Bone morphogenetic protein 2- and estradiol-17ß-induced changes in ovarian transcriptome during primordial follicle formation†.

Estradiol-17ß has been shown to promote primordial follicle formation and to involve bone morphogenetic protein 2 (BMP2) as a downstream effector to promote primordial follicle in hamsters. However, the molecular mechanism whereby these factors regulate ovarian somatic cells to pre-granulosa cells transition leading to primordial follicle formation remains unclear. The objective of this study was to determine whether BMP2 and/or estradiol-17ß would regulate the expression of specific ovarian transcriptome during pre-granulosa cells transition and primordial follicle formation in the mouse ovary. BMP2 mRNA level increased during the period of primordial follicle formation with the concurrent presence of BMP2 protein in ovarian somatic cells. Estradiol-17ß but not BMP2 exposure led to increased expression of ovarian BMP2 messenger RNA (mRNA), and the effect of estradiol-17ß could not be suppressed by 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride (LDN) 193189. BMP2 or estradiol-17ß stimulated primordial follicle formation without inducing apoptosis. Ribonucleic acid-sequence analysis (RNA-seq) of ovaries exposed to exogenous BMP2 or estradiol-17ß revealed differential expression of several thousand genes. Most of the differentially expressed genes, which were common between BMP2 or estradiol-17ß treatment demonstrated concordant changes, suggesting that estradiol-17ß and BMP2 affected the same set of genes during primordial follicle formation. Further, we have identified that estradiol-17ß, in cooperation with BMP2, could affect the expression of three major transcription factors, GATA binding protein 2, GATA binding protein 4 and Early growth response 2, and one serine protease, hepsin, in pre-granulosa cells during primordial follicle formation. Taken together, results of this study suggest that estradiol-17ß and BMP2 may regulate ovarian gene expression that promote somatic cells to pre-granulosa cells transition and primordial follicle formation in the mouse ovary.

Transformation of Wurtzite ZnO to a New Triclinic Nanoporous ZnO Phase via Hydrothermal Treatment with Metformin for Designing Proton Conducting Material.

Zinc oxide is one of the most widely studied semiconductor metal oxides, which predominantly crystallizes as hexagonal wurtzite and often cubic zinc-blende phases. Here we report the transformation of the highly stable wurtzite ZnO to a new triclinic phase NZO-2 by using metformin as a template during post-synthesis hydrothermal treatment. This crystalline phase of the material NZO-2 has been identified through the refinement of the powder XRD data. NZO-2 possesses porous rod like particle morphology consisting of the self-assembly of 3-7 nm size spherical nanoparticles and interparticle nanoscopic voids spaces. NZO-2 has been surface phosphorylated and the resulting material displayed good proton conductivity. Further, NZO-2 displayed ultra-low band gap of 1.74 eV, thereby responsible for red emission under high energy laser excitation and this may open new opportunities in optoelectronic application of ZnO.

Sex differences impact the lung-bone inflammatory response to repetitive inhalant lipopolysaccharide exposures in mice.

Skeletal health consequences associated with inflammatory diseases of the airways significantly contribute to morbidity. Sex differences have been described independently for lung and bone diseases. Repetitive inhalant exposure to lipopolysaccharide (LPS) induces bone loss and deterioration in male mice, but comparison effects in females are unknown. Using an intranasal inhalation exposure model, 8-week-old C57BL/6 male and female mice were treated daily with LPS (100 ng) or saline for 3 weeks. Bronchoalveolar lavage fluids, lung tissues, tibias, bone marrow cells, and blood were collected. LPS-induced airway neutrophil influx, interleukin (IL)-6 and neutrophil chemoattractant levels, and bronchiolar inflammation were exaggerated in male animals as compared to female mice. Trabecular bone micro-CT imaging and analysis of the proximal tibia were conducted. Inhalant LPS exposures lead to deterioration of bone quality only in male mice (not females) marked by decreased bone mineral density, bone volume/tissue volume ratio, trabecular thickness and number, and increased bone surface-to-bone volume ratio. Serum pentraxin-2 levels were modulated by sex differences and LPS exposure. In proof-of-concept studies, ovarectomized female mice demonstrated LPS-induced bone deterioration, and estradiol supplementation of ovarectomized female mice and control male mice protected against LPS-induced bone deterioration findings. Collectively, sex-specific differences exist in LPS-induced airway inflammatory consequences with significant differences found in bone quantity and quality parameters. Male mice demonstrated susceptibility to bone loss and female animals were protected, which was modulated by estrogen. Therefore, sex differences influence the biologic response in the lung-bone inflammatory axis in response to inhalant LPS exposures.

