S-nitrosoglutathione (GSNO) induces necroptotic cell death in K562 cells: Involvement of p73, TSC2 and SIRT1.

BACKGROUND: Nitric oxide and Reactive Nitrogen Species are known to effect tumorigenicity. GSNO is one of the main NO carrying signalling moiety in cell. In the current study, we tried to delve into the effect of GSNO induced nitrosative stress in three different myelogenous leukemic K562, U937 and THP-1 cell lines. METHOD: WST-8 assay was performed to investigate cell viability. RT-PCR and western-blot analysis were done to investigate mRNA and protein expression. Spectrophotometric and fluorimetric assays were done to investigate enzyme activities. RESULT: We found that GSNO exposure led to reduced cell viability and the mode of cell death in K562 was non apoptotic in nature. GSNO promoted impaired autophagic flux and necroptosis. GSNO treatment heightened phosphorylation of AMPK and TSC2 and inhibited mTOR pathway. We observed increase in NAD+/ NADH ratio following GSNO treatment. Increase in both SIRT1 m-RNA and protein expression was observed. While total SIRT activity remained unaltered. GSNO increased tumor suppressor TAp73/ oncogenic ∆Np73 ratio in K562 cells which was correlated with cell mortality. Surprisingly, GSNO did not alter cellular redox status or redox associated protein expression. However, steep increase in total SNO and PSNO content was observed. Furthermore, inhibition of autophagy, AMPK phosphorylation or SIRT1 exacerbated the effect of GSNO. Altogether our work gives insights into GSNO mediated necroptotic event in K562 cells which can be excavated to develop NO based anticancer therapeutics. CONCLUSION: Our data suggests that GSNO could induce necroptotic cell death in K562 through mitochondrial dysfunctionality and PTM of different cellular proteins.

Microfilaria Infection in Metastatic Node in a Case of Breast Carcinoma.

Filariasis is a major public health problem in tropical and subtropical countries such as India. It is caused by nematodes, principally Wuchereria bancrofti and Brugia malayi and is transmitted by the Culex mosquito. W.bancrofti accounts for 90% of cases of lymphatic filariasis. Filaria of the breast with axillary lymphadenopathy and lymphedema, mimicking breast carcinoma has been previously reported. Microfilariae have also been previously observed as coincidental findings with various inflammatory and neoplastic lesions. Few examples are hemangiomas of the liver, meningiomas, intracranial hemangioblastomas, uterine cervix, pharyngeal, urinary bladder, preputial and metastatic carcinomas, melanomas, and leukemia.Here, we report an unusual case of filarial infection of a metastatic axillary node in a case of invasive breast carcinoma, observed in a modified radical mastectomy specimen. Previous cases reported in literature have reported microfilaria in metastatic axillary node detected on cytology specimens; histological diagnosis, though reported, is rare.

Millifluidics for chemical synthesis and time-resolved mechanistic studies.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidics for time-resolved mapping of the growth of gold nanostructures.

Innovative in situ characterization tools are essential for understanding the reaction mechanisms leading to the growth of nanoscale materials. Though techniques, such as in situ transmission X-ray microscopy, fast single-particle spectroscopy, small-angle X-ray scattering, etc., are currently being developed, these tools are complex, not easily accessible, and do not necessarily provide the temporal resolution required to follow the formation of nanomaterials in real time. Here, we demonstrate for the first time the utility of a simple millifluidic chip for an in situ real time analysis of morphology and dimension-controlled growth of gold nano- and microstructures with a time resolution of 5 ms. The structures formed were characterized using synchrotron radiation-based in situ X-ray absorption spectroscopy, 3-D X-ray tomography, and high-resolution electron microscopy. These gold nanostructures were found to be catalytically active for conversion of 4-nitrophenol into 4-aminophenol, providing an example of the potential opportunities for time-resolved analysis of catalytic reactions. While the investigations reported here are focused on gold nanostructures, the technique can be applied to analyze the time-resolved growth of other types of nanostructured metals and metal oxides. With the ability to probe at least a 10-fold higher concentrations, in comparison with traditional microfluidics, the tool has potential to revolutionize a broad range of fields from catalysis, molecular analysis, biodefense, and molecular biology.

Small molecule fluorophore and copolymer RGD peptide conjugates for ex vivo two-photon fluorescence tumor vasculature imaging.

We report the use of small molecule and block copolymer RGD peptide conjugates for deep ex vivo imaging of tumor vasculature in "whole" excised tumors using two-photon fluorescence microscopy (2PFM). The fluorescent probes were administered to mice via tail-vein injection, after which the tumors were excised, fixed, and imaged without further sample preparation. Both RGD conjugates demonstrated specific targeting to tumor blood vessels, and this selectivity imparted excellent contrast in 2PFM micrographs that captured high-resolution 3-D images of the tumor vasculature up to depths of 830 µm in Lewis Lung Carcinoma (LLC) tumors. 2PFM ex vivo fluorescence micrographs clearly revealed tumor vessels, while differences in the sensitivity of tumor vessel imaging were apparent between the small molecule and block copolymer conjugates. Both the small molecule and polymer-based two-photon absorbing probe conjugate are valuable for deep tissue tumor microvasculature imaging.

