In this paper, we have developed an interesting imaging method for intracellular ATP molecules with semi-quantitation. While there has been a lot of work in understanding intracellular events, very few can come close to quantitation or semi-quantitation in living cells. In this work, we made an effective use of nanomaterials, graphene oxides, both as a quencher and a carrier for intracellular delivery. In addition, this graphene oxide also serves as the carrier for reference probes for fluorescent imaging. An ATP aptamer molecular beacon (AAMB) is adsorbed on graphene oxide (GO) to form a double quenching platform. The AAMB/GO spontaneously enters cells, and then AAMB is released and opened by intracellular ATP. The resulting fluorescence recovery is used to perform ATP live-cell imaging with greatly improved background and signaling. Moreover, a control ssDNA, which is released non-specifically from GO by non-target cellular proteins, can serve as an internal reference for ATP semi-quantification inside living cells using the intensity ratio of the AAMB and control. This approach can serve as a way for intracellular delivery and quantitative analysis.
nonspecific desorption; graphene oxide; aptamer molecular beacon; internal reference; ATP; semi-quantification
Rare protein enrichment and sensitive detection hold great potential in biomedical studies and clinical practice. This work describes using aptamer-conjugated gold nanorods for the efficient enrichment of rare proteins from buffer solutions and human plasma. Gold nanorod (AuNR) surfaces were modified with a long PEG chain and a 15-mer thrombin aptamer for protein enrichment and detection. Studies of the effect of surface modification on enrichment efficiency of thrombin showed that a change of only one EG6 linker unit, i.e., from 2EG6 to 3EG6, could increase thrombin protein capture efficiency by up to 47%. Furthermore, a 1-ppm sample of thrombin in buffer could be enriched with around 90% efficiency using a low concentration (0.19 nM) of gold nanorod probe modified with 3EG6 spacer, and with the same probe, effective capture was achieved down to 10 ppb (1 ng) thrombin in plasma samples. In addition to α-thrombin enrichment, prothrombin was also efficiently captured from plasma samples via gold nanorods conjugated with 15-mer thrombin aptamer. Our work demonstrates efficient enrichment of rare proteins using aptamer modified nanomaterials, which can be used in biomarker discovery studies.
Protein Enrichment; Gold Nanorods; Surface Modification; Electrophoresis
Whole body heat stress had detrimental effect on male reproductive function. It's known that the nuclear factor erythroid 2-related factor 2 (Nrf2) activates expression of cytoprotective genes to enable cell adaptation to protect against oxidative stress. However, it’s still unclear about the exactly effects of Nrf2 on the testis. Here, we investigate the protective effect of Nrf2 on whole body heat stress-induced oxidative damage in mouse testis.
Male mice were exposed to the elevated ambient temperature (42°C) daily for 2 h. During the period of twelve consecutive days, mice were sacrificed on days 1, 2, 4, 8 and 12 immediately following heat exposure. Testes weight, enzymatic antioxidant activities and concentrations of malondialdehyde (MDA) and glutathione (GSH) in the testes were determined and immunohistochemical detection of Nrf2 protein and mRNA expression of Nrf2-regulated genes were analyzed to assess the status of Nrf2-antioxidant system.
Heat-exposed mice presented significant increases in rectal, scrotal surface and body surface temperature. The concentrations of cortisol and testosterone in serum fluctuated with the number of exposed days. There were significant decrease in testes weight and relative testes weight on day 12 compared with those on other days, but significant increases in catalase (CAT) activity on day 1 and GSH level on day 4 compared with control group. The activities of total superoxide dismutase (T-SOD) and copper-zinc SOD (CuZn-SOD) increased significantly on days 8 and 12. Moreover, prominent nuclear accumulation of Nrf2 protein was observed in Leydig cells on day 2, accompanying with up-regulated mRNA levels of Nrf2-regulated genes such as Nrf2, heme oxygenase 1 (HO-1), γ-Glutamylcysteine synthetase (GCLC) and NAD (P) H: quinone oxidoreductase 1 (NQO1)) in heat-treated groups.
These results suggest that Nrf2 displayed nuclear accumulation and protective activity in the process of heat treated-induced oxidative stress in mouse testes, indicating that Nrf2 might be a potential target for new drugs designed to protect germ cell and Leydig cell from oxidative stress.
