Bioimaging based on Poly(ethylenimine)-Coated Carbon Dots and Gold Nanoparticles for pH Sensing and Metal Enhanced Fluorescence.

When exposed to specific light wavelengths, carbon dots (CDs), which tend to be fluorescent, can emit colorful light. It provides them with a lot of adaptability for different applications including bioimaging, optoelectronics, and even environmental sensing. Poly(ethylenimine) (PEI) coated carbon dots (PEI-CDs) with a long emission wavelength were synthesized via the hydrothermal method. The resultant CDs show strong fluorescence with quantum yield up to 20.2%. The PEI-CDs exist with distinct pH-sensitive features with pH values in the range of 2-14. The optical characteristics of CDs are pH-responsive due to the presence of different amine groups on PEI, which is a functional polycationic polymer. One of the most widely employed nanoparticles for improving the fluorescence plasmonic characteristics of a nanocomposite is gold. Gold nanoparticles were coupled with PEI-CDs in this assay by using the EDC-NHS coupling to increase the photoluminescence property of the PEI-CDs by using the metal-enhanced fluorescence approach. In the presence of gold nanoparticles, the fluorescence is enhanced 5-6 times. The likely mechanism in our investigation was primarily derived from enhancement of the intrinsic radiative decay rate rather than the local electric field impact. Moreover, PEI-CDs can be used as a bioimaging agent, as these molecules are nontoxic to the cells, and the positively charged PEI-CDs have the potential for nuclear targeting, allowing for electrostatic contact with DNA in the nucleus. This finding will expand the application that the PEI-CDs can be used in the future for targeted imaging applications.

Azithromycin and Sildenafil May Have Protective Effects on Retinal Ganglion Cells via Different Pathways: Study in a Rodent Microbead Model.

Decreased blood flow to the optic nerve (ON) and neuroinflammation are suggested to play an important role in the pathophysiology of glaucoma. This study investigated the potential neuroprotective effect of azithromycin, an anti-inflammatory macrolide, and sildenafil, a selective phosphodiesterase-5 inhibitor, on retinal ganglion cell survival in a glaucoma model, which was induced by microbead injection into the right anterior chamber of 50 wild-type (WT) and 30 transgenic toll-like receptor 4 knockout (TLR4KO) mice. Treatment groups included intraperitoneal azithromycin 0.1 mL (1 mg/0.1 mL), intravitreal sildenafil 3 µL, or intraperitoneal sildenafil 0.1 mL (0.24 µg/3 µL). Left eyes served as controls. Microbead injection increased intraocular pressure (IOP), which peaked on day 7 in all groups and on day 14 in azithromycin-treated mice. Furthermore, the retinas and ON of microbead-injected eyes showed a trend of increased expression of inflammatory- and apoptosis-related genes, mainly in WT and to a lesser extent in TLR4KO mice. Azithromycin reduced the BAX/BCL2 ratio, TGFß, and TNFα levels in the ON and CD45 expression in WT retina. Sildenafil activated TNFα-mediated pathways. Both azithromycin and sildenafil exerted a neuroprotective effect in WT and TLR4KO mice with microbead-induced glaucoma, albeit via different pathways, without affecting IOP. The relatively low apoptotic effect observed in microbead-injected TLR4KO mice suggests a role of inflammation in glaucomatous damage.

Neuroprotective Effect of Azithromycin Following Induction of Optic Nerve Crush in Wild Type and Immunodeficient Mice.