Comparative analysis of endothelial cell loss following phacoemulsification in pupils of different sizes.

PURPOSE: To compare Endothelial cell(EC) loss following Phacoemulsification (PKE) in pupils of different sizes. METHODS: A prospective double masked observational study in which a total of 150 eyes of 150 patients between 50 & 70 years of age with senile cataract of nuclear sclerosis grade II were enrolled. Patients were allocated into three groups of 50 eyes each in Group A (pupil size <5 mm), Group B (pupil size 5-7 mm) and Group C (pupil size >7 mm). Pupillary size was measured by determining the height of slit on slit-lamp biomicroscope examination. PKE was done by the same expert surgeon using vertical chop technique and a foldable intraocular lens was implanted in the capsular bag. Corneal EC count and pachymetry were performed twice and average of 2 readings was taken for the purpose of this study. Measurements were taken preoperatively and postoperatively on day 1, day 7 and day 30. RESULTS: The mean EC count loss on postoperative day 1 in Group A was 19.45%, Group B 14.89%, Group C 10.19% with statistical significant difference between Group A and Group B, as also Group A and Group C. The difference was not significant between Group B and Group C, though there was a fall in EC count in Group C as well. Increase in corneal thickness on postoperative day 1 in group A was 5.43%, Group B 3.55%, Group C 2.14% with statistical significant difference between Group A and Group B, as also Group A and Group C with no difference in Group B and Group C. CONCLUSION: PKE done in eyes with maximal pupillary dilatation of <5 mm causes a greater EC loss and results in thicker corneas postoperatively as compared to eyes with pupillary dilatation of >5 mm at the end of one month.

Stimulation of primordial follicle assembly by estradiol-17ß requires the action of bone morphogenetic protein-2 (BMP2).

Primordial follicle (PF) pool determines the availability of follicles for ovulation in all mammals. Premature depletion of the PF reserve leads to subfertility or infertility. Bone morphogenetic protein 2 (BMP2) promotes PF formation by facilitating oocyte and granulosa cell development. Estradiol-17ß (E2) upregulates PF formation in developing hamster ovaries. However, if BMP2 mediates E2 effect is not known. We hypothesize that E2 facilitates the effect of BMP2 on somatic to granulosa cell transition. BMP2 and E2 together significantly upregulated the percentage of PFs in hamster fetal ovaries in vitro compared with either of the treatments alone. E2 also promoted BMP2 expression in vivo. Inhibition of BMP2 receptors suppressed E2-stimulation of PF formation while knockdown of BMP2 in vitro significantly suppressed the E2 effect. In contrast, estrogen receptor blocker did not affect BMP2 action. Inhibition of the activity of E2 or BMP2 receptors, either alone or combined during the last two days of the culture (C6-C8) resulted in a significant decrease in PF formation by C8, suggesting that both BMP2 and E2 action is essential for somatic cell differentiation for PF formation. Together, the results suggest that E2 activates BMP2-BMPR system leading to the formation of primordial follicles.

Bone morphogenetic protein 2 promotes primordial follicle formation in the ovary.