Whole body heat stress; Nrf2; Oxidative stress; Germ cell; Leydig cell; Mice
We assessed HIV prevalence and associated behaviors and risk factors among men who have sex with men (MSM) in Beijing, China. Five hundred MSM were recruited for a biological and behavioral survey using respondent-driven sampling (RDS) in 2009. Serologic specimens were tested for markers of HIV and syphilis infection. A computer-assisted personal interview (CAPI) administered questionnaire gathered information including demographic characteristics, sexual behaviors, HIV testing, and social norms concerning condom use. The adjusted HIV prevalence was 8.0%, syphilis 22.0%. HIV testing and disclosure was low; only 39.3% had HIV tested in the past 12 months, 49.7% knew their own HIV status and 22.8% knew their last male partner's HIV status. HIV infection was associated with syphilis, ever having sex with a woman, not knowing the HIV status of the most recent male partner, and never buying condoms in the past 12 months. Stronger endorsement of positive social norms around condom use strongly and predicted lower prevalence of HIV infection. Compared to surveys of similar design in the recent past, HIV continues to spread rapidly among Beijing's MSM. Our results identify points of intervention that, if addressed in time, may still alter the course of the epidemic including the promotion of HIV testing and partner disclosure, syphilis control and particularly changing social norms around condom use.
The aim of the present work was to investigate the influence of formulation factors including different grades of Carbopol® matrices and penetration enhancers on the percutaneous permeation of tetrahydropalmatine (THP), rheological properties, and in vitro release; and the correlation behind rheological properties, in vitro release, and percutaneous permeation. Transdermal penetration of THP through excised rabbit skin and in vitro release of THP across transparent Cellophane® were performed by vertical Franz diffusion cell. Rheological analyses were proceeded in terms of “steady flow tests”, “oscillation stress sweep”, and “creep recovery”. The result of percutaneous penetration of THP indicated that, the emulgel prepared with Carbopol® 971P (Cp 971P) as the matrix and N-methyl-2-pyrrolidone (NMP) as the penetration enhancer had the highest cumulative permeation amount (118.19 μg/cm2). All the experimental data showed a good fit to the Casson model in viscosimetric studies no matter what the types of matrices or the kinds of penetration enhancers were. The release profile fitted the zero-order release kinetics model with Cp 971P as the matrix without any penetration enhancers. However, when adding penetration enhancers, in vitro release of THP presented anomalous (non-Fickian) release kinetics. Clarifying the relationship behind percutaneous permeation of THP, rheological properties, and in vitro release will provide us with profound insights and facilitate the design of specific emulgel.
Carbopol®; emulgel; in vitro release; rheological properties; THP
Inhibition of p-glycoprotein under hyperglycemic conditions has been reported in various barrier tissues including blood-brain barrier, intestine, and kidney, and has been linked to significant clinical complications. However, whether this is also true for the outer blood-retinal barrier constituted by retinal pigment epithelium, or has a role in pathogenesis of diabetic retinopathy is not yet clear. In this study, using cultured human retinal pigment epithelium cell line D407, we found that high glucose exposure induced a significant decrease in p-glycoprotein expression both at mRNA and at protein levels, accompanied by an attenuated p-glycoprotein activity determined by intracellular rhodamine 123 retention. In marked contrast, the expressions of both mRNA and protein levels of inducible nitrate oxide synthase (iNOS) increased, and were accompanied by increased extracellular nitrate/nitrite production by Griess reaction. In addition, mRNA levels of nuclear receptors revealed a decreased expression of pregnane X receptor after the exposure of high glucose. However, the subsequent alterations in production of nitrate/nitrite, functional expression of p-glycoprotein, and mRNA levels of pregnane X receptor were partially blocked when pretreated with S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea•2HBr (PBITU), a selective iNOS inhibitor. Moreover, the effects of PBITU were antagonized with the addition of L-arginine, a substrate for NO synthesis. Our in vitro results suggest for the first time that iNOS induction plays a novel role in decreased p-glycoprotein expression and transport function at the human outer blood-retinal barrier under hyperglycemic conditions and further support the concept of inhibiting iNOS pathway as a therapeutic strategy for diabetic retinopathy.
Eight proteins, defects in which are associated with Meckel-Gruber syndrome and nephronophthisis ciliopathies, work together as two functional modules at the transition zone to establish basal body/transition zone connections with the membrane and barricade entry of non-ciliary components into this organelle.
Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and related ciliopathies present with overlapping phenotypes and display considerable allelism between at least twelve different genes of largely unexplained function. We demonstrate that the conserved C. elegans B9 domain (MKS-1, MKSR-1, and MKSR-2), MKS-3/TMEM67, MKS-5/RPGRIP1L, MKS-6/CC2D2A, NPHP-1, and NPHP-4 proteins exhibit essential, collective functions at the transition zone (TZ), an underappreciated region at the base of all cilia characterized by Y-shaped assemblages that link axoneme microtubules to surrounding membrane. These TZ proteins functionally interact as members of two distinct modules, which together contribute to an early ciliogenic event. Specifically, MKS/MKSR/NPHP proteins establish basal body/TZ membrane attachments before or coinciding with intraflagellar transport–dependent axoneme extension and subsequently restrict accumulation of nonciliary components within the ciliary compartment. Together, our findings uncover a unified role for eight TZ-localized proteins in basal body anchoring and establishing a ciliary gate during ciliogenesis, and suggest that disrupting ciliary gate function contributes to phenotypic features of the MKS/NPHP disease spectrum.
The hawthorn fruit (Crataegus pinnatifida Bunge var. typica Schneider) is used as a traditional medicine in Korea. The objective of this study was to understand the mechanisms of the anti-inflammatory effects of the water fractionated portion of hawthorn fruit on a lipopolysaccharide (LPS)-stimulated RAW 264.7 cellular model. The level of nitric oxide (NO) production in the water fraction and LPS-treated RAW 264.7 cells were determined with an ELISA. The cytotoxicity of the water fraction and LPS was measured with an MTT assay. Expression of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, interleukin 6 (IL-6), and interleukin 1β (IL-1β) mRNA were analyzed with a reverse transcription polymerase chain reaction (RT-PCR). The water fraction of hawthorn fruit was determined to be safe and significantly inhibited NO production in LPS-stimulated RAW 264.7 cells and suppressed COX-2, TNF-α, IL-1β, and IL-6 expression. The observed anti-inflammatory effects of the water fraction of hawthorn fruit might be attributed to the down-regulation of COX-2, TNF-α, IL-1β, and IL-6 expression in LPS-stimulated RAW 264.7 cells.
Anti-inflammatory; hawthorn fruit; LPS; RAW 264.7 cell
The purpose of this study was to assess the prevalence and risk factors for syphilis infection among female sex workers (FSWs) in Liuzhou City, Guangxi Zhuang Autonomous Region, southern China. A cross-sectional study recruited FSWs using a venue-based method and subsequent snowball sampling with mapping strategies. Questionnaire-based interviews were conducted to collect demographic and behavioral information. Blood was tested for syphilis (RPR with TPPA confirmation) and HIV (EIA with Western blot confirmation) infections. Of the 362 eligible participants, 81.7% were non-local residents (60.5% from other parts of Guangxi and 21.2% were non-Guangxi residents); 58.0% belonged to non-Han minority ethnic groups; 37.5% reported inconsistent condom use with their clients and 71.2% reported inconsistent condom use with their regular sex partners during the past month. Nearly 10% reported having had sex with drug users. The prevalence rates for HIV and syphilis infections were 2.3% and 11.0%, respectively. Almost half (46.6%) of participants reported having had STD symptoms. Inconsistent condom use with clients in the past month (AOR=5.0; 95%CI=1.8-13.8), less education (OR=2.6; 95% CI=1.1-7.4), and HIV infection (AOR=8.1; 95%CI=1.1-68.5) were independently associated with syphilis infection.
Silks fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl2 and Na2HPO4. These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering.
