Complexes of tubulin oligomers and tau form a viscoelastic intervening network cross-bridging microtubules into bundles.

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. Tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in varying concentrations of Mg2+ or Ca2+ divalent cations in mixtures containing αß-tubulin, full-length tau, and GTP at 37 °C in a physiological buffer. A concentration-time kinetic phase diagram generated by synchrotron SAXS reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. SAXS with TEM of plastic-embedded samples provides evidence of a viscoelastic intervening network (IN) of complexes of tubulin oligomers and tau stabilizing MT bundles. In this model, αß-tubulin oligomers in the IN are crosslinked by tau's MT binding repeats, which also link αß-tubulin oligomers to αß-tubulin within the MT lattice. The model challenges whether the cross-bridging of MTs is attributed entirely to MAPs. Tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau, promoting nucleation and growth of tau fibrils in tauopathies.

Exosomes are secreted at similar densities by M21 and PC3 human cancer cells and show paclitaxel solubility.

Exosomes are cell-secreted vesicles less than ≈150 nm in size that contain gene-encoding and gene-silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind specific cells and deliver intact genetic material to the cytosol of target cells. We isolated extracellular vesicles (EVs), consisting of a mixture of exosomes and microvesicles, from prostate (PC3) and melanoma (M21) cancer cell lines using serial ultracentrifugation. Interrogation via western blot analysis confirmed enrichment of CD63, a widely recognized EV surface protein, in the EV pellet from both cell lines. Nanoparticle tracking analysis (NTA) of EV pellets revealed that the two cell lines produced distinct vesicle size profiles in the ≈30 nm to ≈400 nm range. NTA further showed that the fraction of exosomes to all EVs was constant, suggesting cellular mechanisms that control the fraction of secreted vesicles that are exosomes. Transmission electron microscopy (TEM) images of the unmodified PC3 EVs showed vesicles with cup-like (i.e., nanocapsule) and previously unreported prolate morphologies. The observed non-spherical morphologies for dehydrated exosomal vesicles (size ≈30-100 nm) are most likely related to the dense packing of proteins in exosome membranes. Solubility phase diagram data showed that EVs enhanced the solubility of paclitaxel (PTX) in aqueous solution compared to a water-only control. Combined with their inherent targeting and cytosol delivery properties, these findings highlight the potential advantages of using exosomes as chemotherapeutic drug carriers in vivo.

Hybrid Theranostic Platform for Second Near-IR Window Light Triggered Selective Two-Photon Imaging and Photothermal Killing of Targeted Melanoma Cells.

Despite advances in the medical field, even in the 21st century cancer is one of the leading causes of death for men and women in the world. Since the second near-infrared (NIR) biological window light between 950 and 1350 nm offers highly efficient tissue penetration, the current article reports the development of hybrid theranostic platform using anti-GD2 antibody attached gold nanoparticle (GNP) conjugated, single-wall carbon nanotube (SWCNT) for second near-IR light triggered selective imaging and efficient photothermal therapy of human melanoma cancer cell. Reported results demonstrate that due to strong plasmon-coupling, two-photon luminescence (TPL) intensity from theranostic GNP attached SWCNT materials is 6 orders of magnitude higher than GNP or SWCNT alone. Experimental and FDTD simulation data indicate that the huge enhancement of TPL intensity is mainly due to strong resonance enhancement coupled with the stronger electric field enhancement. Due to plasmon coupling, the theranostic material serves as a local nanoantennae to enhance the photothermal capability via strong optical energy absorption. Reported data show that theranostic SWCNT can be used for selective two-photon imaging of melanoma UACC903 cell using 1100 nm light. Photothermal killing experiment with 1.0 W/cm(2) 980 nm laser light demonstrates that 100% of melanoma UACC903 cells can be killed using theranostic SWCNT bind melanoma cells after just 8 min of exposure. These results demonstrate that due to plasmon coupling, the theranostic GNP attached SWCNT material serves as a two-photon imaging and photothermal source for cancer cells in biological window II.

Antimicrobial Peptide-Conjugated Graphene Oxide Membrane for Efficient Removal and Effective Killing of Multiple Drug Resistant Bacteria.

According to the World Health Organization (WHO), multiple drug-resistant (MDR) bacterial infection is a top threat to human health. Since bacteria evolve to resist antibiotics faster than scientists can develop new classes of drugs, the development of new materials which can be used, not only for separation, but also for effective disinfection of drug resistant pathogens is urgent. Driven by this need, we report for the first time the development of a nisin antimicrobial peptide conjugated, three dimensional (3D) porous graphene oxide membrane for identification, effective separation, and complete disinfection of MDR methicillin-resistant Staphylococcus aureus (MRSA) pathogens from water. Experimental data show that due to the size differences, MRSA is captured by the porous membrane, allowing only water to pass through. SEM, TEM, and fluorescence images confirm that pathogens are captured by the membrane. RT-PCR data with colony counting indicate that almost 100% of MRSA can be removed and destroyed from the water sample using the developed membrane. Comparison of MDR killing data between nisin alone, the graphene oxide membrane and the nisin attached graphene oxide membrane demonstrate that the nisin antimicrobial peptide attached graphene oxide membrane can dramatically enhance the possibility of destroying MRSA via a synergestic effect due to the multimodal mechanism.

Multifunctional biocompatible graphene oxide quantum dots decorated magnetic nanoplatform for efficient capture and two-photon imaging of rare tumor cells.

