To study the effectiveness of freeze-thaw antigens and acid eluted peptide antigens extracted from tumor cell-pulsed dendritic cells (DC) in inducing prostate cancer-specific cytotoxic T lymphocytes (CTL) in vitro.Tumor antigens were extracted from the prostate cancer cell line PC-3 with the repeated freeze-thaw and weak acid elution methods. Peripheral blood mononuclear cells were cultured with recombinant human GM-CSF and IL-4 for inducing DCs in vitro. Then the DCs were pulsed with the two kinds of prostate cancer tumor antigens respectively and cultured with T cells for inducing CTLs. The activity of the tumor-specific CTLs were detected by LDH release assay.The protein content in the tumor antigens obtained from PC-3 (2 x 10(7)) by citric acid-phosphate buffer elution and that by the repeated freeze-thaw method were (212.2 +/- 7.9) microg and (963.0 +/- 25.3) microg, respectively. The two kinds of prostate cancer antigens-pulsed DCs had a significant role in inducing the PC-3 cell-specific CTLs, and the CTLs induced by acid-eluted peptide antigen-pulsed DCs exhibited an even more significant tumor-specific cytotoxicity than those induced by repeated freeze-thaw ([60.4 +/- 5.52]% vs. [43.7 +/- 4.11]%, P < 0.01).Both the weak acid elution and repeated freeze-thaw methods for extracting prostate cancer antigens can be used for in vitro sensitization of DCs. The DCs pulsed by either of the two kinds of antigens can activate CTLs, and the antigens extracted by weak acid elution are even more effective.
Lipid droplet-associated proteins such as perilipin 3 (PLIN3) and coatomer GTPase proteins (GBF1, ARF1, Sec23a, and ARFRP1) are expressed in skeletal muscle but little is known so far as to their regulation of lipolysis. We aimed here to explore the effects of lipolytic stimulation in vitro in primary human myotubes as well as in vivo following an acute exercise bout. In vitro lipolytic stimulation by epinephrine (100 μM) or by a lipolytic cocktail (30 μM palmitate, 4 μM forskolin, and 0.5 μM ionomycin, PFI) resulted in increases in PLIN3 protein content. Coatomer GTPases such as GBF1, ARF1, Sec23a, and ARFRP1 also increased in response to lipolytic stimuli. Furthermore, a long duration endurance exercise bout (20 males; age 24.0±4.5 y; BMI 23.6±1.8 kg/m2) increased PLIN3 protein in human skeletal muscle (p = 0.03) in proportion to ex vivo palmitate oxidation (r = 0.45, p = 0.04) and whole body in vivo fat oxidation (r = 0.52, p = 0.03). Protein content of ARF1 was increased (p = 0.04) while mRNA expression was increased for several other coatomers (GBF1, ARF1, and Sec23a, all p<0.05). These data provide novel observational insight into the possible relationships between lipolysis and PLIN3 along with these coatomoer GTPase proteins in human skeletal muscle.
Ocular angiogenic diseases, such as proliferative diabetic retinopathy (PDR), are often characterized by pathological new vessels and fibrosis formation.Anti-vascular endothelial growth factor (VEGF) therapy, despite of its efficiency to inhibit new vessels, has limitations, including drug resistance and retinal fibrosis.Here, we identified that Gremlin1, a novel angiogenesis and fibrosis inducer, was secreted from Müller glial cells, and its expression increased in the vitreous fluid from patients with PDR.Mechanistically, Gremlin1 triggered angiogenesis by promoting endothelial-mesenchymal transition via the EGFR/RhoA/ROCK pathway.In addition, Gremlin1 activated microglia to present profibrotic and fibrogenic properties.Further, anti-Gremlin1 antibody inhibited ocular angiogenesis and microglia fibrosis in mouse models.Collectively, Gremlin1 could be a potential therapeutic target in the treatment of ocular angiogenic diseases.
