46 research outputs found
Delivery of MicroRNA-10b with Polylysine Nanoparticles for Inhibition of Breast Cancer Cell Wound Healing
Recent studies revealed that micro RNA-10b (mir-10b) is highly expressed in metastatic breast cancer cells and positively regulates breast cancer cell migration and invasion through inhibition of HOXD10 target synthesis. In this study we designed anti-mir-10b molecules and combined them with poly L-lysine (PLL) to test the delivery effectiveness. An RNA molecule sequence exactly matching the mature mir-10b minor antisense showed strong inhibition when mixed with PLL in a wound-healing assay with human breast cell line MDA-MB-231. The resulting PLL-RNA nanoparticles delivered the anti-microRNA molecules into cytoplasm of breast cancer cells in a concentration-dependent manner that displayed sustainable effectiveness
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Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices
Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3[subscript]T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations
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Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms
The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.This is the publisher’s final pdf. The published article is copyrighted by American Chemical Society and can be found at: http://pubs.acs.org/page/asbcd6/about.htmlKeywords: TNT, biarsenical probe, diatom, silica, anthrax, biosensorKeywords: TNT, biarsenical probe, diatom, silica, anthrax, biosenso
ALDH1A1 Deficiency in Gorlin Syndrome Suggests a Central Role for Retinoic Acid and ATM Deficits in Radiation Carcinogenesis
We hypothesize that aldehyde dehydrogenase 1A1 (ALDH1A1) deficiency will result in impaired ataxia-telangiectasia mutated (ATM) activation in a retinoic acid-sensitive fashion. Data supporting this hypothesis include (1) reduced ATM activation in irradiated primary dermal fibroblasts from ALDH1A1-deficient Gorlin syndrome patients (GDFs), relative to ALDH1A1-positive normal human dermal fibroblasts (NHDFs) and (2) increased ATM activation by X-radiation in GDFs pretreated with retinoic acid, however, the impact of donor variability on ATM activation in fibroblasts was not assessed and is a prudent consideration in future studies. Clonogenic survival of irradiated cells showed differential responses to retinoic acid as a function of treatment time. Long-term (5 Day) retinoic acid treatment functioned as a radiosensitizer and was associated with downregulation of ATM protein levels. Short-term (7 h) retinoic acid treatment showed a trend toward increased survival of irradiated cells and did not downregulate ATM protein levels. Using a newly developed IncubATR technology, which defines changes in bulk chemical bond patterns in live cells, we can discriminate between the NHDF and GDF phenotypes, but treatment of GDFs with retinoic acid does not induce reversion of bulk chemical bond patterns associated with GDFs toward the NHDF phenotype. Collectively, our preliminary investigation of the Gorlin phenotype has identified deficient ALDH1A1 expression associated with deficient ATM activation as a possible susceptibility factor that is consistent with the high incidence of spontaneous and radiation-induced carcinogenesis in these patients. The IncubATR technology exhibits sufficient sensitivity to detect phenotypic differences in live cells that may be relevant to radiation health effects
The role of , TNF-α, IL-6, and IL-10 in the development of myocardial ischemia with type 2 diabetes mellitus
This study is to observe the distribution of intestinal flora and the changes of inflammatory factors in elderly patients with myocardial ischemia complicated with type 2 diabetes mellitus. A total of 106 elderly patients with myocardial ischemia complicated with type 2 diabetes mellitus (complicated group), 106 elderly patients with simple type 2 diabetes mellitus (diabetic group), and 106 healthy elderly people (control group) were selected. The fasting blood glucose (FBG), 1-h postprandial blood glucose (1hPG), 2-h postprandial blood glucose (2hPG), 3-h postprandial blood glucose (3hPG), and hemoglobin A1c (HbA1c) in complicated group and the diabetic group were higher than those in the control group ( P < 0.05 or P < 0.01). The duration of diabetes, FBG, 3hPG, and HbA1c in the complicated group were higher than those in the diabetic group, while the 2hPG was lower than that in the diabetic group ( P < 0.05). Compared with control group, the number of Enterobacteria in the diabetic group and complicated group was increased, while the numbers of Bacteroides, Bifidobacteria , and Lactobacillus were decreased ( P < 0.05 or P < 0.01). Compared with the diabetic group, the number of Enterobacteria in complicated group was increased, while the numbers of Bacteroides, Bifidobacteria , and Lactobacillus were decreased ( P < 0.05 or P < 0.01). Compared with control group, the levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 1 beta (IL-1β), and C-reactive protein (CRP) decreased in the diabetic group and complicated group, and the lowest in the complicated group. Conversely, the levels of interleukin 10 (IL-10) and interleukin 12 (IL-2) increased in the diabetic group and complicated group, and the highest in the complicated group ( P < 0.05 or P < 0.01). Multiple logistic regression analysis showed that the duration of diabetes, HbA1c, Enterobacteria , TNF-α, IL-6, and IL-10 were the influencing factors of myocardial ischemia complicated with type 2 diabetes mellitus ( P < 0.05 or P < 0.01). In conclusion, in the elderly patients with myocardial ischemia complicated with type 2 diabetes mellitus, the number of intestinal probiotics and the level of anti-inflammatory factors decreased, and the number of pathogenic bacteria and the level of inflammatory factors increased. Enterobacteria , TNF-α, IL-6, and IL-10 may play an important role in the development of myocardial ischemia in type 2 diabetes mellitus
Optimized Design and Synthesis of a Cell-Permeable Biarsenical Cyanine Probe for Imaging Tagged Cytosolic Bacterial Proteins
To optimize cellular delivery and specific labeling of
tagged cytosolic
proteins by biarsenical fluorescent probes built around a cyanine
dye (Cy3) scaffold, we have systematically varied the polarity of
the N-alkyl chain (i.e., 4–5 methylene groups appended by a
sulfonate or methoxy ester moiety) and arsenic capping reagent (ethanedithiol
versus benzenedithiol). Optimal live-cell labeling and visualization
of tagged cytosolic proteins is reported using an ethanedithiol capping
reagent with the uncharged methoxy ester functionalized N-alkyl chains.
These measurements demonstrate the general utility of this new class
of photostable and highly fluorescent biarsenical probes based on
the cyanine dye scaffold for in vivo labeling of tagged cellular proteins
for live cell imaging measurements of protein dynamics
Synthesis of a Targeted Biarsenical Cy3-Cy5 Affinity Probe for Super-resolution Fluorescence Imaging
Photoswitchable fluorescent probes capable of the targeted
labeling
of tagged proteins are of significant interest due to their ability
to enable <i>in situ</i> imaging of protein complexes within
native biomolecular assemblies. Here we describe the synthesis of
a fluorescent probe (<b>AsCy3Cy5</b>) and demonstrate the targeted
labeling and super-resolution imaging of a tagged protein within a
supramolecular protein complex
Molecular evidence of a toxic effect on a biofilm and its matrix
Shewanella oneidensis MR-1 wild-type and a hyper-adhesive mutant CP2-1-S1 are used as model organisms and Cr(vi) is selected as a toxic chemical to study biofilm and toxic chemical interactions. Biofilms are cultured in a microfluidic device for in situ time-of-flight secondary ion mass spectrometry imaging. This approach is viable for studying biofilms' responses to antimicrobial resistance.Accepted versio