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Carnosine is an endogenous dipeptide composed of ß-alanine and L-histidine, possessing a multimodal pharmacodynamic profile that includes anti-inflammatory and anti-oxidant activities. Carnosine has also shown its ability to modulate cell proliferation, cell cycle arrest, apoptosis, and even glycolytic energy metabolism, all processes playing a key role in the context of cancer. Cancer is one of the most dreaded diseases of the 20th and 21st centuries. Among the different types of cancer, breast cancer represents the most common non-skin cancer among women, accounting for an estimated 15% of all cancer-related deaths in women. The main aim of the present review was to provide an overview of studies on the anti-cancer activity of carnosine, and in particular its activity against breast cancer. We also highlighted the possible advantages and limitations involved in the use of this dipeptide. The first part of the review entailed a brief description of carnosine's biological activities and the pathophysiology of cancer, with a focus on breast cancer. The second part of the review described the anti-tumoral activity of carnosine, for which numerous studies have been carried out, especially at the preclinical level, showing promising results. However, only a few studies have investigated the therapeutic potential of this dipeptide for breast cancer prevention or treatment. In this context, carnosine has shown to be able to decrease the size of cancer cells and their viability. It also reduces the levels of vascular endothelial growth factor (VEGF), cyclin D1, NAD+, and ATP, as well as cytochrome c oxidase activity in vitro. When tested in mice with induced breast cancer, carnosine proved to be non-toxic to healthy cells and exhibited chemopreventive activity by reducing tumor growth. Some evidence has also been reported at the clinical level. A randomized phase III prospective placebo-controlled trial showed the ability of Zn-carnosine to prevent dysphagia in breast cancer patients undergoing adjuvant radiotherapy. Despite this evidence, more preclinical and clinical studies are needed to better understand carnosine's anti-tumoral activity, especially in the context of breast cancer.

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Chronic neuroinflammation has long been considered to be a central factor in accelerating the progression of neurodegenerative diseases such as Alzheimer's diseases, Parkinson's disease and chronic traumatic encephalopathy. Under pathological conditions microglia produce inflammatory signaling molecules, such as nitric oxide (NO), that can damage DNA and proteins and ultimately induce neuronal apoptosis. One strategy for treating neurodegenerative diseases is to specifically target NO production through inhibition of inducible nitric oxide synthase (iNOS). However, accurately measuring changes in microglial NO production in response to potential therapeutics is challenging due to NO's short half-life and microglial heterogeneity. In this paper we report the application of a microfluidic device for the high-throughput measurement of intracellular NO in SIM-A9 microglial cells. NO production was measured in response to treatment with lipopolysaccharides (LPS) and interferon gamma (IFN-γ) with and without a potent iNOS inhibitor (1400 W dihydrochloride). Cells were labeled with a fluorogenic NO probe, 4-amino-5-methylamino-2',7'-difluorofluoescein diacetate (DAF-FM DA), and 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. Separation and quantitation of intracellular NO was achieved using microchip electrophoresis and laser induced fluorescence detection (LIF). Statistical analysis suggests that the populations fit a lognormal distribution and are better represented by their geometric mean values. Comparison of the geometric means indicated a 1.6-fold increase in NO production between untreated and stimulated cells and a decrease by a factor of approximately 0.5 comparing stimulated and inhibited cells. Additionally, we report experimental data demonstrating the improvement in the sensitivity of our integrated optical fiber-based detection system through the use of refractive index matching gel.

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Here we describe in detail the design, fabrication and operation of our automated high-throughput single cell microchip electrophoresis device with laser induced fluorescence detection. Our device features on-board integrated peristaltic pumps that generate flow directly within the microfluidic channels. Additionally, we have incorporated an optical fiber bridge that enables simultaneous fluorescence detection at two points of interest within the device without the need for additional optical components or detectors. The second detection spot is used to detect the intact cell immediately prior to lysis giving a signal at t=0s for each single-cell electropherogram. We can also use this signal to measure the absolute migration time of the separated analytes to confidently determine the identity of each peak. Finally, we demonstrate the application of our device for the measurement of intracellular nitric oxide (NO) levels in T-lymphocytes. Changes in NO levels within cells is associated with a number of chronic diseases including neurodegenerative, cardiovascular and cancers. We show that our system is capable of measuring NO levels under the following conditions: native, lipopolysaccharide stimulation, and inhibition of inducible nitric oxide synthase. It is our hope that the information and procedures described in this chapter may enable others to use or adapt our system for other analyses at the single cell level.

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This chapter provides step-by-step procedures for the fabrication of glass-based microfluidic devices. These procedures include device design, photomask generation, photolithography, channel etching, and high-temperature bonding.

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