High-throughput DNA synthesis for data storage.

With the explosion of digital world, the dramatically increasing data volume is expected to reach 175 ZB (1 ZB = 1012 GB) in 2025. Storing such huge global data would consume tons of resources. Fortunately, it has been found that the deoxyribonucleic acid (DNA) molecule is the most compact and durable information storage medium in the world so far. Its high coding density and long-term preservation properties make itself one of the best data storage carriers for the future. High-throughput DNA synthesis is a key technology for "DNA data storage", which encodes binary data stream (0/1) into quaternary long DNA sequences consisting of four bases (A/G/C/T). In this review, the workflow of DNA data storage and the basic methods of artificial DNA synthesis technology are outlined first. Then, the technical characteristics of different synthesis methods and the state-of-the-art of representative commercial companies, with a primary focus on silicon chip microarray-based synthesis and novel enzymatic DNA synthesis are presented. Finally, the recent status of DNA storage and new opportunities for future development in the field of high-throughput, large-scale DNA synthesis technology are summarized.

Fingerstick blood assay maps real-world NAD+ disparity across gender and age.

Nicotinamide adenine dinucleotide (NAD+ ) level has been associated with various age-related diseases and its pharmacological modulation emerges as a potential approach for aging intervention. But human NAD+ landscape exhibits large heterogeneity. The lack of rapid, low-cost assays limits the establishment of whole-blood NAD+ baseline and the development of personalized therapies, especially for those with poor responses towards conventional NAD+ supplementations. Here, we developed an automated NAD+ analyzer for the rapid measurement of NAD+ with 5 µL of capillary blood using recombinant bioluminescent sensor protein and automated optical reader. The minimal invasiveness of the assay allowed a frequent and decentralized mapping of real-world NAD+ dynamics. We showed that aerobic sport and NMN supplementation increased whole-blood NAD+ and that male on average has higher NAD+ than female before the age of 50. We further revealed the long-term stability of human NAD+ baseline over 100 days and identified major real-world NAD+ -modulating behaviors.

Ratiometric NAD+ Sensors Reveal Subcellular NAD+ Modulators.

Mapping NAD+ dynamics in live cells and human is essential for translating NAD+ interventions into effective therapies. Yet, genetically encoded NAD+ sensors with better specificity and pH resistance are still needed for the cost-effective monitoring of NAD+ in both subcellular compartments and clinical samples. Here, we introduce multicolor, resonance energy transfer-based NAD+ sensors covering nano- to millimolar concentration ranges for clinical NAD+ measurement and subcellular NAD+ visualization. The sensors captured the blood NAD+ increase induced by NMN supplementation and revealed the distinct subcellular effects of NAD+ precursors and modulators. The sensors then enabled high-throughput screenings for mitochondrial and nuclear NAD+ modulators and identified α-GPC, a cognition-related metabolite that induces NAD+ redistribution from mitochondria to the nucleus relative to the total adenine nucleotides, which was further confirmed by NAD+ FRET microscopy.

Discovering Anti-Cancer Drugs via Computational Methods.

New drug discovery has been acknowledged as a complicated, expensive, time-consuming, and challenging project. It has been estimated that around 12 years and 2.7 billion USD, on average, are demanded for a new drug discovery via traditional drug development pipeline. How to reduce the research cost and speed up the development process of new drug discovery has become a challenging, urgent question for the pharmaceutical industry. Computer-aided drug discovery (CADD) has emerged as a powerful, and promising technology for faster, cheaper, and more effective drug design. Recently, the rapid growth of computational tools for drug discovery, including anticancer therapies, has exhibited a significant and outstanding impact on anticancer drug design, and has also provided fruitful insights into the area of cancer therapy. In this work, we discussed the different subareas of the computer-aided drug discovery process with a focus on anticancer drugs.

A biosensor for measuring NAD+ levels at the point of care.