Primordial follicles (PF) are formed when somatic cells differentiate into flattened pregranulosa cells, invaginate into the oocyte nests and encircle individual oocytes. We hypothesize that BMP2 regulates PF formation by promoting the transition of germ cells into oocytes and somatic cells into pregranulosa cells. E15 hamster ovaries were cultured for 8 days corresponding to postnatal day 8 (P8) in vivo, with or without BMP2, and the formation of PF was examined. BMP2 was expressed in the oocytes as well as ovarian somatic cells during development. BMP2 exposure for the first two days or the last two days or the entire 8 days of culture led to increase in PF formation suggesting that BMP2 affected both germ cell transition and somatic cell differentiation. Whereas an ALK2/3 inhibitor completely blocked BMP2-induced PF formation, an ALK2-specific inhibitor was partially effective, suggesting that BMP2 affected PF formation via both ALK2 and ALK3. BMP2 also reduced apoptosis in vitro. Further, more meiotic oocytes were present in BMP2 exposed ovaries. In summary, the results provide the first evidence that BMP2 regulates primordial follicle formation by promoting germ cell to oocyte transition and somatic cell to pre-granulosa cells formation and it acts via both ALK2 and ALK3.

Effect of azaline B on follicular development and functions in the hamster.

The usefulness of azaline B, a GnRH antagonist, in suppressing gonadotropin secretion in the golden hamster was examined by examining follicular development, steroidogenesis and expression of steroidogenic enzymes. Serum levels of P and E declined significantly, while FSH or LH was undetectable in azaline B-treated hamsters. FSH significantly increased serum E levels, whereas LH upregulated serum P levels. The formation of antral follicles ceased in azaline-treated hamsters, but was reversed by FSH with or without LH supplement. FSH also activated the primordial follicle pool resulting in increased formation of primary and preantral follicles. Further, an increasing trend in the formation of preantral follicles in response to E or E + P, and the formation of antral follicles in response to E + P treatment was evident. The level of Cyp11a1 mRNA increased markedly in LH- or LH + FSH-treated hamsters, whereas FSH with or without LH upregulated Cyp17a1, Cyp19a1 and Fshr mRNA expression. E without or with P also upregulated ovarian Cyp19a1 mRNA expression. The expression of enzyme protein corroborated the mRNA data. In summary, azaline B is an efficient GnRH antagonist in the hamster, and will be useful in studying the selective effect of gonadotropins on ovarian functions without disrupting the physiological functions of other hormones in ovarian cells.

Expression of FSH receptor in the hamster ovary during perinatal development.

FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87 kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression.

Malaria burden and control in Bangladesh and prospects for elimination: an epidemiological and economic assessment.

BACKGROUND: Malaria is endemic in 13 of 64 districts in Bangladesh. About 14 million people are at risk. Some evidence suggests that the prevalence of malaria in Bangladesh has decreased since the the Global Fund to Fight AIDS, Tuberculosis and Malaria started to support the National Malaria Control Program (NMCP) in 2007. We did an epidemiological and economic assessment of malaria control in Bangladesh. METHODS: We obtained annually reported, district-level aggregated malaria case data and information about disbursed funds from the NMCP. We used a Poisson regression model to examine the associations between total malaria, severe malaria, malaria-attributable mortality, and insecticide-treated net coverage. We identified and mapped malaria hotspots using the Getis-Ord Gi* statistic. We estimated the cost-effectiveness of the NMCP by estimating the cost per confirmed case, cost per treated case, and cost per person of insecticide-treated net coverage. FINDINGS: During the study period (from Jan 1, 2008, to Dec 31, 2012) there were 285,731 confirmed malaria cases. Malaria decreased from 6.2 cases per 1000 population in 2008, to 2.1 cases per 1000 population in 2012. Prevalence of all malaria decreased by 65% (95% CI 65-66), severe malaria decreased by 79% (78-80), and malaria-associated mortality decreased by 91% (83-95). By 2012, there was one insecticide-treated net for every 2.6 individuals (SD 0.20). Districts with more than 0.5 insecticide-treated nets per person had a decrease in prevalence of 21% (95% CI 19-23) for all malaria, 25% (17-32) for severe malaria, and 76% (35-91) for malaria-associated mortality among all age groups. Malaria hotspots remained in the highly endemic districts in the Chittagong Hill Tracts. The cost per diagnosed case was US$0.39 (SD 0.02) and per treated case was $0.51 (0.27); $0.05 (0.04) was invested per person per year for health education and $0.68 (0.30) was spent per person per year for insecticide-treated net coverage. INTERPRETATION: Malaria elimination is an achievable prospect in Bangladesh and failure to push for elimination nearly ensures a resurgence of disease. Consistent financing is needed to avoid resurgence and maintain elimination goals. FUNDING: None.

