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- Development of lectin-linked immunomagnetic separation for the detection of hepatitis a virus. [Journal Article]
- Viruses 2014; 6(3):1037-48.
The accuracy and sensitivity of PCR-based methods for detection of hepatitis A virus (HAV) are dependent on the methods used to separate and concentrate the HAV from the infected cells. The pH and ionic strength affect the binding affinity of the virus to cells. In this study, we initially investigated the effects of pH (4.0-10.0) and metal ions (Fe2+, Co2+, Cu2+, Mg2+, K+, and Ca2+) on the binding of HAV to oyster digestive cells. The lowest relative binding (RB) of HAV to the cells was found at pH 4.0 and in FeSO4 solution (64.6% and 68.1%, respectively). To develop an alternative to antibody-dependent immunomagnetic separation prior to detection of HAV using RT-PCR, the binding of HAV to five lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin (UEA-1) and soybean agglutinin (SBA), was evaluated using ELISAs. SBA showed significantly higher RB to HAV than the other lectins tested. In addition, HAV could be concentrated within 30 min using SBA-linked magnetic bead separation (SMS) prior to the RT-PCR assay. Our findings demonstrate the feasibility of using SMS combined with RT-PCR to detect HAV at dilutions ranging from 10-1-10-4 of a HAV stock (titer: 104 TCID50/mL).
- A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis. [JOURNAL ARTICLE]
- Development 2014 Mar 5.
The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.
- Construction and validation of a Sambucus nigra biosensor for cancer-associated STn antigen. [JOURNAL ARTICLE]
- Biosens Bioelectron 2014 Feb 15.:254-261.
A label-free electrochemical impedance spectroscopy biosensor for selective detection and discrimination of the cancer-associated sialyl-Tn (STn) antigen was developed by using Sambucus nigra agglutinin type I (SNA-I) as the recognition element. The SNA-I biosensor was constructed by immobilizing the lectin on screen-printed gold electrodes. The formation of a complex between SNA-I and STn-containing glycoproteins (transferrin and bovine submaxillary mucin) was monitored by measuring the impedance increase of the biosensor. The increase in electron transfer resistance was linearly proportional to the concentration of the glycoproteins up to 70ng of transferrin and 40ng of bovine submaxillary mucin, with a limit of detection of 20ng for transferrin. Albumin, the most abundant serum protein, did not interfere in the detection of the STn-glycoproteins up to a concentration of 0.2mgml(-1). The developed lectin-based biosensor was used to evaluate the STn-expression in serum samples and discriminate samples from healthy individuals and patients with different types of malignant tumors, mostly carcinomas, where the increased expression of STn aberrant glycans is well established. This work demonstrates the feasibility of employing SNA-I to selectively recognize the STn epitope in glycoproteins and the use of the constructed biosensor was effective in the analysis of serum samples with the ability to discriminate in a fast way between cancer and healthy status. The proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated STn glycan expression in serum for diagnosis and therapy monitoring.
- Calreticulin activates β1-integrin through fucosylation modification by fucosyltransferase-1 in J82 human bladder cancer cells. [JOURNAL ARTICLE]
- Biochem J 2014 Mar 4.
Fucosylation regulates various pathological events in cells. We previously reported that different levels of calreticulin (CRT) affect cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumor metastasis regulated by CRT remains unclear. Using DNA array, we identified fucosyltransferase-1 (FUT1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation on β1-integrin and this modification was catalyzed by FUT1. To clarify FUT1 roles in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of β1-integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with Ulex europaeus agglutinin I (UEA-1), a lectin recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of β1-integrin. These results indicated that α1,2-fucosylation on β1-integrin were not involved in the integrin-collagen interaction but promoted β1-integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation in 3'-untranslated region (3'-UTR). In conclusion, our findings suggested that CRT stabilized FUT1 mRNA, thereby leading to increase in fucosylation of β1-integrin. Furthermore, increased fucosylation levels activate β1-integrin rather than directly modifying the integrin binding sites.
- Changes in highly sensitive alpha-fetoprotein for the prediction of the outcome in patients with hepatocellular carcinoma after hepatectomy. [JOURNAL ARTICLE]
- Cancer Med 2014 Mar 3.