The cellular level of nicotinamide adenine dinucleotide (NAD+), through its different functions, affects cellular metabolism and signalling1-3. A decrease in the NAD+ content has been associated with various pathologies and physiological aging4,5, while strategies to boost cellular NAD+ levels have been shown to be effective against age-related diseases in many animal models6. The link between decreased NAD+ levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD+, ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD+ levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD+. This NAD+-dependent colour change enables the use of the biosensor in paper-based assays in which NAD+ is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD+ levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD+-dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.

Semisynthetic sensor proteins enable metabolic assays at the point of care.

Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, light-emitting sensor proteins for use in paper-based metabolic assays. The metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color in the presence of the reduced cofactor, enabling metabolite quantification with the use of a digital camera. The approach makes any metabolite that can be oxidized by the cofactor a candidate for quantitative point-of-care assays, as shown for phenylalanine, glucose, and glutamate. Phenylalanine blood levels of phenylketonuria patients were analyzed at the point of care within minutes with only 0.5 microliters of blood. Results were within 15% of those obtained with standard testing methods.

Luciferases with Tunable Emission Wavelengths.

We introduce luciferases whose emission maxima can be tuned to different wavelengths by chemical labeling. The luciferases are chimeras of NanoLuc with either SNAP-tag or HaloTag7. Labeling of the self-labeling tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Luciferases with tunable colors have applications as reporter genes, for the construction of biosensors and in bioimaging.

Bioluminescent Antibodies for Point-of-Care Diagnostics.

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔRmax >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.

Highly Modular Bioluminescent Sensors for Small Molecules and Proteins.

Obtaining patient-specific information through the quantification of small molecules and proteins in bodily fluids is essential for personalized therapies. Point-of-care (POC) diagnostic devices hold the promise of delivering such benefit to a wide range of patients. However, there is a lack of enabling technology, as the majority of newly developed POC devices focus on the same underlying core technologies. Here we provide an overview of a new technology based on highly modular bioluminescent sensors that enables the quantification of small molecules and proteins at the POC with low-cost devices.

On-chip spyhole mass spectrometry for droplet-based microfluidics.

For efficient coupling of droplet-based microfluidics with mass spectrometry (MS), a spyhole drilled on the top of a microchip is used to sample the passing droplets by electrostatic-spray ionization (ESTASI) MS. The technique involves placing an electrode below the chip under the spyhole and applying high-voltage pulses. Electrospray occurs directly from the spyhole, and the droplet content is analyzed by MS without a dilution or oil removal step. To demonstrate the versatility of this technique, we have successfully monitored a droplet-based tryptic digestion, as well as a biphasic reaction between ß-lactoglobulin in water and α-tocopheryl acetate in 1,2-dichloroethane, where the protein extracts the antioxidant from the oil phase and becomes reduced.

An electrochemical and surface plasmon resonance study of adsorption actions of DNA by Escherichia coli.

In this work, we proposed an immobilization scheme targeting Escherichia coli bacterial cells onto gold surface utilizing polypeptide RGD's binding specificity to gold particles. Adsorption kinetics of extracellular Herring Sperm DNA on E. coli was then studied using electrochemical methods along with surface plasmon resonance spectroscopy through this immobilization scheme. The adsorption equilibrium constants of DNA adsorbing to E. coli from electrochemical method and surface plasmon resonance spectroscopy, were (5.596±0.462)×105 L mol(-1) and (1.24±0.361)×105 L mol(-1), accordingly. Importantly, this is the first study that used an electrochemical method to express the adsorptive action of DNA by E. coli.

Mitochondrial dysfunction induced by honokiol.

Honokiol has shown the ability to induce the apoptosis of several different cancer cell lines. Considering that mitochondria are involved in apoptosis, the aim of the present work was to investigate the effects of honokiol on mitochondria. The effects of honokiol on the permeability of H⁺ and K⁺, membrane potential, membrane fluidity, respiration and swelling of mitochondria isolated from the rat liver were assessed. The results show that honokiol can significantly induce mitochondrial swelling, decrease membrane potential and affect the respiration of mitochondria. Meanwhile, honokiol does not have a direct effect on the mitochondrial permeability transition pore.