Antimalarial drug resistance in Bangladesh, 1996-2012.

Malaria remains an important health problem in Bangladesh, with approximately 14 million people at risk. Antimalarial drug resistance is a major obstacle to the control of malaria in endemic countries. In 2012, Bangladesh reported an estimated 29 522 malaria episodes, of which 94% were reported as being caused by Plasmodium falciparum. In this study, we reviewed and summarized antimalarial drug resistance data from Bangladesh published until June 2013. We searched published sources for data referring to any type of P. falciparum drug resistance (in vivo, in vitro, or molecular) and found 169 articles published in peer-reviewed journals. Of these, 143 articles were excluded because they did not meet our inclusion criteria. After detailed review of the remaining 26 articles, 14 were selected for evaluation. Published studies indicate that P. falciparum shows varying levels of resistance to chloroquine, mefloquine and sulfadoxine-pyrimethamine. Combination therapy of chloroquine and primaquine has proven ineffective and combinations of sulfadoxine-pyrimethamine with either quinine or chloroquine have also shown poor efficacy. Recent studies indicate that artemisinin derivatives, such as artesunate, remain highly efficacious in treating P. falciparum malaria. Available data suggest that artemisinins, quinine, doxycyline, mefloquine-artesunate and azithromycin-artesunate combination therapy remain efficacious in the treatment of P. falciparum malaria in Bangladesh.

Expression of ErbB3-binding protein-1 (EBP1) during primordial follicle formation: role of estradiol-17ß.

The formation of primordial follicles involves the interaction between the oocytes and surrounding somatic cells, which differentiate into granulosa cells. Estradiol-17ß (E) promotes primordial follicle formation in vivo and in vitro; however, the underlying mechanisms are poorly understood. The expression of an ERBB3-binding protein 1 (EBP1) is downregulated in 8-day old hamster ovaries concurrent with the increase in serum estradiol levels and the formation of primordial follicles. The objectives of the present study were to determine the spatio-temporal expression and putative E regulation of EBP1 in ovarian cells during perinatal development with respect to primordial follicle formation. Hamster EBP1 nucleic acid and amino acid sequences were more than 93% and 98% similar, respectively, to those of mouse and human, and contained nucleolar localization signal, RNA-binding domain and several phosphorylation sites. EBP1 protein was present in somatic cells and oocytes from E15, and declined in oocytes by P1 and in somatic cells by P5. Thereafter, EBP1 expression increased through P7 with a transient decline on P8 primarily in interstitial cells. EBP1 mRNA levels mirrored protein expression pattern. E treatment on P1 and P4 upregulated EBP1 expression by P8 whereas E treatment on P4 downregulated it by 72 h suggesting a compensatory upregulation due to E pretreatment. Treatment with an FSH-antiserum, which suppressed primordial follicle formation, prevented the decline in EBP1 levels, and the effect was reversed by E treatment. Therefore, the results provide the first evidence that EBP1 may play an important role in mediating the effect of E in the differentiation of somatic cells into granulosa cells during primordial follicle formation.

Expression of estrogen receptor α 36 (ESR36) in the hamster ovary throughout the estrous cycle: effects of gonadotropins.

Estradiol-17ß (E) plays an important role in ovarian follicular development. Evidence indicates that some of the effect of E is mediated by the transmembrane estrogen receptor. In this study, we examined the spatio-temporal expression of recently discovered ERα36 (ESR36), a splice variant of Esr1 and a receptor for non-genomic E signaling, in the hamster ovary during the estrous cycle and the role of gonadotropins and ovarian steroid hormones in ESR36 expression. ESR36 expression was high on estrus (D1:0900 h) and declined precipitously by proestrus (D4:0900 h) and remained low up to D4:1600 h. Immunofluorescence findings corroborated immunoblot findings and revealed that ESR36 was expressed only in the cell membrane of both follicular and non-follicular cells, except the oocytes. Ovarian ESR36 was capable of binding to the E-affinity matrix, and have different molecular weight than that of the ESR1 or GPER. Hypophysectomy (Hx) resulted in a marked decline in ESR36 protein levels. FSH and LH, alone or combined, markedly upregulated ESR36 protein in Hx hamsters to the levels observed in D1 hamsters, but neither E nor P had any effect. Inhibition of the gonadotropin surge by phenobarbital treatment on D4:1100 h attenuated ESR36 expression in D1:0900 h ovaries, but the decline was restored by either FSH or LH replacement on D4 afternoon. This is the first report to show that ESR36, which is distinct from ESR1 or GPER is expressed in the plasma membrane of ovarian follicular and non-follicular cells, binds to E and its expression is regulated directly by the gonadotropins. In light of our previous findings, the results suggest that ovarian cells contain at least two distinct membrane estrogen receptors, such as GPER and ESR36, and strongly suggest for a non-genomic action of E regulating ovarian follicular functions.

