan mineralisaatio oli merkittävästi suurempaa sähköstimuloiduissa PPy-CS-pinnoitteen päällä olevissa soluissa verrattuna vastaavassa ryhmässä olevaan PPy-HA-pinnoitteeseen. Ihmisen rasvan kantasolujen proliferaatio oli merkittävästi suurempaa PPy-pinnoitetuissa PLA kuiturakenteissa verrattuna vastaaviin pinnoittamattomiin rakenteisiin. Sähköstimulaatiolla ei todettu olevan vaikutusta soluihin kummassakaan in vitro-tutkimuksessa verrattuna stimuloimattomiin kontrolleihin. Kovakudoshistologian, digitaalisen röntgenkuvauksen ja tetrasykliinileimauksen perusteella PPy-pinnoitetut biohajoavat ruuvit edistivät merkittävästi uudisluun muodostusta verrattuna vastaaviin pinnoittamattomiin ruuveihin. Lisäksi mitään merkkejä akuuttisesta, systemaattisesta tai kroonisesta toksisuudesta ei havaittu yhdessäkään kanista. Tutkimuksen johtopäätös on, että sähkökemiallisesti valmistettu PPy-CS ja kemiallisesti kondoitiinisulfaatin läsnäollessa valmistettu PPy-pinnoite osoittautuivat sopiviksi substraateiksi ihmisen rasvan kantasoluille niiden morphologian, proliferaation ja luuerilaistumisen perusteella. Tämä osoittaa niiden suuren potentiaalin luun kudosteknologiassa. Kemiallisesti tuotettu PPy-pinnoite todettiin potentiaaliseksi luuimplanttien pinnoitemateriaaliksi myös PLGA-β-TCP-ruuveissa edistäen merkittävästi luun uudismuodostusta in vivo.
Background and aims: Prostate cancer is the most common cancer among men worldwide, and the molecular mechanisms involved in the disease still remain poorly understood. Prostate cancer is commonly associated with genomic copy number alterations that have helped to identify cancer associated target genes. 9p13.3 chromosomal region is recurrently amplified in prostate cancer and associated with the aggressive form of the disease, higher PSA-level, and poor progression-free survival in prostatectomy treated patients. 9p13.3 region may harbor one or more novel oncogenes. NOL6, C9orf23, FANCG, and STOML2 are possible 9p13.3 amplicon target genes that exhibit elevated gene expression in prostate cancer. Our aim was to investigate whether NFX1 and KIF24 genes are also part of this group. The possible effects of target gene silencing on cancer cell proliferation and migration were also studied. Methods: NFX1 and KIF24 gene expression was studied with RT-qPCR in cancer cell lines xenografts with different copy number status. Putative target genes were silenced by trans-fecting cancer cell lines with target gene specific siRNAs. Cancer cell proliferation was as-sessed by AlamarBlue assay and by microscopy and digital image analysis. The effects on cell migration were studied with wound healing assay and digital image analysis. Results: KIF24 exhibited elevated gene expression correlating with increased copy number of 9p13.3 and was therefore selected as putative target gene of the amplicon. The other genes outlined for our study were C9orf23, FANCG, NOL6, and STOML2. Silencing C9orf23 and NOL6 had a negative effect on cancer cell proliferation and migration. On the other hand, FANCG and STOML2 gene silencing had positive effect on cancer cell proliferation. Conclusion: C9orf23 and NOL6 remain as putative target genes of 9p13.3 amplicon. Further investigations of these genes should be carried out in order to conclude their function and contribution to prostate cancer. FANCG and STOML2 genes remain as possible tumor sup-pressors, and their contribution to cancer needs further clarification. Confirmation of the 9p13.3 amplicon target gene may provide new prognostic or diagnostic marker for prostate cancer in the future.
Abstract Objective Computational models of calcium (Ca2+) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer. Methods We combined six essential Ca2+ signaling components into a model: stretch-sensitive Ca2+ channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked. Results Our model was able to explain Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance. Conclusions The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.
The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.
ABSTRACT Background and Aims: Mast cells (MCs) are tissue-dwelling effector cells of innate and adaptive immunity that differentiate in peripheral tissues from committed circulating progenitor cells of bone marrow origin. Human MCs are conventionally classified into two major subtypes based on their neutral protease content, namely the MCT, which contain only tryptase and the MCTC, which contain both tryptase and chymase, as well as carboxypeptidase A3 and cathepsin G. Granzyme B has been identified in cultured human MCs and in MCs of human skin. In vitro studies have helped trace factors that regulate human MC development and have provided a powerful method for studying neutral protease expression during MC development. However, inconsistent information from these studies has made it impossible to establish a consistent concept of what causes MCs to develop into MCT and MCTC: are they two committed subtypes deriving from two distinct progenitors with irreversibly predetermined protease phenotype, or are they functional states that MCs assume under the influence of the local microenvironment? Previous findings of human tissue MCs further imply that the phenotypic heterogeneity of human MCs may be greater than initially suggested. However, an understanding of the complexity of the phenotypic heterogeneity of human MCs, and to what extent this heterogeneity is intrinsically variable, has been hindered. This is because virtually all studies describing the relationship of human MC development and protease expression have focused on the expression of tryptase and/or chymase, whereas the expressions of carboxypeptidase A3, cathepsin G, and granzyme B by human MCs have not been fully considered. The aim of the present Master's thesis was to clarify the contradictions on human MC phenotypes and their development by studying how MCs derived from their circulating progenitors express the various neutral proteases during their development. Methods: MCs were generated from human peripheral blood-derived CD34+ progenitors under the influence of Kit ligand and sequentially added cytokines according to a previously published method. The protease expressions of tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B were investigated on a weekly basis during MC development by flow cytometry and quantitative PCR. Furthermore, immunostainings of the proteases were performed for immunofluorescence microscopy. Results: All investigated proteases were detected in the developing MCs at week 1 of culture and were increasingly expressed beyond that time. By the end of week 6, a single homogeneous population of cells expressing all the investigated proteases was observed. Conclusion: The data of the present study suggest that human MCs derive from a common circulating progenitor cell, which has the potential to express the full complement of the investigated proteases. Thus, the heterogeneity of human MC protease phenotypes reflects microenvironmental regulation of protease expression, rather than the existence of distinct progenitor cells with precommitted protease phenotypes. TIIVISTELMÄ Viljeltyjen humaanisyöttösolujen proteaasi-ilmiasun karakterisointi syöttösolujen erilaistumisen ja kypsymisen aikana Tausta ja Tavoitteet: Syöttösolut ovat ihmisen synnynnäisen ja hankitun immuniteetin keskeisiä toimijasoluja, jotka erilaistuvat kudoksissa luuytimestä peräisin olevista, verenkierrossa kiertävistä esiastesoluista. Ihmisen syöttösolut jaetaan perinteisesti kahteen alatyyppiin niiden sisältämien neutraaliproteaasien perusteella. Nämä ovat MCT syöttösolu, joka sisältää ainoastaan tryptaasia ja MCTC syöttösolu, joka sisältää tryptaasia ja kymaasia. Jälkimmäiseen alatyyppiin kuuluvat syöttösolut sisältävät myös karboksipeptidaasi A3:a ja katepsiini G:tä. Lisäksi ihmisen viljellyt ja ihosta eristetyt syöttösolut ilmentävät grantsyymi B:tä. Ihmisen syöttösolujen erilaistaminen viljelyolosuhteissa on mahdollistanut neutraaliproteaasien ilmentymisen tutkimisen syöttösolujen erilaistumisen ja kasvun aikana. Tästä huolimatta on epäselvää, ovatko MCT ja MCTC peräisin kahdesta eri kehityslinjasta vai ovatko ne ennemminkin syöttösolun toiminnallisia tiloja, jotka määräytyvät ympärillä vallitsevien olosuhteiden mukaan. Viime vuosien löydökset viittaavat lisäksi siihen, että ihmisen syöttösolujen proteaasi-ilmiasujen kirjo on luultua monimuotoisempaa. Se, kuinka kirjavaa tämä monimuotoisuus on, ja missä määrin se on synnynnäisesti vaihtelevaa, on kuitenkin epäselvää. Tämä johtuu pääosin siitä, että valtaosa tutkimuksista on keskittynyt vain tryptaasin ja/tai kymaasin ilmentymiseen, kun taas muiden neutraaliproteaasien ilmentymistä ei ole juurikaan tutkittu. Tässä tutkimuksessa seurattiin kaikkien tunnettujen ihmisen syöttösoluproteaasien ilmentymistä viljeltyjen syöttösolujen erilaistumisen ja kasvun aikana. Tavoitteena oli selventää nykyistä käsitystä ihmisen syöttösolujen proteaasi-ilmiasujen monimuotoisuudesta ja niistä tekijöistä, jotka ovat tämän monimuotoisuuden takana. Menetelmät: Syöttösolut erilaistettiin viljelemällä verenkierrosta eristettyjä CD34+ esiastesoluja tärkeimmän syöttösolujen kasvua edistävän kasvutekijän, Kit ligandin ja jaksoittain lisättyjen sytokiinien, interleukiini (IL)-3:n, IL-9:n ja IL-6:n kanssa. Solujen erilaistumisen ja kasvun aikana tryptaasin, kymaasin, karboksipeptidaasi A3:n, katepsiini G:n ja grantsyymi B:n ilmentymistä tutkittiin viikoittain kvantitatiivisella PCR:lla ja virtaussytometrialla. Lisäksi tehtiin immunovärjäyksiä mikroskopointia varten. Tulokset: Kaikki tutkitut proteaasit tunnistettiin syöttösolujen kehittymisen aikana sekä mRNA että proteiinitasolla jo ensimmäisen viljelyviikon jälkeen. Proteaasien ilmentymistasot nousivat viljelyn edetessä, ja kuuden viikon jälkeen kaikki syöttösolut ilmensivät tutkittuja proteaaseja. Yhdenkään proteaasin kohdalla ei havaittu erillisiä proteaasipositiivisia tai proteaasinegatiivisia alapopulaatioita, vaan jokaisessa tutkitussa aikapisteessä havaittiin yksi syöttösolupopulaatio, joka ilmensi vaihtelevalla tasolla tutkittua proteaasia. Yhteenveto: Tämän tutkimuksen tulokset viittaavat siihen, että ihmisen syöttösolut ovat peräsin yhteisestä esiastesolusta, joka pystyy ilmentämään kaikkia tutkittuja neutraaliporteaaseja. Syöttösolujen proteaasi-ilmiasujen heterogeenisyys on seurausta paikallisessa kudosympäristössä olevista tekijöistä, kuten sytokiineista, joilla on kyky säädellä proteaasien ilmentymistasoja.