osteogenesis; silk scaffold; hydroxyapatite; tissue engineering; stem cells
Bombyx mori silk fibroin self-assembles on surfaces to form ultrathin nanoscale coatings based on our prior studies using layer-by-layer deposition techniques driven by hydrophobic interactions between silk fibroin protein molecules. In the present study, polylactic-co-glycolic acid (PLGA) and alginate microspheres were used as substrates and coated with silk fibroin. The coatings were visualized by confocal laser scanning microscopy using fluorescein-labeled silk fibroin. On PLGA microspheres the coating was ~1 μm and discontinuous, reflecting the porous surface of these microspheres determined by SEM. In contrast, on alginate microspheres the coating was ~10 μm thick and continuous. The silk fibroin penetrated into the alginate gel matrix. The silk coating on the PLGA microspheres delayed PLGA degradation. The silk coating on the alginate microspheres survived ethylenediamine tetraacetic acid (EDTA) treatment used to remove the Ca+2-cross-links in the alginate gels to solubilize the alginate. This suggests that alginate microspheres can be used as templates to form silk microcapsules. Horseradish peroxidase (HRP) and tetramethylrhodamine-conjugated bovine serum albumin (BSA) as model protein drugs were encapsulated in the PLGA and alginate microspheres with and without the silk fibroin coatings. Drug release was significantly retarded by the silk coatings when compared to uncoated microsphere controls, and was retarded further by methanol-treated silk coating when compared to silk water-based coatings on alginate microspheres. Silk coatings on PLGA and alginate microspheres provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. This coating technique has potential for biosensor and drug delivery applications due to the aqueous process employed, the ability to control coating thickness and crystalline content, and the biocompatibility of the silk fibroin protein used in the process.
To assess the predictors and prevalence of HIV infection among injection drug users in highly endemic regions along major drug trafficking routes in three Chinese provinces.
We enrolled participants using community outreach and peer referrals. uestionnaire-based interviews provided demographic, drug use, and sexual behavior information. HIV was tested via ELISA and syphilis by RPR.
Of the 689 participants, 51.8% were HIV-infected, with persons living in Guangxi having significantly lower prevalence (16.4%) than those from Xinjiang and Yunnan (66.8% and 67.1%, respectively). Syphilis seropositivity was noted in 5.4%. Longer duration of IDU, greater awareness of HIV transmission routes, and living in Xinjiang or Yunnan were associated with HIV seropositivity on multivariable analysis. Independent risk factors differed between sites. In Guangxi, being male and having a longer duration of IDU were independent risk factors for HIV infection; in Xinjiang, older age and sharing needles and/or syringes were independent factors; in Yunnan, more frequent drug injection, greater awareness of HIV transmission routes, and higher income were independent predictors of HIV seropositivity.
Prevalence rates of HIV among IDUs in China are more than two out of three in some venues. Risk factors include longer duration of IDU and needle sharing. Also associated with HIV were factors that may indicate some success in education in higher risk persons, such as higher knowledge. A systemic community-level intervention with respect to evidenced-based, population-level interventions to stem the spread of HIV from IDU in China should include needle exchange, opiate agonist-based drug treatment, condom distribution along with promotion, and advocacy for community-based VCT with bridges to HIV preventive services and care.
The synthesis and characterization of thermoresponsive, water-soluble poly-N -isopropyl acrylamide (PNIPAM) derived macroligands displaying cyclosporin A (CsA) and dexamethasone (Dex) for use as novel affinity resins is described. Characterization of these soluble macroligands, including ligand loading and integrity was determined by 1H NMR spectroscopy. One of the CsA-macroligands was used in a protein affinity experiment to capture known binding proteins of CsA, the cyclophilins, from Jurkat T-cell lysates.
Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.
MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.
The transport of protein complexes and associated cargo along microtubule tracks represents an essential eukaryotic process responsible for a multitude of cellular functions, including cell division, vesicle movement to membranes, and trafficking along dendrites, axons, and cilia. The latter organelles are hair-like cellular appendages implicated in cell and fluid motility, sensing and transducing information from their environment, and development. Their biogenesis and maintenance depends on a kinesin- and dynein-mediated motility process termed intraflagellar transport (IFT). In addition to comprising these specialized molecular motors, the IFT machinery consists of large multisubunit complexes whose exact composition and organization has not been fully defined. Here we identify a protein, DYF-11/MIP-T3, that is conserved in all ciliated organisms and is associated with IFT in C. elegans. Disruption of C. elegans DYF-11 results in structurally compromised cilia, likely as a result of IFT motor and subunit misassembly. Animals lacking DYF-11 display chemosensory anomalies, consistent with a role for the protein in cilia-associated sensory processes. In zebrafish, MIP-T3 is essential for gastrulation movements during development, similar to that observed for other ciliary components, including Bardet-Biedl syndrome proteins. In conclusion, we have identified a novel IFT machinery component that is also essential for development in vertebrates.
Sensory cilium biogenesis within Caenorhabditis elegans neurons depends on the kinesin-2–dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs (Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as “modules.” We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegans IFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis.
The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet–Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.