Madhu Biyani

In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH

The aspartic protease cathepsin E has been shown to induce apoptosis in cancer cells under physiological conditions. Therefore, cathepsin E-activity-enhancing peptides functioning in the physiological pH range are valuable potential cancer therapeutic candidates. Here, we have used a general in vitro selection method (evolutionary rapid panning analysis system (eRAPANSY)), based on inverse substrate-function link (SF-link) selection to successfully identify cathepsin E-activity-enhancing peptide aptamers at neutral pH. A successive enrichment of peptide activators was attained in the course of selection. One such peptide activated cathepsin E up to 260%, had a high affinity (KD; ∼300 nM), and had physiological activity as demonstrated by its apoptosis-inducing reaction in cancerous cells. This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method.

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E‐inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E‐activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module‐finding, module‐shuffling, and module‐pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. Copyright

Peptide Aptamer-Based ELISA-Like System for Detection of Cathepsin E in Tissues and Plasma

ELISA (enzyme-linked immunosorbent assay), a highly sensitive and powerful molecular detection tool, has been constructed based on antibodies, as its name denotes. However, it is not so easy to prepare antibodies for any target molecule and there are still problems in their cost and stability. In this study, peptide-based ELISA-like systems (pep- ELISA) were first constructed and shown to be effective. In particular, if the target is an enzyme, such as cathepsin E, that can generate a fluorescent product, the construct of pep-ELISA can be very simple, as its name indicates; i.e., Enzyme-on-Peptide. These constructs, together with peptide-based Sandwich ELISA-like (Sandwich pep-ELISA), were actually constructed and examined using the tissues and blood specimens extracted from cathepsin E-normal/knockout rats. Through these experiments, the sufficient sensitivity (~10µg/ml) and methodological convenience of pep-ELISA were demonstrated.

DEP-On-Go for Simultaneous Sensing of Multiple Heavy Metals Pollutants in Environmental Samples

We describe a simple and affordable “Disposable electrode printed (DEP)-On-Go” sensing platform for the rapid on-site monitoring of trace heavy metal pollutants in environmental samples for early warning by developing a mobile electrochemical device composed of palm-sized potentiostat and disposable unmodified screen-printed electrode chips. We present the analytical performance of our device for the sensitive detection of major heavy metal ions, namely, mercury, cadmium, lead, arsenic, zinc, and copper with detection limits of 1.5, 2.6, 4.0, 5.0, 14.4, and, 15.5 μg·L−1, respectively. Importantly, the utility of this device is extended to detect multiple heavy metals simultaneously with well-defined voltammograms and similar sensitivity. Finally, “DEP-On-Go” was successfully applied to detect heavy metals in real environmental samples from groundwater, tap water, house dust, soil, and industry-processed rice and noodle foods. We evaluated the efficiency of this system with a linear correlation through inductively coupled plasma mass spectrometry, and the results suggested that this system can be reliable for on-site screening purposes. On-field applications using real samples of groundwater for drinking in the northern parts of India support the easy-to-detect, low-cost (<1 USD), rapid (within 5 min), and reliable detection limit (ppb levels) performance of our device for the on-site detection and monitoring of multiple heavy metals in resource-limited settings.

Biomolecular Display Technology: A New Tool for Drug Discovery

Abstract The identification of molecules with desired function is of a great significance in biology and medicine. Display technologies represent a new tool for the drug discovery by facilitating the screening of novel biomolecules against any target of choice. This chapter reviews the development of in vitro display technologies and their application into drug discovery, focusing on challenges and perspective for the rapid and efficient modern drug discovery process.

Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH

Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors.

Instant enumeration of total viable bacterial counts for food quality assurance using ‘DEP-On-Go’ sensor

Food quality has to be constantly monitored from farm-to-table to control foodborne illness. Total viable count (TVC), which estimates the population of live microbial load, can be an extremely useful tool for food quality assurance. This work presents the utility of an electrochemical ‘DEP-On-Go’ sensor to determine TVCs instantly and quantitatively in milk and other food samples, including vegetables, and meat, using the oxygen consumption activity of viable microbes. We present the analytical performance of our device for the sensitive detection of the TVC in packed milk with a detection limit of ≤300 CFU mL−1, which fulfills the acceptable limit (<30 000 CFU mL−1) recommended by Prevention of Food Adulteration (PFA) standards. We detected the TVC in raw or packed milk immediately without any pretreatment or incubation. The calibration curve of the TVC obtained by the standard plate count method and peak current resulting from the oxygen consumption activity of bacteria in milk samples showed linearity with a correlation coefficient of 0.9907 over the range of 1.8–8.7 log CFU mL−1. The ‘DEP-On-Go’ sensor was further utilized to selectively determine the coliform bacteria count in milk samples with a linear coefficient of 0.9455 over the range of 0.6–7.0 log CFU mL−1 within 4 hours of incubation time using selective broth media. Our results support the easy-to-detect, low cost (1–5 USD), rapid and reliable monitoring of microbial contamination using the ‘DEP-On-Go’ device, which can become a powerful mobile platform in modern food industries.