24 research outputs found
Extracellular vesicle glycosylations as novel biomarkers of urological cancers: Nanoparticle-aided glycovariant assay to detect vesicles for the early detection of cancer
Prostate and bladder cancer are common urological malignancies that are associated with major causes of morbidity and mortality worldwide. Current diagnosis options for prostate cancer (PCa) and bladder cancer (BlCa) suffer from a lack of sensitivity and specificity that lead to either over-diagnosis or missed cancers. Moreover, current methods are invasive and painful for patients. Therefore, there is an urgent need for the development of sensitive and specific assays involving non-invasive techniques.
The primary aim of this doctoral thesis was to explore possibilities to develop a simple assay for the detection of cancer-associated extracellular vesicles (EVs) directly from human urine without any extensive preprocessing. This project focused on the development of a europium chelate-doped nanoparticles (NPs)-aided immunocapture approach that uses protein and glycan-based markers and their potential combinations for the detection of urinary EVs of urological cancer patients. Several cancer-associated integrin markers in combination with a panel of lectin library were tested to find our best functional biomarkers and their corresponding potential assays. Then the functional biomarker combinations were characterized and validated using EVs-derived from cancer cell lines as well as urine of PCa and BlCa patients. The best biomarker assay, combining with integrin and lectin, ITGA3- UEA can significantly discriminate BlCa from PCa, benign, and healthy controls.
The results of this project emphasize the importance of systemically screening of lectin library in combination with different cancer-associated integrin markers for the advancement of an immunocapturing glycovariant assay to detect urinary EVs for the diagnosis of urological cancers from unprocessed samples. This assay concept could be used as an open platform for exploring glyco-isoform from the surface of EVs for novel biomarker discovery.Solunulkoisten vesikkeleiden glykosylaatiot urologisten syöpien uusina biomarkkereina
Eturauhas- ja virtsarakkosyöpä ovat yleisiä urologisia sairauksia, jotka aiheuttavat maailmanlaajuisesti merkittävää sairastuvuutta ja kuolleisuutta. Eturauhassyövän (PCa) ja virtsarakkosyövän (BlCa) nykyiset diagnosointivaihtoehdot kärsivät sekä herkkyyden että spesifisyyden puutteesta, mikä johtaa joko ylidiagnosointiin tai syöpien jäämiseen huomaamatta. Nykyiset menetelmät ovat invasiivisia ja kivuliaita potilaille, minkä takia on kiireellisesti kehitettävä herkkiä ja spesifisiä määrityksiä, joissa käytetään ei-invasiivisia tekniikoita.
Tämän väitöskirjan ensisijaisena tavoitteena oli tutkia mahdollisuuksia kehittää yksinkertainen immunomääritys syöpään liittyvien solunulkoisten vesikkeleiden (EV) havaitsemiseksi suoraan ihmisen virtsasta ilman mitään esikäsittelyä. Työssä keskityttiin kehittämään europiumkelaatteja sisältävien nanopartikkeleiden (NP:t) avustamaa immunokaappausmenetelmää, jossa hyödynnetään proteiini- ja glykaanipohjaisia biomarkkereita ja niiden mahdollisia yhdistelmiä syöpäpotilaiden virtsan EV:iden havaitsemiseksi. Syöpään liittyvien integriinimarkkereiden sekä lektiinikirjaston paneelin yhdistelmiä testattiin toimivien biomarkkereiden sekä niitä vastaavien mahdollisten määritysten löytämiseksi. Tämän jälkeen toimivien biomarkkereiden yhdistelmät karakterisoitiin ja validoitiin käyttämällä syöpäsolulinjoista sekä PCa- ja BlCa-potilaiden virtsasta peräisin olevia EV:itä. Paras määritys, joka yhdisti integriini ITGA3:n ja lektiini UEA:n, kykeni luotettavasti erottamaan BlCa:n PCa:sta, sekä hyvänlaatuisista- ja terveistä kontrolleista.