This study evaluated the potential neuroprotective effect of azithromycin (AZ) intraperitoneal injections in male C57Bl/6 (wild type, WT) and female NOD scid gamma (NSG) mice subjected to optic nerve crush (ONC) as a model for optic neuropathy. Histologically, reduced apoptosis and improved retinal ganglion cell (RGC) preservation were noted in the AZ-treated mice as shown by TUNEL staining-in the WT mice more than in the NSG mice. The increased microglial activation following ONC was reduced with the AZ treatment. In the molecular analysis of WT and NSG mice, similar trends were detected regarding apoptosis, as well as stress-related and inflammatory markers examining BCL2-associated X (Bax), heme oxygenase 1 (Ho-1), interleukin 1 beta (Il1ß), superoxide dismutase 1 (Sod1), and nuclear factor-kappa B (Nfkb) levels. In the optic nerve, AZ increased the levels of expression of Sod1 and Nfkb only in the WT mice and decreased them in the NSG mice. In the retinas of the WT and NSG mice, the Bax and Ho-1 levels of expression decreased following the AZ treatment, while the Sod1 and Nfkb expression decreased only in the WT mice, and remained stable near the baseline in the NSG mice. Il1ß remained at the baseline in WT mice while it decreased towards the baseline in AZ-treated NSG mice. The neuroprotective effects demonstrated by the reduced RGC apoptosis in AZ-treated WT mice retinae, and in the optic nerves as stress-related and inflammatory gene expression increase. This did not occur in the immunodeficient NSG mice. AZ modulated the inflammatory reaction and microglial activation. The lack of an effect in NSG mice supports the assumption that AZ acts by immunomodulation, which is known to play a role in ONC damage. These findings have implications for the development and repurposing of drugs to preserve RGCs after acute optic neuropathies.

Histological and molecular characterization of glaucoma model induced by one or two injections of microbeads to the anterior chamber of mice.

PURPOSE: To characterize glaucoma-induced damage following injections of plastic microbeads into the anterior chamber of mice. METHODS: Mice were divided into three groups: a single plastic microbeads injection (n = 21); two consecutive plastic microbead injections to the right eye at 1-week intervals, 4 of which with two consecutive saline injections in the left eye (n = 15); and an additional control group of two consecutive saline injections at 1-week intervals (n = 6). Intraocular pressure (IOP) was measured weekly. Retinal thickness, ganglion cells (RGCs) and axonal loss, inflammatory and gliosis reactions were measured at week four. Molecular analysis using qRT-PCR in the microbeads injection groups focused on expression levels of inflammation and glaucoma-related genes. RESULTS: Mean IOP following single injection at 4 weeks was significantly elevated compared to baseline in injected eyes (14.5 ± 3.3 mmHg vs. 11.1 ± 2.5 mmHg, respectively, p = 0.003) and not in fellow eyes (13.2 ± 2.9 mmHg vs. 12.2 ± 2.9, respectively, NS). Six (35.3%) bead-injected eyes had IOP ≥ 17 mmHg compared with 2 (11.8%) saline-injected control eyes. Retinal thickness in injected and fellow eyes was 193.7 ± 15.5 µm and 223.9 ± 15.5 µm, respectively (p = 0.03). RGC loss in injected and fellow eyes was 16.0 ± 0.5 and 17.6 ± 0.7 cells per 200 µm, respectively (p = 0.005). Retinal gliosis, axonal loss and inflammatory cell infiltration to the bead-injected eyes were noted. Molecular analysis following double injection showed STAT3 expression decreased in the glaucoma-induced optic nerves (0.69 ± 0.3 vs. 1.16 ± 0.3, p = 0.04), but increased in the glaucoma-induced retinae (p = 0.05) versus saline; retinal IL-1ß decreased significantly (0.04 ± 0.04 vs. 0.36 ± 0.2, p = 0.02). TNF-α, NFkB and SOD-1 expression did not change. CONCLUSION: One/two injections of microbeads elevated IOP, with measurable neuronal damage. An inflammatory response was detected in the injured retina and optic nerve. The therapeutic significance of these findings should be explored.

Dysregulation of anti-Mullerian hormone expression levels in mural granulosa cells of FMR1 premutation carriers.

FMR1 premutation (55-200 CGG repeats) results in fragile X-associated primary ovarian insufficiency (FXPOI). We evaluated expression levels of folliculogenesis-related mediators, follicle-stimulating hormone (FSH) receptor and anti-Mullerian hormone (AMH), to gain insights into the mechanisms underlying the reduced ovarian function. Mural granulosa cells (MGCs) were collected from FMR1 premutation carriers and noncarriers undergoing IVF treatments. At baseline, MGCs of carriers demonstrated significantly higher mRNA expression levels of AMH (3.5 ± 2.2, n = 12 and 0.97 ± 0.5, n = 17, respectively; p = 0.0003) and FSH receptor (5.6 ± 2.8 and 2.7 ± 2.8, respectively; p = 0.02) and higher AMH protein expression on immunostaining. Accordingly, FMR1 premutation-transfected COV434 cells exhibited higher AMH protein expression than COV434 cells transfected with 20 CGG repeats. We conclude that FMR1 premutation may lead to dysregulation of AMH expression levels, probably due to a compensatory mechanism. Elucidating the pathophysiology of FXPOI may help in early detection of ovarian dysfunction and tailoring IVF treatments to FMR1 premutation carriers.

Does gonadotropin-releasing hormone agonist cause luteolysis by inducing apoptosis of the human granulosa-luteal cells?

OBJECTIVES: To evaluates the effect of different modes of final follicular maturation triggering on the degree of apoptosis of granulosa cells (GCs) and the potential effect on progesterone secretion. METHODS: Thirty patients undergoing controlled ovarian hyperstimulation for IVF who received hCG, GnRH agonist, or dual trigger for final follicular maturation were included in the study. Granulosa cells were obtained at the time of oocyte retrieval. The proportion of apoptotic cells was evaluated via TUNEL and immunohistochemistry. RESULTS: The proportion of apoptotic cells was significantly higher in the GnRH agonist-alone group compared to hCG-alone and the dual trigger groups (13.5 ± 1.5% vs. 7.8% ± 1.8 vs. 10.1% ± 2, respectively, P < 0.01). Moreover, the expression of active-caspase-3 was also significantly increased in the GnRH agonist-alone group compared with the hCG-alone and the dual trigger groups (15.5% ± 2.9 vs. 8.4% ± 1.6 vs. 12.7% ± 2.6, respectively, P < 0.01). The progesterone levels measured in the granulosa-luteal cell culture medium after 24 h of incubation were similar between the three groups. CONCLUSIONS: The levels of apoptosis are increased after GnRH agonist/dual trigger. The increased apoptosis might be one of the culprit of the subsequent premature demise of the corpus luteum post GnRH agonist trigger.

Gene-Related Response of Basal Cell Carcinoma to Biologic Treatment with Vismodegib.

We aimed to characterise the response of locally advanced basal cell carcinoma (BCC) to systemic treatment with Vismodegib, a Hedgehog pathway inhibitor, by changes in the expression levels of Hedgehog pathway genes. Data were collected prospectively on 12 patients treated systemically for locally advanced BCC. Biopsy samples taken on admission and after treatment cessation were analysed pathologically and with the NanoString nCounter system to quantify the expression of 40 Hedgehog signaling pathway genes. Findings were compared before and after treatment, between complete and partial responders, and with localised BCC samples from 22 patients. Sixteen Hedgehog pathway genes changed significantly from before to after treatment. GAS1 was the only gene with a significantly different expression at baseline between complete responders (6 patients) and partial responders (4 patients) to Vismodegib (P = 0.014). GAS, GLIS2 and PRKACG1 showed different expression before treatment between the locally advanced and localised BCCs. The baseline expression level of GAS1 appears to be predictive of the response of locally advanced BCC to systemic Vismodegib treatment. A change in expression of many Hedgehog pathway genes, albeit expected by the known activity of Vismodegib, may nevertheless serve as an indicator of the response potential of the tumour.

Pathophysiology Mechanisms in Fragile-X Primary Ovarian Insufficiency.

Women who carry the FMR1 premutation may suffer from ongoing deterioration of ovarian function. The lucidity of the molecular mechanism of FXTAS is emerging and findings from research in the field of FXTAS could elucidate the pathogenesis of FXPOI. To date there are three possible mechanisms for ovarian dysfunction in FMR1 permutation carriers. The first is the RNA toxic gain-of-function mechanism initiating loss of function of over 30 specific RNA-binding proteins. The second is associated to the formation of an abnormal polyglycine-containing protein (FMRpolyG), and the third is related to novel lncRNAs, named FMR4 and FMR6. Herein we describe our laboratory methodology, focusing on the culturing and manipulation of granulosa cells from human female premutation carriers, trying to reveal the actual possible mechanisms liable to FXPOI. Detecting the precise pathways in premutation carrier might facilitate in offering these women the opportunity to make an informed decision regarding their reproductive and family planning.