We investigated changes in highly sensitive lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein (hsAFP-L3) measured using a novel method and its predictive ability for prognosis in patients with hepatocellular carcinoma (HCC) who underwent curative hepatectomy, comparing to other HCC tumor markers, that is, AFP, des-gamma-carboxy prothrombin (DCP), and AFP-L3 measured with conventional method (cAFP-L3). AFP, DCP, and AFP-L3 including both cAFP-L3 and hsAFP-L3 were measured before and after curative hepatectomy in 187 patients. The percentage of patients with elevated tumor marker levels pre- and postoperatively was compared, and recurrence-free and overall survival rates were analyzed based on changes in tumor markers. The percentages of patients with elevated AFP, DCP, and cAFP-L3 decreased postoperatively. In contrast, the percentage of patients with elevated hsAFP-L3 did not decrease postoperatively. Both recurrence-free and overall survival rates were significantly lower in patients whose tumor marker levels remained elevated postoperatively than patients without tumor marker elevation postoperatively. Recurrence-free and overall survival rates of patients in whom hsAFP-L3 became elevated postoperatively despite normal preoperative hsAFP-L3 levels were significantly lower than those of patients with normal hsAFP-L3 postoperatively, and were similar to those of patients with persistent elevation. Preoperative elevations of AFP, DCP, and cAFP normalized in many patients postoperatively, but not for hsAFP-L3. The elevation of hsAFP-L3 identifies patients with poor prognosis despite the normalization of AFP and DCP.
- Evaluation of TH Agglutinin Titres of Widal test in the Diagnosis of Typhoid Fever. [Journal Article]
- Mymensingh Med J 2014 Jan; 23(1):1-6.
This prospective study was evaluated the role of TH agglutinins and reevaluate the diagnostic value of Widal test in the diagnosis of typhoid fever by identifying the S. typhi specific antibody. The study was carried out in the department of Microbiology, Mymensingh Medical College, Mymensingh between July 2010 and June 2011, including 200 individuals of different ages and sexes. Widal test and blood culture were performed for each of the cases and controls. Among 150 blood samples from the suspected cases, 23(15.3%) were culture positive for S. typhi and 67(44.7%) had high titres of ≥1:160 for 'O' or 'H' or both agglutinins. Out of 23 culture positive cases, 3(13%) had TO ≥:160, 5(21.7%) had significant TH ≥1:160. Based on the above findings, it was concluded that judicious interpretation of Widal test play an essential role in diagnosis of typhoid fever, especially in the resource poor countries like Bangladesh.
- GALNT2 enhances migration and invasion of oral squamous cell carcinoma by regulating EGFR glycosylation and activity. [JOURNAL ARTICLE]
- Oral Oncol 2014 Feb 27.
Oral squamous cell carcinoma (OSCC) is one of the leading cancers worldwide. Aberrant glycosylation affects many cellular properties in cancers, including OSCC. This study aimed to explore the role of N-acetylgalactosaminyltransferase 2 (GALNT2) in OSCC.Immunohistochemistry was performed to study the expression of GALNT2 in an OSCC tissue microarray. Effects of GALNT2 overexpression and knockdown on cell migration and invasion were analyzed in SAS cells by transwell migration assay and matrigel invasion assay, respectively. The Vicia villosa agglutinin (VVA) pull down assay was conducted to detect changes in O-glycans on acceptor substrates of GALNT2. Cell signaling was analyzed by Western blotting.GALNT2 was overexpressed in 73% (35/48) of OSCC tissues. Moreover, GALNT2 expression was localized in the invasive front and increased in high grade OSCC. GALNT2 overexpression enhanced migration and invasion of SAS cells triggered by fetal bovine serum (FBS) and epidermal growth factor (EGF). In contrast, GALNT2 knockdown inhibited SAS cell migration and invasion. Furthermore, GALNT2 overexpression enhanced VVA binding to epidermal growth factor receptor (EGFR) and EGF-induced phosphorylation of EGFR and AKT. Conversely, GALNT2 knockdown decreased VVA binding and suppressed activity of EGFR and AKT.GALNT2 is frequently overexpressed in OSCC, especially in the carcinoma cells at the invasive front. GALNT2 overexpression enhances the invasive potential of OSCC cells via modifying O-glycosylation and activity of EGFR. These findings suggest that GALNT2 plays an important role in the invasive behavior of OSCC and that targeting GALNT2 could be a promising approach for OSCC therapy.
- Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte. [JOURNAL ARTICLE]
- Reprod Biomed Online 2013 Dec 4.
In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles. Vesicles are artificially prepared, small lipid membrane-enclosed sacks that can store substances. As vesicles encapsulate an aqueous solution inside a partly hydrophobic membrane, they can carry both hydrophobic and hydrophilic molecules. Due to their unique properties, liposomes are often used as model systems to create or study artificial cells, as well as drug delivery vehicles. In order to transport nucleic acids, proteins or other cargo, the vesicles should exhibit mobility. In the present paper we propose a new delivery strategy, using the motion of spermatozoa to transport attached vesicles into the oocyte during fertilization. To use spermatozoa as a vesicle-transport system, we have to accomplish three goals. One, the lipid composition of the vesicle membranes needs to be created such that they can bind to the sperm cells. Two, the spermatozoa should still be able to swim with the extra loaded vesicle cargo. And three, the bound vesicles should not inhibit the capability of sperm cells to fertilize. In this study, vesicles started to show binding after 20mol% neutral lipids were substituted with either positive, negative, cerebroside or ganglioside lipids. A new 'hybrid' delivery system has been created in which vesicles are bound to spermatozoa in physiological conditions with no significant alterations in sperm motility and function. Moreover, with IVF experiments in the mouse model, it was confirmed that spermatozoa remained capable of fertilizing while delivering vesicles simultaneously.
- Platelet cold agglutinins and thrombocytopenia: A diagnostic dilemma in the intensive care unit. [Journal Article]
- J Anaesthesiol Clin Pharmacol 2014 Jan; 30(1):89-90.
We report a case of pseudo-thrombocytopenia due to cold agglutinins against platelets. These cold agglutinins were the cause for diagnostic confusion and resulted in extensive workup and unnecessary therapeutic precautions. A thirty two year old female with Guillain-Barre syndrome was admitted in the ICU and serial work-up showed markedly low levels of platelets. The patient had no symptoms of bleeding and patient was investigated extensively for deciphering the etiology of low platelet count. In-vitro clumping of platelets was suspected and in-vitro studies showed marked clumping of platelets with ethylene-diamine-tetra-acetic acid, citrate and heparinized samples. The manual platelet count was found to be within normal limits. Thrombocytopenia as a result of platelet cold agglutinins is a rare cause of in-vitro low platelet counts. No clinical problems have been reported due to the same.
- Lens culinaris agglutinin-reactive α-fetoprotein decline after transcatheter arterial chemoembolization in patients with hepatocellular carcinoma predicts survival. [JOURNAL ARTICLE]
- Clin Chim Acta 2014 Feb 22.
Lens culinaris agglutinin-reactive α-fetoprotein (AFP-L3) is a fucosylated fraction of AFP that is highly specific for hepatocellular carcinoma (HCC). We studied the relationship between AFP-L3 response and treatment outcome in terms of radiologic response and overall survival in patients undergoing transcatheter arterial chemoembolization (TACE).We retrospectively analyzed 152 patients with advanced HCC undergoing TACE. Serum AFP-L3 and AFP levels were measured simultaneously with a novel lectin dual-label time-resolved immunofluorometric assay (lectin dual-label TRFIA). AFP-L3 response was defined as a ≥20% reduction in AFP-L3 level after a minimum of 2cycles of chemotherapy.A total of 47 AFP-L3 responders had improved median overall survival of 42.9months compared with 15.4months in nonresponders (P<0.0001), and AFP-L3 response was strongly associated with radiologic response (P<0.0001). The combination of AFP-L3 response and serum AFP response provided further prognostic information. On multivariate analysis, the prognostic value of AFP-L3 response was independent of maximum tumor diameter and BCLC stage.A significant reduction in AFP-L3 in patients with advanced HCC is an important predictor of survival. Achieving an AFP-L3 response should be one of the therapeutic intents of TACE.