Circulating tumor cells (CTCs) are extremely rare cells in blood containing billions of other cells. The selective capture and identification of rare cells with sufficient sensitivity is a real challenge. Driven by this need, this manuscript reports the development of a multifunctional biocompatible graphene oxide quantum dots (GOQDs) coated, high-luminescence magnetic nanoplatform for the selective separation and diagnosis of Glypican-3 (GPC3)-expressed Hep G2 liver cancer tumor CTCs from infected blood. Experimental data show that an anti-GPC3-antibody-attached multifunctional nanoplatform can be used for selective Hep G2 hepatocellular carcinoma tumor cell separation from infected blood containing 10 tumor cells/mL of blood in a 15 mL sample. Reported data indicate that, because of an extremely high two-photon absorption cross section (40530 GM), an anti-GPC3-antibody-attached GOQDs-coated magnetic nanoplatform can be used as a two-photon luminescence platform for selective and very bright imaging of a Hep G2 tumor cell in a biological transparency window using 960 nm light. Experimental results with nontargeted GPC3(-) and SK-BR-3 breast cancer cells show that multifunctional-nanoplatform-based cell separation, followed by two-photon imaging, is highly selective for Hep G2 hepatocellular carcinoma tumor cells.

D-Glucose-Induced Cytotoxic, Genotoxic, and Apoptotic Effects on Human Breast Adenocarcinoma (MCF-7) Cells.

INTRODUCTION: Glucose is a simple sugar that plays an important role in energy production in biological systems. However, it has been linked to many long-term health problems including the risk of heart disease and stroke, erectile dysfunction in men and pregnancy complications in women, and damage to the kidneys, nerves, eye and vision. Also, the underlying mechanisms of diabetic complications are poorly understood. METHODS: In the present study, D-glucose-induced cytotoxic, genotoxic, and apoptotic effects were studied using MCF-7 cells as an in vitro test model. Cell viability was determined by MTT assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet) assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay). RESULTS: The results of MTT assay indicated that D-glucose significantly reduces the viability of MCF-7 cells in a dose and time-dependent manner. Similar trend was obtained with the trypan blue exclusion test. Data obtained from the Comet assay indicated that D-glucose causes DNA damage in MCF-7 cells in a dose-dependent manner. The flow cytometry assessment (Annexin V FITC/PI) showed a strong dose-response relationship between D-glucose exposure and annexin V positive MCF-7 cells undergoing early apoptosis. CONCLUSION: Taking together, these data provide clear evidence that D-glucose induces cytotoxic, genotoxic, and apoptotic effects on MCF-7 cells. This finding represents the basis for further studies addressing the pathophysiological mechanisms of action of glucose overdose.

N-acetyl-cysteine protects against DNA damage associated with lead toxicity in HepG2 cells.

Lead toxicity has been associated with its ability to interact and damage DNA. However, its molecular mechanisms of action are not fully understood. In vitro studies in our laboratory indicated that lead nitrate (PbNO3) induces cytotoxicity and oxidative stress to human liver carcinoma (HepG2) cells in a dose-dependent manner. In this research, we hypothesized that n-acetyl-cysteine (NAC), a known antioxidant compound, affords protection against lead-induced cell death associated with genotoxic damage. To test this hypothesis, HepG2 cells were treated either with a physiologic dose of NAC, NAC plus PbNO3, or PbNO3 alone, followed by incubation in humidified 5% CO2 incubator at 37 degrees C for 48 hr. The cell viability was determined by trypan blue exclusion test. The degree of DNA damage was detected by micro gel electrophoresis (comet) assay. Our results showed that lead exposure induces a substantial cytotoxicity as well as a significant genotoxicity to HepG2 cells. However, co-treatment with a physiologic dose (500 microM) of NAC slightly increases cell viability, and significantly reduced (P < .05) the degree of DNA damage. Hence, NAC treatment may be a promising therapeutic candidate for chemoprevention against lead toxicity, based on its ability to scavenge free radicals.

Ascorbic Acid Potentiation of Arsenic Trioxide Anticancer Activity Against Acute Promyelocytic Leukemia.

INTRODUCTION: Acute promyelocytic leukemia (APL) is a malignant disorder of the white blood cells. Arsenic trioxide (As(2)O(3)) has been used as a therapeutic agent to treat APL and other tumors. Studies suggest that ascorbic acid (AA) supplementation may improve the clinical outcome of As(2)O(3) for APL patients. Our aim was to use human leukemia (HL-60) APL-cells as an in vitro test model to evaluate the effect of physiologic doses of AA on As(2)O(3)-induced toxicity and apoptosis of HL-60 cells. METHODS: HL-60 cells were treated either with a pharmacologic dose of As(2)O(3) alone and with several physiologic doses of AA. Cell survival was determined by trypan blue exclusion test. The extent of oxidative cell/tissue damage was determined by measuring lipid hydroperoxide concentration by spectrophotometry. Cell apoptosis was measured by flow cytometry using Annexin-V and propidium iodide (PI) staining. RESULTS: AA treatment potentiates the cytotoxicity of As(2)O(3) in HL-60 cells. Viability decreased from (58 +/- 3)% in cells with As(2)O(3) alone to (47 +/- 2)% in cells treated with 100 microM AA and 6 microg/mL As(2)O(3) with P < 0.05. There was a significant (P < 0.05) increase in lipid hydroperoxide concentrations in HL-60 cells co-treated with AA compared to As(2)O(3) alone. Flow cytometry assessment (Annexin V FITC/PI) suggested that AA co-treatment induces more apoptosis of HL-60 cells than did As(2)O(3) alone, but this was not statistically significant. Taken together, our experiment indicates that As(2)O(3) induced in vitro cell death and apoptosis of HL-60 cells. Administration of physiologic doses of AA enhanced As(2)O(3)-induced cytotoxicity, oxidative cell/tissue damage, and apoptosis of HL-60 cells through externalization of phosphatidylserine. CONCLUSIONS: These suggest that AA may enhance the cytotoxicity of As(2)O(3), suggesting a possible future role of AA/As(2)O(3) combination therapy in patients with APL.