Simultaneous and multiplexed phenotyping of circulating exosomes with the orthogonal CRISPR-Cas platform.

Simultaneous and multiplexed exosome protein profiling via an orthogonal CRISPR-Cas platform was achieved in this work. Aptamers were recruited to translate exosome surface protein information into Cas12a/Cas13a cleavage activity. The established multiplexed platform performed robustly with biological matrixes and could profile exosome proteins in clinical serum samples.

All Roads Lead to Rome: Comparing Nanoparticle- and Small Molecule-Driven Cell Autophagy.

Autophagy, vital for removing cellular waste, is triggered differently by small molecules and nanoparticles. Small molecules, like rapamycin, non-selectively activate autophagy by inhibiting the mTOR pathway, which is essential for cell regulation. This can clear damaged components but may cause cytotoxicity with prolonged use. Nanoparticles, however, induce autophagy, often causing oxidative stress, through broader cellular interactions and can lead to a targeted form known as "xenophagy." Their impact varies with their properties but can be harnessed therapeutically. In this review, the autophagy induced by nanoparticles is explored and small molecules across four dimensions: the mechanisms behind autophagy induction, the outcomes of such induction, the toxicological effects on cellular autophagy, and the therapeutic potential of employing autophagy triggered by nanoparticles or small molecules. Although small molecules and nanoparticles each induce autophagy through different pathways and lead to diverse effects, both represent invaluable tools in cell biology, nanomedicine, and drug discovery, offering unique insights and therapeutic opportunities.

Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping.

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.

Threats of per- and poly-fluoroalkyl pollutants to susceptible populations.

Environmental exposure to per- and poly-fluoroalkyl substances (PFAS) has raised significant global health concerns due to potential hazards in healthy adults. However, the impact of PFAS on susceptible populations, including pregnant individuals, newborns, the older people, and those with underlying health conditions, has been overlooked. These susceptible groups often have physiological changes that make them less resilient to the same exposures. Consequently, there is an urgent need for a comprehensive understanding of the health risks posed by PFAS exposure to these populations. In this review, we delve into the potential health risks of PFAS exposure in these susceptible populations. Equally important, we also examine and discuss the molecular mechanisms that underlie this susceptibility. These mechanisms include the induction of oxidative stress, disruption of the immune system, impairment of cellular metabolism, and alterations in gut microbiota, all of which contribute to the enhanced toxicity of PFAS in susceptible populations. Finally, we address the primary research challenges and unresolved issues that require further investigation. This discussion aims to foster research for a better understanding of how PFAS affect susceptible populations and to pave the way for strategies to minimize their adverse effects.

Split G-quadruplex based PfAgo sensing platform for nucleotide mutation discrimination and human genotyping.

A PfAgo-G4 sensing platform exploiting G4 as a signal reporter was proposed, validated, and optimized. By introducing two mismatches at the Link strand, a universal nucleotide design rule was established for accurate single nucleotide polymorphism discrimination with PfAgo-G4. The FUT2 gene was then successfully and accurately genotyped using human buccal swab samples.

Unraveling the complex dynamics of signaling molecules in cellular signal transduction.

Signaling molecules in cellular responses to foreign stimuli are described as static up- or down-concentration changes during signal transduction. This is because analytical methods for transducing molecules are much slower than the signaling events. In this study, we develop a dynamic cell model and reveal the temporal regulation of signal transduction events in response to reactive oxygen species (ROS). The model contained a set of 10 batches of redox-modified cells that mimic the temporal ROS accumulation events. Validating this dynamic cell model, we discover that cells survive early ROS attacks by activating the Nrf2/polysulfide/p62/CDK1 pathway. Nearly all signaling molecules exhibit time-dependent V-shape or inverse V-shape activation/feedback regulation dynamics in response to ROS accumulation. The results show that the dynamic cell model approach is invaluable for revealing complex signal intensity- and time-dependent cell signaling events.

Modulating the toxicity of engineered nanoparticles by controlling protein corona formation: Recent advances and future prospects.