An acoustic coupling scheme largely determines the performance of optical-resolution photoacoustic microscopy (OR-PAM), including practicability, sensitivity, and stability. In this study, we propose OR-PAM based on a local-flexible acoustic coupling scheme, which includes a well-designed combiner connecting a set of circulating systems. The combiner integrates an objective lens and an ultrasonic transducer, controls the water level, restricts the flow rate, and drains bubbles. The circulating system provides sustained and steady flowing water. The flowing water constrained in the combiner and the circulating system forms a flexible and stable local contact between the sample and the transducer. Phantom experiments demonstrate that the proposed method can maintain high optical resolution but improve the detection sensitivity by approximately 1.9 times in comparison to dry coupling. In vivo imaging experiments of the mouse eyeground are conducted to examine the practicability of the proposed system in biomedicine. Moreover, in vivo experiments show that OR-PAM based on local-flexible coupling can reveal more details of eyeground microvasculatures, benefiting from its enhanced sensitivity. These merits promise that OR-PAM based on local-flexible coupling may have broad applications in biomedical fields.
Gram-negative bacteria contain a double membrane system which is made up of the inner membrane (IM) and the outer membrane (OM). The OM, the asymmetric membrane, is an important contributor towards resistance against various toxic molecules which provides extensive protection of cell viability. The OM consists of lipopolysaccharides (LPS) at the outer leaflet and glycerophospholipid (GPL) at the inner leaflet. LPSisa large amphipathic moleculethatis essential for most Gram-negative bacteria and is composed of three moieties: lipid A, core oligosaccharide and O-antigen. Precursors of LPS, the rough LPS and O-antigen units, are synthesized at the inner leaflet of the IM and are assembled into mature LPS at the outer leaflet of the IM. Seven proteins, lipopolysaccharide transporters (LptA-G), form a periplasmic bridge and transport mature LPS from the IM tothe outer leaflet of the OM, where LPS functions as an impermeable barrier. The inner leaflet of the OM is also an obligate componentfor the stability and functionality of OM. GPL molecules consist of phosphatidylethanolamine (PE), phosphatidylglycerols (PG) and cardiolipin (CL) in an approximately 75: 20: 5 ratio in most bacterial membranes. The biogenesis and transport of LPS and GPL are often the highlights of novel drug development; however little is known about the mechanisms of the actionsof the proteins involved in these processes.
Protein X-ray crystallography is a technique that determinesthe molecular structure of a protein or a protein complex from the diffraction data of a protein crystal generated underX-ray beam. Knowing a protein’s molecular structure provides us with the informationwhich may include possible molecular motion and conformational changes or interactions between residues, substrates and domains. These important details may provide hints into how to perform further studies on broad-ranged specific targets.
This thesis includes four Chapters related to the X-ray crystal determination of proteins involved in biosynthesis, modificationand transport of LPS and the transport of CL. In Chapter 1 I have included the successful cloning, expression and structure determination of the ABC transporter LptB2FG, which is responsible for LPS extraction from the inner membrane. Also, the corresponding in vivoand in vitroassays are reported to support the LPS extraction and transportation functionality of LptB2FG. Chapter 2 contains the successful expression of glycosyl transferase WaaB and the determination of its structure. Mutagenesis and enzyme activity assays have also been performed to determine the galactosyl transferase activity site of WaaB(work by Gareth James Asheworth). Chapter 3 describesthe successful cloning and structural determination of LapB, which may controlan intermediated step of lipid A biosynthesis via balancing the level of LpxC & FtsH, Finally, Chapter 4 reveals the protein expression and structural determination of the globular domain of PbgA. PbgA is regarded as the cardiolipin transportation protein and we have also performed in vivo mutagenesis functional assays to identify the important residues for its functionality.