Toxicity of CdTe quantum dots on yeast Saccharomyces cerevisiae.

Along with the widespread development of their bioapplications, concerns about the biosafety of quantum dots (QDs) have increasingly attracted intensive attention. This study examines the toxic effect and subcellular location of cadmium telluride (CdTe) QDs with different sizes against yeast Saccharomyces cerevisiae. The innovative approach is based on the combination of microcalorimetric, spectroscopic, electrochemical, and microscopic methods, which allows analysis of the toxic effect of CdTe QDs on S. cerevisiae and its mechanism. According to the values of the half inhibitory concentration (IC(50)), CdTe QDs exhibit marked cytotoxicity in yeast cells at concentrations as low as 80.81 nmol L(-1) for green-emitting CdTe QDs and 17.07 nmol L(-1) for orange-emitting CdTe QDs. QD-induced cell death is characterized by cell wall breakage and cytoplasm blebbing. These findings suggest that QDs with sizes ranging from 4.1 to 5.8 nm can be internalized into yeast cells, which then leads to QD-induced cytotoxicity. These studies provide valuable information for the design and development of aqueous QDs for biological applications.

Spectroscopic, structural and thermodynamic properties of chlorpyrifos bound to serum albumin: A comparative study between BSA and HSA.

Chlorpyrifos (CPF) is a widely used organophosphate insecticide which could bind with human serum albumin (HSA) and bovine serum albumin (BSA). The binding behavior was studied employing fluorescence, three-dimensional fluorescence, Circular dichroism (CD) spectroscopy, UV-vis absorption spectroscopy, electrochemistry and molecular modeling methods. The fluorescence spectra revealed that CPF causes the quenching of the fluorescence emission of serum albumin. Stern-Volmer plots were made and quenching constants were thus obtained. The results suggested the formation of the complexes of CPF with serum albumins, which were in good agreement with the results from electrochemical experiments. Association constants at 25°C were 3.039 × 10(5) mol L(-1) for HSA, and 0.3307 × 10(5) mol L(-1) for BSA, which could affect the distribution, metabolism, and excretion of pesticide. The alterations of protein secondary structure in the presence of CPF were confirmed by the evidences from UV and CD spectra. Site competitive experiments also suggested that the primary binding site for CPF on serum albumin is close to tryptophan residues 214 of HSA and 212 of BSA, which was further confirmed by molecular modeling.

Microcalorimetric, spectroscopic and microscopic investigation on the toxic effects of CdTe quantum dots on Halobacterium halobium R1.

The biological effect of CdTe quantum dots (QDs) on Halobacterium halobium R1 (H. halobium R1) growth was analyzed by a microcalorimetric technique. By using a TAM air eight channels microcalorimeter, the thermogenic curves of H. halobium R1 growth were obtained at 37 °C. To analyze the results, the maximum heat power (P(m)) and the growth rate constants (k) were determined, which showed that they were correlated to the concentration of QDs. The addition of quantum dots caused a gradual increase of P(m) and k at low concentrations of QDs, and a conspicuous decrease at high concentrations. For confirmation, the turbidity (OD(600)) and respiratory rate at different concentrations of QDs were studied. The morphology of H. halobium R1 cells both in the absence and presence of QDs was examined by transmission electron microscopy (TEM). The results of these studies were corroborated with ones derived from microcalorimetry. In this work, the mechanism of cytotoxicity of QDs was explored through fluorescence spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and microcalorimetry. It was clear that metabolic mechanism of H. halobium R1 growth was changed by the addition of QDs. To the best of our knowledge, the thermokinetics and toxicology of CdTe QDs against H. halobium R1 were obtained for the first time by microcalorimetry.