With the rapid development and widespread application of engineered nanoparticles (ENPs), understanding the fundamental interactions between ENPs and biological systems is essential to assess and predict the fate of ENPs in vivo. When ENPs are exposed to complex physiological environments, biomolecules quickly and inevitably adsorb to ENPs to form a biomolecule corona, such as a protein corona (PC). The formed PC has a significant effect on the physicochemical properties of ENPs and gives them a brand new identity in the biological environment, which determines the subsequent ENP-cell/tissue/organ interactions. Controlling the formation of PCs is therefore of utmost importance to accurately predict and optimize the behavior of ENPs within living organisms, as well as ensure the safety of their applications. In this review, we provide an overview of the fundamental aspects of the PC, including the formation mechanism, composition, and frequently used characterization techniques. We comprehensively discuss the potential impact of the PC on ENP toxicity, including cytotoxicity, immune response, and so on. Additionally, we summarize recent advancements in manipulating PC formation on ENPs to achieve the desired biological outcomes. We further discuss the challenges and prospects, aiming to provide valuable insights for a better understanding and prediction of ENP behaviors in vivo, as well as the development of low-toxicity ENPs.

Nanomaterial-Based Drug Delivery Systems for Pain Treatment and Relief: From the Delivery of a Single Drug to Co-Delivery of Multiple Therapeutics.

The use of nanomaterials in drug delivery systems for pain treatment is becoming increasingly common. This review aims to summarize how nanomaterial-based drug delivery systems can be used to effectively treat and relieve pain, whether via the delivery of a single drug or a combination of multiple therapeutics. By utilizing nanoformulations, the solubility of analgesics can be increased. Meanwhile, controlled drug release and targeted delivery can be realized. These not only improve the pharmacokinetics and biodistribution of analgesics but also lead to improved pain relief effects with fewer side effects. Additionally, combination therapy is frequently applied to anesthesia and analgesia. The co-encapsulation of multiple therapeutics into a single nanoformulation for drug co-delivery has garnered significant interest. Numerous approaches using nanoformulation-based combination therapy have been developed and evaluated for pain management. These methods offer prolonged analgesic effects and reduced administration frequency by harnessing the synergy and co-action of multiple targets. However, it is important to note that these nanomaterial-based pain treatment methods are still in the exploratory stage and require further research to be effectively translated into clinical practice.

Polysaccharides in contact lenses: From additives to bulk materials.

As the number of applications has increased, so has the demand for contact lenses comfort. Adding polysaccharides to lenses is a popular way to enhance comfort for wearers. However, this may also compromise some lens properties. It is still unclear how to balance the variation of individual lens parameters in the design of contact lenses containing polysaccharides. This review provides a comprehensive overview of how polysaccharide addition impacts lens wear parameters, such as water content, oxygen permeability, surface wettability, protein deposition, and light transmittance. It also examines how various factors, such as polysaccharide type, molecular weight, amount, and mode of incorporation into lenses modulate these effects. Polysaccharide addition can improve some wear parameters while reducing others depending on the specific conditions. The optimal method, type, and amount of added polysaccharides depend on the trade-off between various lens parameters and wear requirements. Simultaneously, polysaccharide-based contact lenses may be a promising option for biodegradable contact lenses as concerns regarding environmental risks associated with contact lens degradation continue to increase. It is hoped that this review will shed light on the rational use of polysaccharides in contact lenses to make personalized lenses more accessible.

Split G-quadruplex-programmed label-free CRISPR-Cas12a sensing system.

A split G-quadruplex (G4)-programmed Cas12a platform was established, validated, and optimized. The split G4 motif was recruited as substrate for Cas12a, and the label-free sensing platform provided a concentration-dependent response towards the input target. Furthermore, exosomal surface proteins from cultured cancer cells and clinical samples were detected and profiled.

RNA extraction-free workflow integrated with a single-tube CRISPR-Cas-based colorimetric assay for rapid SARS-CoV-2 detection in different environmental matrices.

On-site environmental surveillance of viruses is increasingly important for infection prevention and pandemic control. Herein, we report a facile single-tube colorimetric assay for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from environmental compartments. Using glycerol as the phase separation additive, reverse transcription recombinase polymerase amplification (RT-RPA), CRISPR-Cas system activation, G-quadruplex (G4) cleavage, and G4-based colorimetric reaction were performed in a single tube. To further simplify the test, viral RNA genomes used for the one-tube assay were obtained via acid/base treatment without further purification. The whole assay from sampling to visual readout was completed within 30 min at a constant temperature without the need for sophisticated instruments. Coupling the RT-RPA to CRISPR-Cas improved the reliability by avoiding false positive results. Non-labeled cost-effective G4-based colorimetric systems are highly sensitive to CRISPR-Cas cleavage events, and the proposed assay reached the limit of detection of 0.84 copies/µL. Moreover, environmental samples from contaminated surfaces and wastewater were analyzed using this facile colorimetric assay. Given its simplicity, sensitivity, specificity, and cost-effectiveness, our proposed colorimetric assay is highly promising for applications in on-site environmental surveillance of viruses.

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection.

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 µL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

CRISPR-Cas12a-Based Aptasensor for On-Site and Highly Sensitive Detection of Microcystin-LR in Freshwater.

On-site monitoring of trace organic pollutants with facile methods is critical to environmental pollutant prevention and control. Herein, we proposed a CRISPR-Cas12a-based aptasensor platform (named as MC-LR-Casor) for on-site and sensitive detection of microcystin-LR (MC-LR). After hybridization with blocker DNA, the MC-LR aptamers were conjugated to magnetic beads (MBs) to get the MB aptasensor. In the presence of MC-LR, their interactions with aptamers were triggered and the specific binding caused the release of blocker DNA. Using the programmability of the CRISPR-Cas system, the released blocker DNA was designed to activate a Cas12a-crRNA complex. Single strand DNA reporters were rapidly cleaved by the complex. Signal readout could be achieved by fluorometer or lateral flow strips, which were positively correlated to MC-LR concentration. Benefiting from the CRISPR-Cas12a amplification system, the proposed sensing platform exhibited high sensitivity and reached the limit of detection of ∼3 × 10-6 µg/L (fluorescence method) or 1 × 10-3 µg/L (lateral flow assay). In addition, the MC-LR-Casor showed excellent selectivity and good recovery rates, demonstrating their good applicability for real water sample analysis. During the whole assay, only two steps of incubation at a constant temperature were required and the results could be visualized when employing flow strips. Therefore, the proposed assay offered a simple and convenient alternative for in situ MC-LR monitoring, which may hold great promise for future environmental surveillance.

Safety Assessment of 2D MXenes: In Vitro and In Vivo.

MXenes, representing a new class of two-dimensional nanomaterial, have attracted intense interest in a variety of fields as supercapacitors, catalysts, and sensors, and in biomedicine. The assessment of the safety of MXenes and related materials in biological systems is thus an issue that requires significant attention. In this review, the toxic effects of MXenes and their derivatives are summarized through the discussion of current research into their behaviors in mammalian cells, animals and plants. Numerous studies have shown that MXenes have generally low cytotoxicity and good biocompatibility. However, a few studies have indicated that MXenes are toxic to stem cells and embryos. These in vitro and in vivo toxic effects are strongly associated with the dose of material, the cell type, the mode of exposure, and the specific type of MXene. In addition, surface modifications alter the toxic effects of MXenes. The stability of MXenes must be considered during toxicity evaluation, as degradation can lead to potentially toxic byproducts. Although research concerning the toxicity of MXenes is limited, this review provides an overview of the current understanding of interactions of MXenes with biological systems and suggests future research directions.

Predicting cytotoxicity of binary pollutants towards a human cell panel in environmental water by experimentation and deep learning methods.

Biological assays are useful in water quality evaluation by providing the overall toxicity of chemical mixtures in environmental waters. However, it is impossible to elucidate the source of toxicity and some lethal combination of pollutants simply using biological assays. As facile and cost-effective methods, computation model-based toxicity assessments are complementary technologies. Herein, we predicted the human health risk of binary pollutant mixtures (i.e., binary combinations of As(III), Cd(II), Cr(VI), Pb(II) and F(I)) in water using in vitro biological assays and deep learning methods. By employing a human cell panel containing human stomach, colon, liver, and kidney cell lines, we assessed the human health risk mimicking cellular responses after oral exposures of environmental water containing pollutants. Based on the experimental cytotoxicity data in pure water, multi-task deep learning was applied to predict cellular response of binary pollutant mixtures in environmental water. Using additive descriptors and single pollutant toxicity data in pure water, the established deep learning model could predict the toxicity of most binary mixtures in environmental water, with coefficient of determination (R2) > 0.65 and root mean squared error (RMSE) < 0.22. Further combining the experimental data on synergistic and antagonistic effects of pollutant mixtures, deep learning helped improve the predictive ability of the model (R2 > 0.74 and RMSE <0.17). Moreover, predictive models allowed us identify a number of toxicity source-related physiochemical properties. This study illustrates the combination of experimental findings and deep learning methods in the water quality evaluation.