The present study was designed to explore the clinical values of microRNA-129 (miR-129) expression in peripheral blood mononuclear cells for prostate cancer patients and the role of miR-129 in the proliferation of prostate cancer.The peripheral blood mononuclear cells were isolated form blood simple from 98 patients confirmed with prostate cancer and 56 matched healthy volunteers. Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression level of miR-129 in peripheral blood mononuclear cells. Cox proportional hazards regression models and Kaplan-Meier analysis were used to evaluate the association of miR-129 expression with clinical and pathological characteristics of prostate cancer patients. The effect of miR-129 on the proliferation of prostate cancer cells in vitro was also determined.Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the expression of miR-129 was dramatically down-regulated in peripheral blood mononuclear cells for prostate cancer patients in comparison with healthy controls (P<0.05). The decrease in miR-129 expression in peripheral blood mononuclear cells was significantly associated with aggressive clinical pathological features such as histological grade (P=0.010), high preoperative PSA level (P=0.002), pathological stage (P=0.011), high Gleason score (P=0.005), lymph node metastasis (P=0.002), angiolymphatic invasion (P=0.004), biochemical recurrence (P=0.001). The prostate cancer patients with a low miR-129 expression in peripheral blood mononuclear cells had an obviously shorter BCR-free survival compared with high miR-129 expression (P<0.001). The Cox multivariate analysis established that the miR-129 expression may be an independent prognostic factor for biochemical recurrence (BCR)-free survival prostate cancer patients (P=0.000). The results of in vitro CCK-8 assays, as well as proliferating cell nuclear antigen (PCNA) and phosphorylated histone-3 (P-H3) (markers of proliferation) indicated that miR-129 overexpression markedly retarded the proliferation of PC-3 and DU-145 cells.Our results provide the first evidence that the miR-129 is significantly downregulated in prostate cancer patients and multivariate analysis confirmed that miR-129 is a novel independent prognostic factor for prostate cancer. Overexpression of miR-129 exerts tumor suppressive functions and abrogates prostate cancer growth.
Purpose: To compare the efficacy of a modified perfluorocarbon liquid–assisted inverted internal limiting membrane (ILM) flap technique with the standard ILM peeling for the treatment of macular hole retinal detachment in highly myopic eyes. Methods: This was a retrospective, consecutive, nonrandomized comparative study. Forty-two macular hole retinal detachment eyes of 42 patients were included into either a perfluorocarbon liquid–assisted inverted ILM flap technique group (n = 22, inverted group) or standard ILM removal group (n = 20, peeling group). Outcomes measured were macular hole closure, retinal reattachment, and best-corrected visual acuity at least 6 months after surgery. Results: Macular hole closure was achieved in 20 eyes (90.9%) in the inverted group and in eight eyes (40%) in the peeling group ( P < 0.01). Reattachment rates were 100% in the inverted group and 95% in the peeling group ( P = 0.476). The mean best-corrected visual acuity improvement from baseline was 27.4 ± 19.9 Early Treatment Diabetic Retinopathy Study letters in the inverted group while the best-corrected visual acuity improvement was 13.6 ± 22.5 Early Treatment Diabetic Retinopathy Study letters in the peeling group ( P = 0.044). Conclusion: The perfluorocarbon liquid–assisted inverted ILM flap technique was effective in sealing the macular hole, reattaching retina, and improving visual function postoperatively in highly myopic macular hole retinal detachment.
Retinal diseases, the leading causes of vison loss and blindness, are associated with complicated pathogeneses such as angiogenesis, inflammation, immune regulation, fibrous proliferation, and neurodegeneration. The retina is a complex tissue, where the various resident cell types communicate between themselves and with cells from the blood and immune systems. Exosomes, which are bilayer membrane vesicles with diameters of 30-150 nm, carry a variety of proteins, lipids, and nucleic acids, and participate in cell-to-cell communication. Recently, the roles of exosomes in pathophysiological process and their therapeutic potential have been emerging. Here, we critically review the roles of exosomes as possible intracellular mediators and discuss the possibility of using exosomes as therapeutic agents in retinal diseases.
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