This thesis work builds on the recent discovery by Stergachis and his colleagues, which describes the process the genome uses to write genetic code. This work extends the previous vision in genome research, which states, that codons and regulatory elements work independently. Codons are a triplet of nucleotides that encode amino acids; and regulatory elements are responsible for regulation of the gene expression. However, as discovered by Stergachis and his colleagues in 2013, around 15% of codons within 85% of human genes are occupied by transcription factor binding sites (TFBSs) (see Stergachis et al., 2013). Consequently, these type of codons encode two types of information. They were labelled ‘duons’ and described as highly conserved entities with low levels of genetic variation. Overall, regulatory proteins bind to the same stretches of As, Cs, Ts and Gs and influence the process of gene expression, and also specify the amino acids of the protein that is made. This work applies Stergachis findings of ‘duons’ to analyse a variant data. An interesting fact of Stregachis work is that a mutation may occur without affecting a protein. This happens due to the ability of some amino acids to be encoded by a multiple combination of nucleotides (codons). Obviously, if an alteration occurs in the codon, which still encodes for the same amino acid, the functionality of the produced protein remains the same. In this case, transcription factors (TFs) bind to an altered (mutated) region, implicating a change of activity of TFs due to the fact, that the genetic pattern has been modified. As a result, wrong instructions are given to the expression of a gene, as Stergachis and his colleagues discovered (Stergachis et al., 2013). His discoveries led to the finding, that 13% of the deoxyribonucleic acid (DNA) mutations leading to a disease development are located in ‘duons’. Thus it is important to investigate disease-associated variants within ‘duons’ that increase the risk of disrupting both regulatory and protein-structural function. A finding by Kircher in 2014 - the application of a method that aimed at the interpretation of pathogenicity of human genetic variations – lead to a new method. This method developed by Kircher in 2014 is called the combined annotation dependent depletion (CADD) tool. It uses a single C score to annotate a variant as pathogenic. In contrast to other methods the CADD takes into consideration regulatory elements, thus the CADD tool was selected for this project work. These two research findings are used in the thesis work. The goal of this work was therefore the extraction and recording of variants from provided data, which have potential for ‘duons’. To achieve this goal, the thesis applied the techniques of the C score, the position weight matrix (PWMs), and p value estimation. The aim of this study was to apply the PWMs framework, and C score on provided data, in order to extract and record those variants from the data that have potential for’ duons’. Thus they could be putative causes of a disease development. First of all, the provided data was filtered to identify pathogenic variants based on C score. Afterwards, the above presented concept was used to compute the TFBSs for original reference and mutated nucleotide sequences, where the maximum and minimum difference between these scores were found and used as a criteria for computing p value. Eventually, the resulting set of genes was submitted to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database and analysed for correlation of mutations to the type of a disease. The outcome of the KEGG database analysis represents the main pathways where resulting genes are involved into metabolic, cancer, and neuroactive ligand-receptor interaction pathways.
Background and aims: Long QT syndrome (LQTS) is an inherent cardiac disorder causing severe arrhythmias due to mutations in cardiac ion channel genes. Four founder mutations causing LQTS have been identified from Finland and they disrupt functions of cardiac potassium ion channels. The aim of this study was to characterize expressions of ion channel genes or their allelic expression variation in three different experiments by using qPCR. In cell line characterization experiment was evaluated applicability of cell lysis -based genotyping method to characterize cardiac cell lines. The aim of the second experiment was to evaluate impacts of allelic expression differences on disease phenotype variation and development. The last experiment was used for examining capacity of single-cell qPCR method to detect specific gene expression within single cardiomyocytes. Methods: qPCR was used to detect mRNA levels of ion channel genes or structural genes within LQTS-specific cardiomyocytes. The TaqMan® Sample-to-SNP™ Kit was tested in order to characterize KCNQ1 mutated heterozygous cardiac cell lines. Allelic imbalance determination was performed using plasmid-derived standard curve method for each Finnish founder mutations. The Single cell-to-CT™ Kit was used to detect expression of TNNT2 at the single cell level and cell population level. Results: The used cell lysis -based genotyping method succeeded to characterize cardiac cell lines and any disruption was not detected during analyses. Allelic imbalance determination study revealed that KCNQ1-FinA and KCNQ1-FinB specific cardiomyocytes expressed alleles of KCNQ1 with allelic ratios 3:1 indicating that expression of wild type allele was three-fold as compared to expression of mutant allele within these LQT1-specific cells. The Single cell-to-CT™ Kit did not manage to detect gene expression of TNNT2 within single cell samples and its expression at population level was also reduced. Conclusions: The detected allelic ratio 3:1 (WT:MUT) within KCNQ1-FinA and KCNQ1-FinB mutated cardiomyocytes could result in milder phenotypic effects due to that higher expression of wild type alleles may suppress effects of mutations. Milder phenotypic effects have been thought to be reason for unique enrichment of founder mutations among Finnish population. Therefore, allelic imbalance occurring in disease-causing genes can be a significant disease phenotype modifier. To validate this hypothesis, larger study population of mutation carriers with high phenotypic variation is needed. If correlation between allelic imbalance and phenotypic variation is detected, the allelic expression variation might be confirmed to be one explaining genetic factor to affect phenotypic variation. TIIVISTELMÄ: qPCR-menetelmän hyödyntäminen indusoiduista pluripotenteista kantasoluista erilaistettujen LQTS-spesifisten sydänlihassolujen karakterisoinnissa Tutkimuksen tausta ja tavoitteet: Pitkä QT oireyhtymä on vakavia rytmihäiriöitä aiheuttava perinnöllinen sairaus, joka johtuu mutaatioista sydänlihassolun ionikanavageeneissä. Suomessa vallitsee neljä pitkä QT oireyhtymää aiheuttavaa perustajamutaatiota, jotka heikentävät sydänlihassolujen kalium-ionikanavien toimintaa. Tämän tutkimuksen tavoitteena oli karakterisoida LQTS-spesifisten sydänlihassolujen ionikanavageenien ilmentymistä tai niiden alleeli-ilmentymiseroja hyödyntämällä qPCR-menetelmää kolmessa erillisessä työssä. Ensimmäisessä työssä arvioitiin solulyysaukseen perustuvan genotyyppausmenetelmän soveltuvuutta sydänsolulinjojen karakterisointiin. Toisen työn tavoitteena oli arvioida alleeli-ilmentymiserojen vaikutuksia tautifenotyypin vaihteluun ja kehittymiseen. Kolmannen työn tarkoituksena oli tutkia yksisolu-qPCR-menetelmän tehokkuutta havainnoida yksittäisten sydänlihassolujen geeni-ilmentymistä. Menetelmät: qPCR-menetelmää käytettiin jokaisessa työssä mittaamaan ionikanavageenien tai rakennegeenien mRNA-tasoja LQTS-spesifisistä sydänlihassoluista. Ensimmäisessä työssä käytettiin TaqMan® Sample-to-SNP™ Kit -menetelmää karakterisoimaan heterotsygoottisia sydänsolulinjoja, joissa oli KCNQ1-geenin mutaatio. Alleeli-ilmentymisvaihteluun perustuva tutkimus suoritettiin plasmideista tuotetun standardikuvaajan avulla jokaiselle Suomessa vallitsevalle perustajamutaatiolle. Viimeisessä työssä käytettiin Single cell-to-CT™ Kit -menetelmää, jonka avulla mitattiin TNNT2-geenin ilmentymistä sekä yksittäissolu- että solupopulaatiotasolla. Tulokset: Ensimmäisen työn tuloksista pääteltiin, että solulyysaukseen perustuva genotyyppausmenetelmä soveltuu sydänsolulinjojen karakterisointiin, eikä analyysiä häiritseviä tekijöitä havaittu. Alleeli-ilmentymiseen perustuva tutkimus paljasti, että KCNQ1-FinA ja KCNQ1-FinB spesifisissä sydänlihassoluissa KCNQ1-geenin alleelit ilmentyivät 3:1-suhteessa, joka tarkoitti sitä, että villityyppialleelien ilmentyminen oli kolminkertainen verrattuna mutanttialleelien ilmentymiseen näissä LQT1-spesifisissä soluissa. Single cell-to-CT™ Kit -menetelmällä ei onnistuttu havainnoimaan TNNT2-geenin ilmentymistä yksittäissolutasolla ja solupopulaatiotasolla sen ilmentyminen oli myös heikkoa. Johtopäätökset: Havaittu alleeli-ilmentymissuhde 3:1 (WT:MUT) KCNQ1-FinA ja KCNQ1-FinB mutatoituneissa sydänlihassoluissa voisi johtaa mutaatioiden heikentyneisiin fenotyyppivaikutuksiin, koska villityyppialleelin voimakkaampi ilmentyminen saattaa heikentää mutaatioiden vaikutuksia. Aiemmin on arveltu, että perustajamutaatioiden heikompi fenotyyppivaikutus on johtanut niiden yleistymiseen Suomessa. Tällöin taudinaiheuttajageenien alleelien ilmentymiserot saattavat vaikuttaa merkittävästi tautifenotyyppiin. Tämän varmistamiseksi on kuitenkin tutkittava alleeli-ilmentymiserot suuremmasta populaatiosta mutaationkantajia, joilla on havaittu suuri fenotyyppivaihtelu. Jos alleeli-imbalanssin ja fenotyypin välillä havaitaan korrelaatiota, voitaisiin alleeli-imbalanssi varmistaa yhdeksi fenotyyppivaihtelua selittäväksi geneettiseksi tekijäksi.
Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine
BACKGROUND: Germline loss-of-function mutations in PALB2 are known to confer a predisposition to breast cancer. However, the lifetime risk of breast cancer that is conferred by such mutations remains unknown. METHODS: We analyzed the risk of breast cancer among 362 members of 154 families who had deleterious truncating, splice, or deletion mutations in PALB2. The age-specific breast-cancer risk for mutation carriers was estimated with the use of a modified segregation-analysis approach that allowed for the effects of PALB2 genotype and residual familial aggregation. RESULTS: The risk of breast cancer for female PALB2 mutation carriers, as compared with the general population, was eight to nine times as high among those younger than 40 years of age, six to eight times as high among those 40 to 60 years of age, and five times as high among those older than 60 years of age. The estimated cumulative risk of breast cancer among female mutation carriers was 14% (95% confidence interval [CI], 9 to 20) by 50 years of age and 35% (95% CI, 26 to 46) by 70 years of age. Breast-cancer risk was also significantly influenced by birth cohort (P<0.001) and by other familial factors (P=0.04). The absolute breast-cancer risk for PALB2 female mutation carriers by 70 years of age ranged from 33% (95% CI, 25 to 44) for those with no family history of breast cancer to 58% (95% CI, 50 to 66) for those with two or more first-degree relatives with breast cancer at 50 years of age. CONCLUSIONS: Loss-of-function mutations in PALB2 are an important cause of hereditary breast cancer, with respect both to the frequency of cancer-predisposing mutations and to the risk associated with them. Our data suggest the breast-cancer risk for PALB2 mutation carriers may overlap with that for BRCA2 mutation carriers. (Funded by the European Research Council and others.).