Tämän työn tulokset korostavat lektiinikirjaston ja syöpään liittyvien integriinimarkkereiden yhdistelmien systeemisen seulonnan tärkeyttä, erityisesti ajatellen immunokaappaukseen perustuvaa glykovariantti-määritystä virtsan EV:iden havaitsemiseksi ja urologisten syöpien diagnosoimiseksi käsittelemättömistä näytteistä. Kyseistä määrityskonseptia voisi käyttää avoimena alustana glykoisoformien tutkimiseen EV:iden pinnalta uusien biomarkkerien löytämiseksi
TUKAB: An Efficient NAT Traversal Scheme on Security of VoIP Network System Based on Session Initiation Protocol
Voice over Internet Protocol (VoIP) is subject to many security threats unique to both telephony and traditional Internet data transmission. As adoption of Session Initiation Protocol (SIP) based telephony increases, concerns are rising over risks to system confidentiality, integrity and availability. Currently, several VoIP security tools are available to detect vulnerabilities and protect against attacks. In this paper we present various issues concerning the security of VoIP. A brief discussion of the SIP protocol is presented based on its operating principle. Finally we proposed a solution for the Network Address Translation (NAT) traversal problem of SIP based networks. This solution supports all types of NAT and maintains the current VoIP architecture. Based on our experiment, we examined the latency, buffer size and voice packet loss under various network conditions. We found that it is possible to establish a call from outside the NAT to inside maintaining the quality issues of VoIP call. With this approach it is possible to use the current network architecture with having few changes in the registrar server. Hence we evaluate our model showing the QoS conditions that achieves both high efficiency and secure voice transmission. Sufficient simulation results are presented to verify our model
Detection of bladder cancer with aberrantly fucosylated ITGA3
We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay
Antinociceptive and Antioxidant Activity of Zanthoxylum budrunga
Different parts of the medicinal plant Zanthoxylum budrunga Wall enjoy a variety of uses in ethnobotanical practice in Bangladesh. In the present study, a number of phytochemical and pharmacological investigations were done on the ethanol extract of Z. budrunga seeds (ZBSE) to evaluate its antinociceptive and antioxidant potential. ZBSE was also subjected to HPLC analysis to detect the presence of some common antioxidants. In acetic acid induced writhing test in mice, ZBSE showed 65.28 and 74.30% inhibition of writhing at the doses of 250 and 500 mg/kg and the results were statistically significant (P<0.001). In hot-plate test, ZBSE raised the pain threshold significantly (P<0.001) throughout the entire observation period. In DPPH scavenging assay, the IC50 of ZBSE was observed at 82.60 μg/mL. The phenolic content was found to be 338.77 mg GAE/100 g of dried plant material. In reducing power assay, ZBSE showed a concentration dependent reducing ability. HPLC analysis indicated the presence of caffeic acid with a concentration of 75.45 mg/100 g ZBSE. Present investigation supported the use of Zanthoxylum budrunga seed in traditional medicine for pain management. Constituents including caffeic acid and other phenolics might have some role in the observed activity
Detection of bladder cancer with aberrantly fucosylated ITGA3
We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-dopednanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCaderived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay.</p
A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles
The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumorassociated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145-and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa-cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs
Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum
Background Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. Aim Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. Methods and results The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3-doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. Conclusions These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.</p
Anti-Inflammatory and Antioxidant Activity of Acalypha hispida Leaf and Analysis of its Major Bioactive Polyphenols by HPLC.
PURPOSE
Inflammation and oxidative stress can lead to different chronic diseases including cancer and atherosclerosis. Many medicinal plants have the potential to show as anti-inflammatory activity. Present investigation was performed to investigate anti-inflammatory, antioxidant activity, and quantification of selected bioactive plant polyphenols of the ethanol (EAH) and aqueous (AAH) extracts of Acalypha hispida (Euphorbiaceae) leaves.
METHODS
Anti-inflammatory activity was evaluated by carragenan and histamine induced rat paw edema models while antioxidant capacity was evaluated by DPPH free radical scavenging, Fe+2 chelating ability, reducing power, NO scavenging, total phenolic and total flavonoid content assay. Identification and quantification of bioactive polyphenols was done by HPLC.
RESULTS
At the doses of 200 and 400 mg/kg, both EAH and AAH showed statistically significant inhibition of paw volume in the anti-inflammatory activity test. Both the extracts showed DPPH scavenging (IC50: 14 and 17 µg/ml, respectively), Fe+2 ion chelating (IC50: 40 and 46 µg/ml, respectively), NO scavenging activity (65.49 and 60.66% inhibition at 100 µg/ml), and concentration dependent reducing power ability. For EAH and AAH, flavonoid content was 126.30 and 149.72 mg QE/g dry extract, while phenolic content was 130.51 and 173.80 mg GAE/g dry extract, respectively. HPLC analysis of EAH and AAH indicated the presence of high content of ellagic acid along with other phenolic constituents.
CONCLUSION
High content of ellagic acid along with other phenolic constituents might have played an important role in the observed anti-inflammatory and antioxidant activity
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Pharmacological and Ethnomedicinal Overview of Heritiera fomes: Future Prospects.
Mangrove plants are specialized woody plants growing in the swamps of tidal-coastal areas and river deltas of tropical and subtropical parts of the world. They have been utilized for medicinal and other purposes by the coastal people over the years. Heritiera fomes Buch. Ham. (family: Sterculiaceae) commonly known as Sundari (Bengali) is a preeminent mangrove plant occurring in the Sundarbans forest located in the southern part of Bangladesh and adjoining West Bengal province of India. The plant has applications in traditional folk medicine as evidenced by its extensive use for treating diabetes, hepatic disorders, gastrointestinal disorders, goiter, and skin diseases by the local people and traditional health practitioners. A number of investigations indicated that the plant possesses significant antioxidant, antinociceptive, antihyperglycemic, antimicrobial, and anticancer activities. Phytochemical analyses have revealed the presence of important chemical constituents like saponins, alkaloids, glycosides, tannins, steroids, flavonoids, gums, phytosterols, and reducing sugars. The present study is aimed at compiling information on phytochemical, biological, pharmacological, and ethnobotanical properties of this important medicinal plant, with a view to critically assess the legitimacy of the use of this plant in the aforementioned disorders as well as providing directions for further research.Peer Reviewe
A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles
The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2–10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs