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.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

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.

Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells.

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

Gel shift selection of translation enhancer sequences using messenger RNA display.

We designed a new approach for selection of translation enhancer sequences that enables efficient protein synthesis in cell-free systems. The selection is based on a gel shift assay of a messenger RNA (mRNA)-protein fusion product that is synthesized in a cell-free translation system using an mRNA display method. A library of randomized 20-nt-long sequences, with all possible combinations of the four nucleotides, upstream of a coding region was screened by successive rounds of screening in which the translation time of the succeeding round was reduced compared with the previous round. An efficient translation enhancer sequence capable of more rapid initiation of cell-free protein synthesis, with a minimal translation time of 5 min, than a natural longer enhancer sequence (Xenopus ß-globin 5'UTR) was selected using rabbit reticulocyte extract as a model cell-free translation system. Furthermore, a successful screening of cap-independent translation enhancer sequence and a significant sequence similarity of the selected candidates validated the efficiency of the combined mRNA display and gel shift assay method for the rapid development of advanced cell-free translation systems.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses.

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.

PEP-on-DEP: A competitive peptide-based disposable electrochemical aptasensor for renin diagnostics.

Antibody-based immunosensors are relatively less accessible to a wide variety of unreachable targets, such as low-molecular-weight biomarkers that represent a rich untapped source of disease-specific diagnostic information. Here, we present a peptide aptamer-based electrochemical sensor technology called 'PEP-on-DEP' to detect less accessible target molecules, such as renin, and to improve the quality of life. Peptide-based aptamers represent a relatively smart class of affinity binders and show great promise in biosensor development. Renin is involved in the regulation of arterial blood pressure and is an emerging biomarker protein for predicting cardiovascular risk and prognosis. To our knowledge, no studies have described aptamer molecules that can be used as new potent probes for renin. Here, we describe a portable electrochemical biosensor platform based on the newly identified peptide aptamer molecules for renin. We constructed a randomized octapeptide library pool with diversified sequences and selected renin specific peptide aptamers using cDNA display technology. We identified a few peptide aptamer sequences with a KD in the µM binding affinity range for renin. Next, we grafted the selected peptide aptamers onto gold nanoparticles and detected renin in a one-step competitive assay using our originally developed DEP (Disposable Electrochemical Printed) chip and a USB powered portable potentiostat system. We successfully detected renin in as little as 300ngmL(-1) using the PEP-on-DEP method. Thus, the generation and characterization of novel probes for unreachable target molecules by merging a newly identified peptide aptamer with electrochemical transduction allowed for the development of a more practical biosensor that, in principle, can be adapted to develop a portable, low-cost and mass-producible biosensor for point-of-care applications.

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.

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 (K(D); ∼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.