AIMS: A fast, non-invasive and observer-independent method to analyze the homogeneity and maturity of human pluripotent stem cell (hPSC) derived retinal pigment epithelial (RPE) cells is warranted to assess the suitability of hPSC-RPE cells for implantation or in vitro use. The aim of this work was to develop and validate methods to create ensembles of state-of-the-art texture descriptors and to provide a robust classification tool to separate three different maturation stages of RPE cells by using phase contrast microscopy images. The same methods were also validated on a wide variety of biological image classification problems, such as histological or virus image classification. METHODS: For image classification we used different texture descriptors, descriptor ensembles and preprocessing techniques. Also, three new methods were tested. The first approach was an ensemble of preprocessing methods, to create an additional set of images. The second was the region-based approach, where saliency detection and wavelet decomposition divide each image in two different regions, from which features were extracted through different descriptors. The third method was an ensemble of Binarized Statistical Image Features, based on different sizes and thresholds. A Support Vector Machine (SVM) was trained for each descriptor histogram and the set of SVMs combined by sum rule. The accuracy of the computer vision tool was verified in classifying the hPSC-RPE cell maturation level. DATASET AND RESULTS: The RPE dataset contains 1862 subwindows from 195 phase contrast images. The final descriptor ensemble outperformed the most recent stand-alone texture descriptors, obtaining, for the RPE dataset, an area under ROC curve (AUC) of 86.49% with the 10-fold cross validation and 91.98% with the leave-one-image-out protocol. The generality of the three proposed approaches was ascertained with 10 more biological image datasets, obtaining an average AUC greater than 97%. CONCLUSIONS: Here we showed that the developed ensembles of texture descriptors are able to classify the RPE cell maturation stage. Moreover, we proved that preprocessing and region-based decomposition improves many descriptors' accuracy in biological dataset classification. Finally, we built the first public dataset of stem cell-derived RPE cells, which is publicly available to the scientific community for classification studies. The proposed tool is available at https://www.dei.unipd.it/node/2357 and the RPE dataset at http://www.biomeditech.fi/data/RPE_dataset/. Both are available at https://figshare.com/s/d6fb591f1beb4f8efa6f.
According to international human genome sequencing consortium 2004[43], it was known that only less than 2% of the total human genome code for proteins. This ignited quite a surprise in the scientific community. Since then, a lot of researchers are attracted towards the noncoding part of the genome. There are explosion of researches addressing the role of the 98% of the human untranslated regions of the genome. This shows that the transcription is not only limited to the protein coding regions of the genome rather more than 90% of the genome are likely to be transcribed. [43] This will result in the transcription of tens and thousands of the long noncoding RNAs (lncRNAs) with little or no coding potential. However, the molecular mechanism and function of long noncoding RNAs are still an open research topic. Although the functions of limited lncRNAs are identified, there is still a gap in identifying the function of novel lncRNAs. This project implements different computational methods to predict the function of novel lncRNAs identified from TCGA glioblastoma multiforme samples. The methods used in this functional prediction include both expression and sequence-based analysis approach. In expression-based analysis, the co-expressing genes with lncRNAs are used to predict the possible functional relation. In sequence based analysis, the gene-protein and lncRNA-protein interactions together with miRNA-lncRNA interactions are considered towards the possible functional predictions. The result from the integrated functional prediction on the novel lncRNAs show that TCGA_gbm3-153501 novel lncRNA which is co-expressed together with the THBS1 gene with correlation coefficient of more that 0.5 is predicted to function in cell-cell and cell-to-matrix interactions, platelet aggregation, angiogenesis, and tumorigenesis. [202] MSI1, RBM3 and RBM8A are RNA binding proteins (RBPs) that have binding site on both the first top five differentially expressed lncRNAs which are TCGA_gbm-2-104096501, TCGA_gbm-3-153501, TCGA_gbm-5-63687001 and TCGA_gbm-17-10671251 and IGF2 which is among the top 10 differentially expressed genes. Therefore, these lncRNAs are predicted to have functional role in cell proliferation and maintenance of stem cells in the central nervous system.
Background It is generally acknowledged that a functional understanding of a biological system can only be obtained by an understanding of the collective of molecular interactions in form of biological networks. Protein networks are one particular network type of special importance, because proteins form the functional base units of every biological cell. On a mesoscopic level of protein networks, modules are of significant importance because these building blocks may be the next elementary functional level above individual proteins allowing to gain insight into fundamental organizational principles of biological cells. Results In this paper, we provide a comparative analysis of five popular and four novel module detection algorithms. We study these module prediction methods for simulated benchmark networks as well as 10 biological protein interaction networks (PINs). A particular focus of our analysis is placed on the biological meaning of the predicted modules by utilizing the Gene Ontology (GO) database as gold standard for the definition of biological processes. Furthermore, we investigate the robustness of the results by perturbing the PINs simulating in this way our incomplete knowledge of protein networks. Conclusions Overall, our study reveals that there is a large heterogeneity among the different module prediction algorithms if one zooms-in the biological level of biological processes in the form of GO terms and all methods are severely affected by a slight perturbation of the networks. However, we also find pathways that are enriched in multiple modules, which could provide important information about the hierarchical organization of the system.
Biotinylated bait molecules can be immobilized on biotinylated sensor chips by formation of biotin–avidin–biotin bridges which are very stable when using wild-type (strept)avidin. Stable immobilization of biotinylated baits is important for monitoring reversible binding and dissociation of prey molecules. For measurements with another bait molecule, however, it is desirable to replace all immobilized proteins by fresh (strept)avidin and new biotinylated bait. In this study, five avidin mutants have been characterized with respect to their ability to form switchable biotin–avidin–biotin bridges on biotinylated chip surfaces, as needed for complete chip regeneration. All five mutants formed stable biotin–avidin–biotin bridges at pH 7, were more or less stable at pH 2–3, and required the combination of pH 2 with SDS for quantitative removal from the chip surface. Mutant #3 (“switchavidin”) showed the best combination of properties, i.e., low nonspecific adsorption of protein and nucleic acids, high binding capacity, and good stability at pH 2–3, as typically used for quantitative removal of prey molecules in repeated measurement cycles.
