Point-of-care impedimetric aptasensor to detect the luteinizing hormone.

Luteinizing hormone (LH) is a useful biomarker for identifying ovulation events in the cows to predict the time of ovulation to achieve a high success rate of conception following artificial insemination. Although antibody-based radioimmunoassay and enzyme-linked immunosorbent assay are being used for LH measurement, these techniques are expensive, time-consuming, and require expertise and sophisticated laboratory facilities. So, there is a need for a field-applicable, affordable, easy-to-use method for LH detection. For developing such a specific, quantitative, and inexpensive system, an aptamer-based smartphone-enabled aptasensor has been investigated. The aptamer was used instead of the antibody as a biorecognition element due to its comparative stability at ambient temperature, ease of synthesis, and cost-effectiveness. Electrochemical impedance spectroscopy has been used to obtain label-free detection of LH within 20 min in ~ 20 µL sample volume. The screen-printed gold electrode is compatible with a smartphone-enabled miniaturized device (Sensit Smart; Palmsens BV, The Netherlands) and was fabricated with the aptamer to detect LH in biological fluids (limit of detection 0.80 and 0.61 ng/mL in buffer and undiluted/unprocessed serum, respectively, with the dynamic range of detection of 0.01 to 50 ng/mL). All the data were obtained in the 10 kHz to 0.10 Hz frequency range at a bias potential of 0.30 V with an alternating potential of 10 mV. The clinical relevance of the sensor was evaluated in 10 serum samples collected from dairy animals which established a high correlation with standard LH-ELISA (κ > 0.87). The aptasensor can be stored at room temperature for 30 days without any significant loss in electrochemical sensing ability.

Development of an Impedimetric Aptasensor for Detection of Progesterone in Undiluted Biological Fluids.

A cost-effective, deployable, and quantitative progesterone biosensor is desirable for regular progesterone sensing in biological and environmental samples to safeguard public health. Aptasensors have been shown to be affordable as compared to antibody-based sensors, but so far, none of the progesterone aptamers could detect it in undiluted and unprocessed biological samples. Thus, to select an aptamer suitable for biosensing in unprocessed biological samples, a modified magnetic bead-based approach with counter-selection in milk and serum was performed. G-quadruplex forming progesterone aptamers were preferentially screened through in silico, gold nanoparticle-based adsorption-desorption assay and circular dichroism spectroscopy. GQ5 aptamer showed extended stability and a high progesterone binding affinity (K D 5.29 ± 2.9 nM) as compared to any other reported progesterone aptamers (P4G11 and P4G13). Under optimized conditions, GQ5 aptamer was coated on the gold electrode to develop an impedimetric aptasensor (limit of detection: 0.53, 0.91, and 1.9 ng/mL in spiked buffer, undiluted milk, and serum, respectively, with the dynamic range of detection from 0.1 to 50 ng/mL in buffer and 0.1 to 30 ng/mL in both milk and serum). The aptasensor exhibited a very high level of κ value (>0.9) with ELISA to detect progesterone in milk and serum. The aptasensor could be regenerated three times and can be stored for up to 10 days at 4 °C. Therefore, GQ5 may be used to develop a portable impedimetric aptasensor for clinical and on-site progesterone sensing in various biological and environmental samples.

Leukaemia inhibitory factor mediated proliferation of HTR-8/SVneo trophoblast cells is dependent on activation of extracellular signal-regulated kinase 1/2.

Leukaemia inhibitory factor (LIF) is one of the cytokines that is indispensable for embryo implantation. The aim of the present study was to investigate the role of activation of extracellular signal-regulated kinase (ERK) 1/2 in LIF-mediated proliferation of HTR-8/SVneo cells. Stimulation of HTR-8/SVneo cells with LIF (50 ng mL(-1)) resulted in an increase in cell proliferation (P < 0.05) via increased transition of cells to the G(2)/M phase of cell cycle. Stimulation with LIF resulted in the activation of both signal transducer and activator of transcription (STAT) 3 Tyr(705) and ERK1/2, but inhibition of ERK1/2 signalling by pretreatment of cells with U0126 (10 µM) for 2h resulted in abrogation of LIF-mediated increases in G(2)/M transition, with a significant decrease (P < 0.05) in absolute cell numbers compared with control. Although STAT3 silencing had no effect on LIF-dependent proliferation of HTR-8/SVneo cells, it did result in an increase in cell apoptosis, which increased further upon inhibition of ERK1/2 activation irrespective of LIF stimulation. Stimulation of cells with LIF increased the Bcl-2/Bax ratio, whereas ERK1/2 inhibition decreased the Bcl-2/Bax ratio, even after LIF stimulation. Hence, it can be inferred that ERK1/2 activation is essential for LIF-mediated increases in proliferation and that both STAT3 and ERK1/2 activation are important for the survival of HTR-8/SVneo cells.

Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Colorimetric aptasensor for on-site detection of oxytetracycline antibiotic in milk.

Oxytetracycline (OTC), one of the largely used antibiotic in veterinary practice has been banned due to its potential side effects. Development of a field applicable and affordable kit to detect OTC will help to eliminate such milk from human consumption. An aptamer has been designed (27 nt; Kd = 29.2 ± 19.4 nM) through rational truncation. OTC interacts with this aptamer in G rich regions as confirmed by molecular modelling and circular dichroism spectroscopy. To develop a lateral flow based aptasensor, OTC was conjugated with a 7 kDa carrier protein to immobilize onto the nitrocellulose membrane. Using 0.125 µM aptamer-gold conjugate, assay could visually detects upto 5 ng/mL of OTC in spiked milk within 10 mins [Limit of quantitation (LOQ)-0.254 ± 1.62 ng/mL; permissible limit 100 ng/mL]. It showed no cross reactivity with components of milk and data correlated with analysis done through HPLC.

Prospects of electrochemical immunosensors for early diagnosis of preeclampsia.

Preeclampsia is a vascular multisystem disorder that accounts for varying degree of morbidity and mortality of mother and the fetus. This can be significantly averted if diagnosed at an early (18-20 weeks) stage of gestation, as there is no known way to prevent preeclampsia. In spite of extensive work on biomarker discovery, the existing method for its detection is mostly based on colorimetric immunoassays whose sensitivity is ranging in nanomolar range. Further, it has also been observed that change in the expression of a single biomarker is not sufficient to diagnose this condition. So, for early diagnosis (by 18-20 weeks), an immuno-diagnostic platform with detection limits in picomolar range and beyond along with the ability to do simultaneous detection of multiple analyte would be of great importance. A nano-immunosensors with an electrochemical readout system can be a potential alternative that promises for the ultrasensitive detection of analyte with high specificity as well as suitability for on-site analysis. Coupling the lateral flow technology with immunosensors would make it feasible to detect more than one biomarker simultaneously on a microchip. This review intends to summarize the potential preeclampsia biomarkers, limitations of existing diagnostic methods along with the recent advancements, and prospects to develop electrochemical immunosensors for early clinical diagnosis.

Decrease in Expression of HOXA10 in the Decidua After Embryo Implantation Promotes Trophoblast Invasion.

An important step toward successful pregnancy involves invasion of the trophoblast cells into the decidua for placentation. Herein, we show that in the human and baboon decidua HOXA10 expression is downregulated after implantation and that this reduction is most prominent in the decidual cells juxtaposed to the invading placental villi. The supernatants derived from HOXA10-depleted human decidual cells increase the invasiveness of the trophoblast cell lines ACH-3P and JEG3 in vitro; this increase is due to higher expression and activity of matrix metalloproteases (MMPs) and reduced expression of tissue inhibitors of MMPs in both the cell lines. The proinvasive ability of HOXA10-depleted decidual cells is due to increased levels and secretion of leukemia inhibitor factor (LIF) and interleukin (IL)-6. Both these cytokines individually promote invasion of ACH-3P and JEG3 cell by increasing the activities of MMPs and decreasing mRNA levels of TIMPs. Finally, we demonstrate that the supernatants derived from HOXA10-depleted decidual cell-phosphorylated STAT3 (Tyr 705) and knocking down STAT3 in ACH-3P and JEG3 cells restrained the invasion mediated by supernatants derived from HOXA10-depleted decidual cells. These results imply that STAT3 activity is essential and sufficient to promote invasion in response to downregulation of HOXA10 in decidual cells. We propose that downregulation of HOXA10 in the decidual cells promotes the expression of LIF and IL-6, which, in a paracrine manner, activates STAT3 in the trophoblast cells, leading to an increase in MMPs to facilitate invasion.

Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation.

The aim of the present study is to delineate the role of human chorionic gonadotropin (hCG) in trophoblast fusion. In this direction, using shRNA lentiviral particles, α- and ß-hCG silenced 'BeWo' cell lines were generated. Treatment of both α- and ß-hCG silenced BeWo cells with either forskolin or exogenous hCG showed a significant reduction in cell fusion as compared with control shRNA treated cells. Studies by qRT-PCR, Western blotting and immunofluorescence revealed down-regulation of fusion-associated proteins such as syncytin-1 and syndecan-1 in the α- and ß-hCG silenced cells. Delineation of downstream signaling pathways revealed that phosphorylation of PKA and CREB were compromised in the silenced cells whereas, no significant changes in p38MAPK and ERK1/2 phosphorylation were observed. Moreover, ß-catenin activation was unaffected by either α- or ß-hCG silencing. Further, inhibition of PKA by H89 inhibitor led to a significant decrease in BeWo cell fusion but had no effect on ß-catenin activation suggesting the absence of non-canonical ß-catenin stabilization via PKA. Interestingly, canonical activation of ß-catenin was associated with the up-regulation of Wnt 10b expression. In summary, this study establishes the significance of hCG in the fusion of trophoblastic BeWo cells, but there may be additional factors involved in this process.

Recent advances in immunosensor for narcotic drug detection.

INTRODUCTION: Immunosensor for illicit drugs have gained immense interest and have found several applications for drug abuse monitoring. This technology has offered a low cost detection of narcotics; thereby, providing a confirmatory platform to compliment the existing analytical methods. METHODS: In this minireview, we define the basic concept of transducer for immunosensor development that utilizes antibodies and low molecular mass hapten (opiate) molecules. RESULTS: This article emphasizes on recent advances in immunoanalytical techniques for monitoring of opiate drugs. Our results demonstrate that high quality antibodies can be used for immunosensor development against target analyte with greater sensitivity, specificity and precision than other available analytical methods. CONCLUSION: In this review we highlight the fundamentals of different transducer technologies and its applications for immunosensor development currently being developed in our laboratory using rapid screening via immunochromatographic kit, label free optical detection via enzyme, fluorescence, gold nanoparticles and carbon nanotubes based immunosensing for sensitive and specific monitoring of opiates.

Chrysin rich Scutellaria discolor Colebr. induces cervical cancer cell death via the induction of cell cycle arrest and caspase-dependent apoptosis.

AIMS: Scutellaria discolor Colebr. has been extensively used in traditional medicine against several diseases. The purpose of this study was to investigate the anticancer potential of S. discolor and to isolate the bioactive principle responsible for the anticancer activity. METHODS: Cytotoxicity experiments were performed on cancer and normal cells using MTT assay. The mechanism of cell death was evaluated using real time PCR array, fluorescence microscopy, flow cytometry and Western blotting. MTT assay guided isolation (partition and column chromatography) was performed to identify the antiproliferative principle. Quantification of the active principle was done using HPLC. KEY FINDINGS: Acetone extract of S. discolor (SDE) inhibited the growth and survival of cancer cells to varying degree, but the inhibition was found to be maximum in cervical cancer cell lines. There was no significant toxicity induced to normal cells. The cell death was mediated through apoptosis. There was increased mitochondrial membrane depolarization, expression of Bax, caspase-9, caspase-3 and cleaved-PARP indicating that SDE-induced caspase dependent apoptosis in HeLa cells. Moreover, SDE caused cell cycle arrest in G2 phase in HeLa cells. Cytotoxicity guided fractionation of SDE led to the isolation of chrysin as the active principle responsible for the antiproliferative activity for cervical cancer cells. Interestingly, chrysin was the major phytochemical constituent present in S. discolor. SIGNIFICANCE: S. discolor is an important anticancer plant and a new source of chrysin.

STAT3 and ERK1/2 cross-talk in leukaemia inhibitory factor mediated trophoblastic JEG-3 cell invasion and expression of mucin 1 and Fos.

PROBLEM: The aim of this study was to investigate the relative importance of STAT3 and ERK1/2 activation in leukaemia inhibitory factor (LIF)-mediated invasion of JEG-3 cells. METHOD OF STUDY: Matrigel matrix-based invasion assay; Western blot; cDNA microarray; quantitative RT-PCR; gene silencing by siRNA. RESULTS: Leukaemia inhibitory factor treatment led to the activation of STAT3 and ERK1/2 signaling pathways which was followed by changes in the expression of several invasion-associated molecules such as mucin1 (MUC1), Fos, Jun, etc. Abrogation of either STAT3 or ERK1/2 signaling reduced (P < 0.05) the LIF-mediated invasion of JEG-3 cells. It was associated with a significant reduction in the expression of both MUC1 and Fos, suggesting a common denominator in LIF-STAT3-ERK1/2 signaling. To this effect, we observed a decrease in LIF-mediated p-STAT3 (Ser727) upon blocking STAT3 or ERK1/2 signaling. CONCLUSIONS: ERK1/2 as well as JAK-STAT-mediated STAT3 (Ser727) phosphorylation play an important role in LIF-mediated JEG-3 trophoblastic cell invasion and gene expression.

Prospects and advancements in C-reactive protein detection.

C-reactive protein (CRP) is one of the earliest proteins that appear in the blood circulation in most systemic inflammatory conditions and this is the reason for its significance, even after identification of many organ specific inflammatory markers which appear relatively late during the course of disease. Earlier methods of CRP detection were based on the classical methods of antigen-antibody interaction through precipitation and agglutination reactions. Later on, CRP based enzymatic assays came into the picture which were further modified by integration of an antigen-antibody detection system with surface plasma spectroscopy. Then came the time for the development of electrochemical biosensors where nanomaterials were used to make a highly sensitive and portable detection system based on silicon nanowire, metal-oxide-semiconductor field-effect transistor/bipolar junction transistor, ZnS nanoparticle, aptamer, field emission transmitter, vertical flow immunoassay etc. This editorial attempts to summarize developments in the field of CRP detection, with a special emphasis on biosensor technology. This would help in translating the latest development in CRP detection in the clinical diagnosis of inflammatory conditions at an early onset of the diseases.

Leukemia inhibitory factor increases the invasiveness of trophoblastic cells through integrated increase in the expression of adhesion molecules and pappalysin 1 with a concomitant decrease in the expression of tissue inhibitor of matrix metalloproteinases.

OBJECTIVE: To identify the invasion-associated molecules during leukemia inhibitory factor-(LIF-)mediated increase in the invasion of trophoblast cells. DESIGN: Experimental study. SETTING: Research institution. PATIENT(S): None. INTERVENTION(S): Cultured trophoblastic HTR-8/SVneo cells were treated with LIF. MAIN OUTCOME MEASURE(S): Matrigel matrix-based invasion assay of HTR-8/SVneo cells. Signaling molecules associated with LIF-mediated increase in invasion were investigated by Western blot, cDNA microarray, quantitative reverse transcriptase polymerase chain reaction, immunofluorescence, immunohistochemistry, and gene silencing by siRNA. RESULT(S): Treatment of HTR-8/SVneo cells with LIF (50 ng/mL) led to a significant increase in invasion. Treatment with LIF also led to an increase in nuclear localization of activated STAT1 and STAT3. Among 237 differentially expressed genes after LIF treatment, expression of pappalysin 1, SERPINB3, podoplanin, integrin ß3, ID1, ICAM1, and so on went up, while tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2, and TIMP3 went down significantly. The presence of several of these proteins has also been demonstrated in human trophoblast cells. Silencing of pappalysin 1 led to a significant reduction in basal as well as LIF-mediated invasiveness of HTR-8/SVneo cells. CONCLUSION(S): Identification of novel molecules associated with a LIF-mediated increase in trophoblastic cell invasion may facilitate our understanding of implantation biology.

LIF-STAT signaling and trophoblast biology.

Leukemia inhibitory factor (LIF) is a pleiotropic growth factor that regulates several biological functions. This review focuses on the LIF-dependent STAT activation and its impact on modulation of trophoblast functions during embryo implantation. LIF is mainly produced by the maternal endometrium at the time of implantation while its receptors are present both on the endometrium and trophoblasts. It might influence blastocyst attachment through STAT3 activation and expression of integrins. After attachment of the blastocyst, trophoblasts undergo proliferation and differentiation into invasive EVTs and non-invasive STBs. Under in vitro conditions, LIF regulates all these processes through activation of STAT- and MAPK-dependent signaling pathways. The observations that LIF and STAT3 knockout mice are infertile further strengthen the notion about the critical involvement of LIF-mediated signaling during embryo implantation. Hence, a better understanding of LIF-STAT signaling would help in improving fertility as use of LIF in in vitro blastocyst culture improves the implanting ability of blastocyst after IVF.

Endometrial biology during trophoblast invasion.

Attainment of successful implantation depends upon the synchronized changes in the endometrium before and after the arrival of blastocyst into the uterine cavity. The cues obtained from the receptive endometrium helps in proliferation and differentiation of the trophoblast cells. During the course of invasive differentiation, the trophoblast cells undergo several morphological, biochemical and molecular changes to gain the invasive capabilities. In turn, close apposition of the developing embryo brings out functional and morphological changes into the hormone primed receptive endometrium. Global gene expression profiling of the endometrium in response to the developing embryo or in response to the pregnancy hormone, human chorionic gonadotropin, in primate and human models, suggest that the endometrial-embryo cross-talk mainly influences three biological processes. Biological processes getting influenced by the blastocyst "signals" are associated with immunomodulation, biosensing and invasion. Pro- and anti-invasive paracrine factors expressed by different endometrial cell populations regulate the trophoblast invasion through activation of diverse signaling pathways. Identification of the gene signatures involved in embryo-endometrial dialogue would enhance our understanding about the pathologies like miscarriages and endometriosis.

AP-1 transcription factors, mucin-type molecules and MMPs regulate the IL-11 mediated invasiveness of JEG-3 and HTR-8/SVneo trophoblastic cells.

This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo cells.

Relevance of syndecan-1 in the trophoblastic BeWo cell syncytialization.

PROBLEM: To investigate the role of syndecan-1 in the differentiation of the BeWo cells into syncytiotrophoblast. METHOD OF STUDY: BeWo cells were stimulated with forskolin to form syncytia, and the expression of syndecan-1, desmoplakin I+II, human chorionic gonadotrophin (hCG) and angiogenesis-associated factors was analyzed. Syndecan-1 was silenced by siRNA to evaluate its involvement in the forskolin-mediated syncytia formation. RESULTS: Treatment of the BeWo cells with forskolin led to a significant increase in the syncytia formation. It was associated with an increase in the expression of syndecan-1 with a concomitant decrease in the expression of desmoplakin I+II. Forskolin treatment of the BeWo cells also led to an increase in the secretion of soluble endoglin, whereas no change was observed in the soluble fms-like tyrosine kinase-1. Silencing of the syndecan-1 expression in BeWo cells led to a significant decrease in cell fusion both in the presence and in the absence of forskolin. It was associated with a significant decrease in hCG level in the conditioned medium. CONCLUSION: Syndecan-1 is up-regulated in BeWo cells during differentiation and its silencing inhibits syncytialization and thus could be a useful biomarker for syncytiotrophoblast formation.

Decidualized endometrial stromal cell derived factors promote trophoblast invasion.

OBJECTIVE: To evaluate the effects of decidua-derived factors on trophoblast invasion. DESIGN: Experimental study. SETTINGS: Research institute. PATIENT(S): In vitro decidualized human endometrial cells, trophoblast cell lines JEG-3, and ACH-3P. INTERVENTION(S): The effect of decidual conditioned medium (DCM) on the invasion of trophoblast cells lines via JEG-3 and ACH-3P was investigated using a Matrigel invasion assay. The changes in expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrins in response to DCM in the trophoblast cells was also evaluated. MAIN OUTCOME MEASURE(S): Response of the trophoblast cells to the conditioned medium from decidual cells in terms of their invasive capability, and expression on invasion related molecules was measured. RESULT(S): DCM increased the invasion of both the cell lines by approximately 1.8-2.2-fold, compared with control condition medium. The increase in invasion was associated with elevated levels of MMP2, MMP3, and MMP9 mRNA and increased activity of MMP2 and MMP9 in DCM-treated ACH-3P, but not JEG-3 cells. DCM treatment led to a reduction in TIMP1 and TIMP3 and increased TIMP2 mRNA in JEG-3, cells but not ACH-3P cells. Compared with CCM-treated controls, DCM treatment led to a significant increase in the mRNA expression of integrin α5 and α6, but not integrin αV subunit in both cell lines. CONCLUSION(S): Decidua-derived factors increase the invasiveness of trophoblast cell lines and alter the expression of integrins, MMPs, and TIMPs.

Contraceptive vaccines based on the zona pellucida glycoproteins for dogs and other wildlife population management.

Zona pellucida (ZP) glycoproteins, by virtue of their critical role in fertilization, have been proposed as candidate antigens for the development of contraceptive vaccines. In this review, the potential of a ZP-based contraceptive vaccine for the management of wildlife population, with special reference to street dogs, is discussed. Immunization of various animal species, including female dogs, with native porcine ZP led to inhibition of fertility, which was associated with the ovarian dysfunction. Immunization of female dogs with Escherichia coli-expressed recombinant dog ZP glycoprotein-3 (ZP3) either coupled to diphtheria toxoid or expressed as fusion protein with 'promiscuous' T non-B-cell epitope of tetanus toxoid also led to inhibition of fertility. To improve the contraceptive efficacy of ZP-based contraceptive vaccine, various groups are working on improving the immunogen, use of DNA vaccine as prime-boost strategy, and delivering the zona proteins/peptides presented on either virus-like particles or entrapped in microsphere. Host-specific live vectors such as ectromelia virus and cytomegalovirus have also been used to deliver mouse ZP3 in mice. Various studies show the enormous potential of the ZP-based vaccine for the management of wildlife population, where permanent sterilization may be desirable.

Role of regulatory and angiogenic cytokines in invasion of trophoblastic cells.

PROBLEM: Trophoblast invasion is a temporally and locally restricted process, which regulates implantation and oxygen arrival to the embryo through the dialog with spiral artery endothelium. Trophoblast factors with angiogenic potential are activated by hypoxia. Their capacities to induce proliferation, migration, and invasion of trophoblastic cells have been investigated. METHOD OF STUDY: The expression of interleukin (IL)-6, CD126, CD130, vascular endothelial growth factor (VEGF), and hypoxia inducible factor-1alpha (HIF-1alpha) has been silenced in JEG-3 choriocarcinoma cells by using siRNA. Silencing efficacy has been assessed by ELISA, PCR or Western blotting. Proliferation has been measured by flow cytometry, migration by a transwell assay, and invasion by a Matrigel assay. RESULTS: Proliferation was significantly reduced by silencing of CD126 or CD130, migration by silencing of IL-6, VEGF, or HIF-1alpha, and invasion by silencing of IL-6 and HIF-1alpha. CONCLUSION: The expression of IL-6, VEGF, and HIF-1alpha in trophoblastic cells is involved in the control of trophoblast invasion and migration.