Developing a millifluidic platform for the synthesis of ultrasmall nanoclusters: ultrasmall copper nanoclusters as a case study.

The future of lab-on-a-chip devices for the synthesis of nanomaterials hinges on the successful development of high-throughput methods with better control over their size. While significant effort in this direction mainly focuses on developing "difficult to fabricate" complex microfluidic reactors, scant attention has been paid to the "easy to fabricate" and simple millifluidic systems that could provide the required control as well as high throughput. By utilizing numerical simulation of fluids within the millifluidic space at different flow rates, the results presented here show velocity profiles and residence time distributions similar to the case of microfluidics. By significantly reducing the residence time and residence time distribution, a continuous flow synthesis of ultrasmall copper nanoclusters (UCNCs) with exceptional colloidal stability is achieved. In-situ synchrotron-radiation-based X-ray absorption spectroscopy (XAS) reveal that the as-prepared clusters are about 1 nm, which is further supported by transmission electron microscopy and UV-vis spectroscopy studies. The clusters reported here are the smallest ever produced using a lab-on-a-chip platform. When supported on silica, they are found to efficiently catalyze C-H oxidation reactions, hitherto unknown to be catalyzed by Cu. This work suggests that a millifluidic platform can be an inexpensive, versatile, easy-to-use, and powerful tool for nanoparticle synthesis in general, and more specifically for ultrasmall nanoclusters (UNCs).

Two-photon absorption enhancement of polymer-templated porphyrin-based J-aggregates.

Supramolecular structures based on organized assemblies of macrocyclic chromophores, particularly porphyrin-based dyes, have attracted widespread interest as components of molecular devices with potential applications in molecular electronics, artificial light harvesting, and pharmacology. We report the formation of J-aggregates of two porphyrin-based dyes, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TSPP, 4) and an amino tris-sulfonate analogue (5) in water using a functionalized norbornene-based homopolymer, synthesized by ring-opening metathesis polymerization (ROMP). Ionic interactions of the cationic side chains (ammonium groups) of the polymer under acidic conditions with the negatively charged sulfonate groups of the porphyrins facilitated polymer template enhanced J-aggregation of the porphyrin dyes. J-Aggregation behavior was investigated photophysically by UV-vis absorption along with steady-state and time-resolved fluorescence studies. Two-photon absorption (2PA) was enhanced by about an order of magnitude for the J-aggregated TSPP relative to its free base. Significantly, the 2PA cross section of the polymer-templated TSPP J-aggregate was up to three times higher than the J-aggregated TSPP in the absence of the polymer template while the 2PA cross section for polymer-templated J-aggregates of 5 increased substantially, up to ca. 10,000 GM, suggesting a prominent role of polymer-templating to facilitate porphyrin aggregation and greatly enhance nonlinear absorption.

Size evolution of gold nanoparticles in a millifluidic reactor.

The size evolution of gold nanoparticles in a millifluidic reactor is investigated using spatially resolved transmission electron microscopy (TEM). The experimental data is supported by numerical simulations, carried out to study the residence-time distribution (RTD) of tracers that have the same properties as Au ions. Size and size distribution of the particles within the channels are influenced by the mixing zones as well as the RTD. However, the Au nanoparticles obtained show a broader size distribution even at the shortest investigated residence time of 3.53 s, indicating that in addition to surface growth reaction kinetics also plays an important role. The comparison of time resolved particle growth within the millifluidic channel with flask-based reactions reveals that the particle size can be controlled better within millifluidic channels. Overall, the results indicate potential opportunities to utilize easy to fabricate millifluidic reactors for the synthesis of nanoparticles, as well as as for carrying out time resolved kinetic studies.

Integrin-targeting block copolymer probes for two-photon fluorescence bioimaging.

Targeted molecular imaging with two-photon fluorescence microscopy (2PFM) is a powerful technique for chemical biology and, potentially, for noninvasive diagnosis and treatment of a number of diseases. The synthesis, photophysical studies, and bioimaging are reported for a versatile norbornene-based block copolymer multifunctional scaffold containing biocompatible (PEG), two-photon fluorescent dyes (fluorenyl) and targeting (cyclic-RGD peptide) moieties. The two bioconjugates, containing two different fluorenyl dyes and cRGDfK covalently attached to the polymer probe, formed a spherical micelle and self-assembled structure in water, for which size was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cell viability and 2PFM imaging of human epithelial U87MG cell lines that overexpress α(v)ß(3) integrin was performed via incubation with the new probes, along with negative control studies using MCF-7 breast cancer cells and blocking experiments. 2PFM microscopy confirmed the high selectivity of the biocompatible probe in the integrin-rich area in the U87MF cells while blocking as well as negative control MCF-7 experiments confirmed the integrin-targeting ability of the new probes.