Background and aims: Breast cancer is the second most common cancer in the world after lung cancer, and the most common cancer among women. The risk factors for breast cancer include different hormonal factors and consequently, estrogen has been found to promote breast cancer pathogenesis. Bone morphogenetic proteins (BMP) are a group of growth factors that have been connected to different cancers, including breast cancer. BMPs have been noted to have different effects on breast cancer cells, decreasing proliferation in some, and in contrast, increasing migration and invasion in others. The estrogen and BMP signaling pathways have been shown to have a connection, and they can influence each other's functions. In this study, the objectives are to examine the effect of estrogen on BMP gene expression in six breast cancer cell lines, and to study the effects of estrogen and BMP4 on cell proliferation in two breast cancer cell lines. Methods: The breast cancer cell lines (estrogen receptor positive BT-474, MCF-7, MDA-MB-361, T-47D and ZR-75-30, and the estrogen receptor negative MDA-MB-231) were cultured in estrogen free medium for three days before treatment with 17ẞ-estradiol (E2, 100 nM) or vehicle control for 24 and 48 hours. The gene expression of BMP4 and BMP7, and the positive control genes GREB1 and TFF1 was assessed with qRT-PCR by using a SYBR Green I dye based assay. For examining the cell proliferation, BT-474 and T-47D cells were cultured for three days in estrogen free conditions, and treated with E2 (100 nM), human recombinant BMP4 (100 ng/ml), both, or vehicle control. The cell proliferation was followed by microscopy and imaging, and finally by counting the cells at six (T-47D) and seven (BT-474) days after the treatments. Results: The expression of positive control genes GREB1 and TFF1 increased after estrogen treatment in most estrogen receptor positive cell lines, as expected. Treatment with E2 had a variable effect on BMP4 and BMP7 expression depending on the cell line. BMP4 expression was notably decreased in BT-474, MCF-7 and MDA-MB-361 cell lines, but not in others. The expression of BMP7 was notably decreased in BT-474, T-47D and ZR-75-30, but again, there was no significant change in the others. The cell proliferation experiment showed that as expected, E2 increased cell growth both in BT-474 and T-47D cell line, and that BMP4 was able to inhibit the effect of E2 on cell growth. Conclusions: Estrogen has different effects on BMP4 and BMP7 expression depending on the cell line. In some it decreases BMP expression, and in others it has no significant effect. BMP4 was able to diminish the estrogen induced cell proliferation. This is an important finding that may have clinical applicability for the treatment of breast cancer patients.
Celiac disease (CD) is a complex disorder with no bona fide animal model that would exhibit all the features of the disorder. The small-intestinal mucosal biopsies commonly used to study the disease present a limiting factor, which is the survival of the specimen for only 24-48 hours. The goal of this project was to develop a system to support the biopsy using mice as carriers for a long-term culture and to subsequently use this system to test potential therapeutic compounds. We obtained biopsies from CD and control patients and implanted them under the skin of athymic mice for the duration of 8 days. To determine if certain key features of CD were maintained in the biopsy during the extended incubation time, we performed various immunofluorescent stains. We found abundant, well developed vessels in the control and reduced size and functionality in the CD vessels, consistent with the situation in actual patients. We also confirmed the presence of B-cells, and found evidence of IgA/transglutaminase 2 colocalization, both important features of the disease. To test the effectiveness of different compounds at ameliorating the decreased vascularization seen in CD samples, we applied statin class drugs, shown to increase angiogenesis, to HUVEC cells in the presence and absence of celiac disease-specific transglutaminase 2 targeting autoantibodies. We found that the compound α-amino-γ-butyrolactone hydrobromide had the ability to accelerate the growth of the HUVEC cells even in the inhibiting presence of the CD autoantibodies, while the drug Atorvastatin show variable and inconclusive results. We proceeded with the application of both of these compounds to mice implanted with CD and control biopsies. The samples from these final mouse experiments have yet to be analyzed but we feel confident that our organ culture model provides a valid option for extending the life of human biopsies as well as an opportunity to test novel compounds.
Tuberculosis (TB) is a global health emergency. Up to one-third of the world’s population is infected with Mycobacterium tuberculosis, and the pathogen continues to kill 1.5 million people annually. Currently, the means for preventing, diagnosing, and treating TB are unsatisfactory. One of the main reasons for the poor progress in TB research has been a lack of good animal models to study the latency, dormancy, and reactivation of the disease. Although sophisticated in vitro and in silico methods suitable for TB research are constantly being developed, they cannot reproduce the complete vertebrate immune system and its interplay with pathogens and vaccines. However, the zebrafish has recently emerged as a useful alternative to more traditional models, such as mice, rabbits, guinea pigs, and non-human primates, for studying the complex pathophysiology of a mycobacterial infection. The model is based on the similarity between Mycobacterium marinum – a natural fish pathogen – and M. tuberculosis. In both zebrafish larvae and adult fish, an infection with M. marinum leads to the formation of macrophage aggregates and granulomas, which resemble the M. tuberculosis infections in humans. In this review, we will summarize the current status of the zebrafish model in TB research and highlight the advantages of using zebrafish to dissect mycobacterial virulence strategies as well as the host immune responses elicited against them. In addition, we will discuss the possibilities of using the adult zebrafish model for studying latency, dormancy, and reactivation in a mycobacterial infection.
Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.
To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response.
In this paper, we study the problem of feature selection in cancer-related machine learning tasks. In particular, we study the accuracy and stability of different feature selection approaches within simplistic machine learning pipelines. Earlier studies have shown that for certain cases, the accuracy of detection can easily reach 100% given enough training data. Here, however, we concentrate on simplifying the classification models with and seek for feature selection approaches that are reliable even with extremely small sample sizes. We show that as much as 50% of features can be discarded without compromising the prediction accuracy. Moreover, we study the model selection problem among the ℓ1 regularization path of logistic regression classifiers. To this aim, we compare a more traditional cross-validation approach with a recently proposed Bayesian error estimator.
A straightforward solution-based method to modify the biofunctionality of stainless steel (SS) using heterobifunctional silane-polyethylene glycol (silane-PEG) overlayers is reported. Reduced nonspecific biofouling of both proteins and bacteria onto SS and further selective biofunctionalization of the modified surface were achieved. According to photoelectron spectroscopy analyses, the silane-PEGs formed less than 10 Å thick overlayers with close to 90% surface coverage and reproducible chemical compositions. Consequently, the surfaces also became more hydrophilic, and the observed non-specific biofouling of proteins was reduced by approximately 70%. In addition, the attachment of E. coli was reduced by more than 65%. Moreover, the potential of the overlayer to be further modified was demonstrated by successfully coupling biotinylated alkaline phosphatase (bAP) to a silane-PEG-biotin overlayer via avidin-biotin bridges. The activity of the immobilized enzyme was shown to be well preserved without compromising the achieved antifouling properties. Overall, the simple solution-based approach enables the tailoring of SS to enhance its activity for biomedical and biotechnological applications.
Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischaemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes that provide non-proton-translocating bypasses for respiratory complexes I (single-subunit reduced nicotinamide adenine dinucleotide dehydrogenases, e.g. Ndi1 from yeast) or III + IV [alternative oxidase (AOX)], under conditions of respiratory stress or overload. In previous studies, it was shown that transfer of yeast Ndi1 or Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complex I or IV. Here, we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype, which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits produced a distinct residual phenotype of delayed development, bang sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, while indicating important limitations thereof.
Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.
Circulating insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are associated with prostate cancer. Using genetic variants as instruments for IGF peptides, we investigated whether these associations are likely to be causal. We identified from the literature 56 single nucleotide polymorphisms (SNPs) in the IGF axis previously associated with biomarker levels (8 from a genome-wide association study [GWAS] and 48 in reported candidate genes). In ∼700 men without prostate cancer and two replication cohorts (N ∼ 900 and ∼9,000), we examined the properties of these SNPS as instrumental variables (IVs) for IGF-I, IGF-II, IGFBP-2 and IGFBP-3. Those confirmed as strong IVs were tested for association with prostate cancer risk, low (< 7) vs. high (≥ 7) Gleason grade, localised vs. advanced stage, and mortality, in 22,936 controls and 22,992 cases. IV analysis was used in an attempt to estimate the causal effect of circulating IGF peptides on prostate cancer. Published SNPs in the IGFBP1/IGFBP3 gene region, particularly rs11977526, were strong instruments for IGF-II and IGFBP-3, less so for IGF-I. Rs11977526 was associated with high (vs. low) Gleason grade (OR per IGF-II/IGFBP-3 level-raising allele 1.05; 95% CI: 1.00, 1.10). Using rs11977526 as an IV we estimated the causal effect of a one SD increase in IGF-II (∼265 ng/mL) on risk of high vs. low grade disease as 1.14 (95% CI: 1.00, 1.31). Because of the potential for pleiotropy of the genetic instruments, these findings can only causally implicate the IGF pathway in general, not any one specific biomarker.
BACKGROUND: The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study. METHODS: We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant. RESULTS: For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p=7.1×10-5), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p=6.9×10-8) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p=0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p≤0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p=0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p=0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants.
BACKGROUND: The harvesting of a tooth as a candidate for tooth autotransplantation requires that the delicate dental tissues around the tooth be minimally traumatized. This is especially so for the periradicular tissues of the tooth root and the follicular tissues surrounding the crown. The aim of this report is to describe the use of piezosurgery as an attempt at morbidity reduction in the harvesting of teeth for autotransplantation. METHODS: A piezosurgical handpiece and its selection of tips were easily adapted to allow the harvesting and delivery of teeth for autotransplantation purposes. RESULTS: Twenty premolar teeth were harvested using a piezosurgical device. The harvested teeth were subsequently successfully autotransplanted. All twenty teeth healed in a satisfactory manner without excessive mobility or ankyloses. CONCLUSIONS: Piezosurgery avoids some of the traumatic aspects of harvesting teeth and removing bone which are associated with thermal damage from the use of conventional rotary instruments or saws. Piezosurgery can be adapted to facilitate the predictable harvesting of teeth for autotransplantation purposes.