Characterizing the spatial and temporal variation of malaria incidence in Bangladesh, 2007.

BACKGROUND: Malaria remains a significant health problem in Bangladesh affecting 13 of 64 districts. The risk of malaria is variable across the endemic areas and throughout the year. A better understanding of the spatial and temporal patterns in malaria risk and the determinants driving the variation are crucial for the appropriate targeting of interventions under the National Malaria Control and Prevention Programme. METHODS: Numbers of Plasmodium falciparum and Plasmodium vivax malaria cases reported by month in 2007, across the 70 endemic thanas (sub-districts) in Bangladesh, were assembled from health centre surveillance reports. Bayesian Poisson regression models of incidence were constructed, with fixed effects for monthly rainfall, maximum temperature and elevation, and random effects for thanas, with a conditional autoregressive prior spatial structure. RESULTS: The annual incidence of reported cases was 34.0 and 9.6 cases/10,000 population for P. falciparum and P. vivax respectively and the population of the 70 malaria-endemic thanas was approximately 13.5 million in 2007. Incidence of reported cases for both types of malaria was highest in the mountainous south-east of the country (the Chittagong Hill Tracts). Models revealed statistically significant positive associations between the incidence of reported P. vivax and P. falciparum cases and rainfall and maximum temperature. CONCLUSIONS: The risk of P. falciparum and P. vivax was spatially variable across the endemic thanas of Bangladesh and also highly seasonal, suggesting that interventions should be targeted and timed according to the risk profile of the endemic areas. Rainfall, temperature and elevation are major factors driving the spatiotemporal patterns of malaria in Bangladesh.

In vitro culture of fetal ovaries: a model to study factors regulating early follicular development.

Follicular development commences with the formation of primordial follicles, which begins with the differentiation of pluripotent ovarian somatic cells into early granulosa cells and their apposition to the oocytes in the egg nest. The process of primordial follicle morphogenesis and factors affecting the formation and development of primordial follicles can be examined in vitro using fetal ovaries in organ culture. The functions of candidate genes involved in primordial folliculogenesis can be examined using siRNA or shRNA, which can knockdown specific mRNA targets at specific time points. Here, we describe the organ culture protocol for fetal hamster ovary with GPR30 siRNA as an example. The method to morphologically analyze follicular development is also discussed.

Changes in ovarian protein expression during primordial follicle formation in the hamster.

Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and their interaction with the oocytes.

Neonatal diethylstilbestrol exposure disrupts female reproductive tract structure/function via both direct and indirect mechanisms in the hamster.

We assessed neonatal diethylstilbestrol (DES)-induced disruption at various endocrine levels in the hamster. In particular, we used organ transplantation into the hamster cheek pouch to determine whether abnormalities observed in the post-pubertal ovary are due to: (a) a direct (early) mechanism or (b) an indirect (late) mechanism that involves altered development and function of the hypothalamus and/or pituitary. Of the various disruption endpoints and attributes assessed: (1) some were consistent with the direct mechanism (altered uterine and cervical dimensions/organization, ovarian polyovular follicles, vaginal hypospadius, endometrial hyperplasia/dysplasia); (2) some were consistent with the indirect mechanism (ovarian/oviductal salpingitis, cystic ovarian follicles); (3) some were consistent with a combination of the direct and indirect mechanisms (altered endocrine status); and (4) the mechanism(s) for one (lack of corpora lutea) was uncertain. This study also generated some surprising observations regarding vaginal estrous assessments as a means to monitor periodicity of ovarian function in the hamster.

Expression of E-cadherin and N-cadherin in perinatal hamster ovary: possible involvement in primordial follicle formation and regulation by follicle-stimulating hormone.

We examined the expression and hormonal regulation of E-cadherin (CDH1) and N-cadherin (CDH2) with respect to primordial follicle formation. Hamster Cdh1 and Cdh2 cDNA and amino acid sequences were more than 90% similar to those of the mouse, rat, and human. Although CDH1 expression remained exclusively in the oocytes during neonatal ovary development, CDH2 expression shifted from the oocytes to granulosa cells of primordial follicles on postnatal day (P)8. Subsequently, strong CDH2 expression was restricted to granulosa cells of growing follicles. Cdh2 mRNA levels in the ovary decreased from embryonic d 13 through P10 with a transient increase on P7, which was the day before the appearance of primordial follicles. Cdh1 mRNA levels decreased from embryonic d 13 through P3 and then showed a transient increase on P8, coinciding with the formation of primordial follicles. CDH1 and CDH2 expression were consistent with that of mRNA. Neutralization of FSH in utero impaired primordial follicle formation with an associated decrease in Cdh2 mRNA and CDH2, but an increase in Cdh1 mRNA and CDH1 expression. The altered expression was reversed by equine chorionic gonadotropin treatment on P1. Whereas a CDH2 antibody significantly reduced the formation of primordial and primary follicles in vitro, a CDH1 antibody had the opposite effect. This is the first evidence to suggest that primordial follicle formation requires a differential spatiotemporal expression and action of CDH1 and CDH2. Further, FSH regulation of primordial follicle formation may involve the action of CDH1 and CDH2.

Surface-functionalized nanoparticles for targeted gene delivery across nasal respiratory epithelium.

The objective of this study was to determine whether surface-modified nanoparticles enhance permeability across nasal mucosa, while retaining the effectiveness of the payload. The uptake and permeability of polystyrene nanoparticles (PS-NPs; FluoSpheres) was evaluated across the various regions of the bovine nasal epithelia following conjugation with deslorelin and transferrin. Uptake and transport of PS-NPs, deslorelin-PS-NPs, and transferrin-PS-NPs exhibited regional differences in the order: inferior turbinate posterior (ITP) > medium turbinate posterior (MTP) > medium turbinate anterior (MTA). Uptake and transport also exhibited directionality and temperature dependence in these tissues. Further, uptake as well as transport of functionalized nanoparticles could be inhibited by excess free functionalizing ligand. Confocal microscopy indicated the presence of functionalized nanoparticles in respiratory epithelial cells, as well as other cell types of the nasal tissue. We chose the ITP region for further studies with deslorelin or transferrin-conjugated poly-l-lactide-co-glycolide nanoparticles (PLGA-NPs) encapsulating an anti-VEGF intraceptor (Flt23k) plasmid. Transport of the nanoparticles, as well as the plasmid from the nanoparticles, exhibited the following order: transferrin-PLGA-NPs > deslorelin-PLGA-NPs > PLGA-NPs >> plasmid. The ability of the nanoparticles transported across the nasal tissue to retain the effectiveness of the Flt23k plasmid was evaluated by measuring transfection efficiency (percentage of cells expressing GFP) and VEGF inhibition in LNCaP and PC-3 prostate cancer cells. Transfection efficiencies and VEGF inhibition in LNCaP and PC-3 cells exhibited the following trend: transferrin-PLGA-NPs >or= deslorelin-PLGA-NPs > PLGA-NPs >> plasmid. Further, functionalized nanoparticles exhibited transfection efficiencies and VEGF inhibition significantly superior compared with the routinely used transfecting agent, lipofectamine. Formulating plasmids into nanoparticulate delivery systems enhances the transnasal delivery and gene therapy at remote target cancer cells, which can be further enhanced by nanoparticle functionalization with deslorelin or transferrin.

Disruption of estrogen receptor signaling enhances intestinal neoplasia in Apc(Min/+) mice.

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.