Background and aims: Typically, an X-ray image forms as the applied ray becomes attenuated by the imaged sample. In biological and biomaterial samples, the common imaging issue is the low density and its homogeneous distribution. Without externally supplied heavy elements, studied features may not be visible at all, or the micro-computational tomography (µCT) imaging may take unreasonable amount of time resources. The goal of this study was to test and optimize different background and specific contrast enhancement methods for overcoming these imaging issues. Materials and methods: The used background contrasting methods were based on iodine and phosphotungstic acid. Each staining method were iterated until satisfactory contrasting results were gained for examining the three dimensional anatomy of adult zebra fish. The ethanol based iodine was also used for studying the structural dissimilarities between dystrophic and healthy rat retinas. Stem cell monolayer samples were specifically stained by antibodies which were used for localizing metallic silver in the immediate proximity of the examined antigens. Results: During the research it was shown that all of the used contrasting methods were capable for enhancing the studied features in the treated samples. All of the background contrasting methods successfully enhanced the anatomical features of the adult zebra fish. Furthermore, the ethanol based iodine contrasting preserved the general morphology of the rat eyes and made possible for distinguishing clear structural dissimilarities between dystrophic and control retinas. The specific contrasting method enhanced successfully the target actin and lamin antigens in the examined stem cell monolayer samples. Conclusions: The used contrasting methods gave good and comprehensive base for the following research for examining wide variety of biological samples with µCT.
Nanobioteknologia ja bionanoteknologia ovat suhteellisen uusia, nopeasti kehittyviä tieteenaloja, jotka hyödyntävät biomolekyylejä. Biomolekyylien luonnolliset ominaisuudet ja toiminta täytyy tuntea hyvin, jotta niitä voidaan hyödyntää moderneissa teknisissä sovelluksissa. Biomolekyylien ominaisuuksia täytyy myös usein muokata joko geneettisesti tai kemiallisesti, jotta ne soveltuvat tiettyyn käyttökohteeseen. Tämän tutkimuksen tavoitteena oli karakterisoida ja muokata biomolekyylien ominaisuuksia, jotta niistä voitaisiin valmistaa molekyylityökaluja nanobioteknologian ja bionanoteknologian tarpeisiin. Avidiini-proteiinit valittiin muokattaviksi sen vuoksi, että niitä käytetään jo laajalti bioteknologian sovelluksissa, sillä ne sitovat poikkeuksellisen voimakkaasti pientä vitamiini-molekyyliä, D-biotiinia (Kd ~10-15 M). Tässä työssä muokattiin aiemmin kehitetyn kaksoisketjuavidiinin (dcAvd) toista ligandin sitoutumispaikkaa muodostamaan kovalentin sidoksen ligandin kanssa. Näin aikaansaatu uusi proteiini, dcAvd-Cys, kykenee sitoutumaan kahdenlaisten molekyylien, tioli-reaktiivisten sekä biotinyloitujen molekyylien kanssa samanaikaisesti, joten sitä voitaisiin hyödyntää molekyylien hallitussa immobilisoinnissa. Työssä määritettiin kahden avidiinin kiderakenteet suurella tarkkuudella. Rakenteen, biokemiallisen ja biofysikaalisen karakterisoinnin perusteella näiden proteiinien käyttäytyminen poikkeaa aiemmin tutkituista avidiineista. Tetrameerisen villityypin bradavidiinin C-terminaaliset aminohappotähteet vuorovaikuttavat viereisen monomeerin ligandinsitomistaskun kanssa, käyttäytyen kuten monomeerien välinen sisäinen ligandi. Tämä havainto johti bradavidiini-spesifisen Brad-tag:n kehittämiseen, ja Brad-tag:n osoitettiin olevan käyttökelpoinen affiniteettikahva (Kd ~2,5 × 10-5 M). Suurin osa tutkituista (strept)avidiineista on tetrameerisia tai dimeerisiä proteiineja, kun taas bradavidiini II:n oligomeerisuusaste osoittautui vaihtelevan ympäröivien olosuhteiden mukaan. Bradavidiini II:n heikkoa oligomerisoitumispyrkimystä voitaisiin hyödyntää fuusioproteiinien kehittämisessä. Avidiinien ohella tutkittiin kahta erilaista biomolekyylien muodostamaa kompleksia. Ensimmäiseksi hyödynnettiin deoksiribonukleiinihappojen (DNA) ominaisuuksia kehitettäessä määrätyn kokoinen, itsejärjestyvä DNA-rakenne (B–A–B-kompleksi). Kompleksi ohjattiin kullasta valmistettujen nanoelektrodien väliin dielektroforeesin ja tioli-kulta sidoksen avulla. Streptavidiinin avulla osoitettiin, että kompleksi voidaan funktionalisoida muilla molekyyleillä. B–A–B-kompleksin mitattu johtavuus oli lähes olematonta. Kehitetty kompleksi voisi toimia alustana, johon kyettäisiin tarkasti sijoittamaan muita molekyylielektroniikan komponentteja. Toiseksi hyödynnettiin kitosaanin ja DNA:n sähköstaattista vuorovaikutusta valmistettaessa kitosaani-DNA nanopartikkeleita geenien kuljettamiksi. Nanopartikkelit funktionalisoitiin fluoresoivilla leimoilla ja kohdentavilla peptideillä. Soluviljelyanalyysissä nanopartikkelit kulkeutuivat kohdesolujen sisään. Kokonaisuudessaan tässä työssä tuotettiin monipuolisia molekyylityökaluja, jotka perustuvat muokattuihin avidiineihin, itsejärjestäytyvään DNA-rakenteeseen ja kitosaani-DNA nanopartikkeleihin. Vaikka kehitettyjen työkalujen sovelluskohteet vaihtelevat biosensoripinnoista proteiinien tunnistamiseen ja molekyylitason elektroniikasta geenien kuljettamiseen, eri osatutkimuksissa käytettiin monia samoja menetelmiä ja materiaaleja. Tutkitut ja kehitetyt biomolekyylit lisäävät tietoa, jota tarvitaan parempien molekyylityökalujen valmistamisessa.