Pretreatment with metformin prevents microcystin-LR-induced tau hyperphosphorylation via mTOR-dependent PP2A and GSK-3ß activation.

Microcystin-leucine-arginine (MC-LR) is a toxin secreted by freshwater cyanobacteria that is considered a potential environmental risk factor for Alzheimer's disease (AD). A previous study indicated that tau protein hyperphosphorylation via protein phosphatase 2A (PP2A) and GSK-3ß inhibition was the mechanism by which MC-LR induces neurotoxicity; however, how MC-LR-induced neurotoxicity can be effectively prevented remains unclear. In this study, the reversal effect of metformin on MC-LR-induced neurotoxicity was investigated. The results showed that metformin effectively prevented tau hyperphosphorylation at Ser202 caused by MC-LR through PP2A and GSK-3b activity. The effect of metformin on PP2A activity was dependent on the inhibition of mTOR in MC-LR-treated SH-SY5Y cells. Metformin prevented spatial memory deficits in rats caused by intrahippocampal MC-LR administration. In sum, the results suggested that metformin can ameliorate the MC-LR-induced AD-like phenotype by preventing tau phosphorylation at Ser202, which was mainly mediated by mTOR-dependent PP2A and GSK-3ß activation.

Cytotoxicity Induction by the Oxidative Reactivity of Nanoparticles Revealed by a Combinatorial GNP Library with Diverse Redox Properties.

It is crucial to establish relationship between nanoparticle structures (or properties) and nanotoxicity. Previous investigations have shown that a nanoparticle's size, shape, surface and core materials all impact its toxicity. However, the relationship between the redox property of nanoparticles and their toxicity has not been established when all other nanoparticle properties are identical. Here, by synthesizing an 80-membered combinatorial gold nanoparticle (GNP) library with diverse redox properties, we systematically explored this causal relationship. The compelling results revealed that the oxidative reactivity of GNPs, rather than their other physicochemical properties, directly caused cytotoxicity via induction of cellular oxidative stress. Our results show that the redox diversity of nanoparticles is regulated by GNPs modified with redox reactive ligands.

Protein corona precoating on redox-responsive chitosan-based nano-carriers for improving the therapeutic effect of nucleic acid drugs.

Spontaneous formation of protein corona on chitosan-based nano-carriers is inevitable once they enter the blood, which is considered to be an important factor that weakens the delivery efficiency and therapeutic effect of nucleic acid drugs. For this, cyclic RGDyK peptide (cRGD) modified bovine serum albumin (BSA) was designed as a corona to precoat on redox-responsive chitosan-based nano-carriers (TsR NPs) before administration. The effects of the precoating corona on the pharmaceutical properties and delivery efficiency of the nano-carriers and the therapeutic effect of model siRNA (siVEGF) were investigated. The results showed that BSA-cRGD formed steady corona around TsR NPs, which enhanced targeting ability to cancer cells and reduced serum proteins adsorption. The Bc corona improved the stability and biocompatibility of TsR NPs, increased the intracellular uptake, facilitated the lysosomal escape and maintained their redox-sensitive responsiveness, resulting in enhanced gene silencing efficiency and anti-tumor proliferation effects both in vitro and in vivo.

In vivo Protein Corona Formation: Characterizations, Effects on Engineered Nanoparticles' Biobehaviors, and Applications.

Understanding the basic interactions between engineered nanoparticles (ENPs) and biological systems is essential for evaluating ENPs' safety and developing better nanomedicine. Profound interactions between ENPs and biomolecules such as proteins are inevitable to occur when ENPs are administered or exposed to biological systems, for example, through intravenous injection, oral, or respiration. As a key component of these interactions, protein corona (PC) is immediately formed surrounding the outlayer of ENPs. PC formation is crucial because it gives ENPs a new biological identity by altering not only the physiochemical properties, but also the biobehaviors of ENPs. In the past two decades, most investigations about PC formation were carried out with in vitro systems which could not represent the true events occurring within in vivo systems. Most recently, studies of in vivo PC formation were reported, and it was found that the protein compositions and structures were very different from those formed in vitro. Herein, we provide an in-time review of the recent investigations of this in vivo PC formation of ENPs. In this review, commonly used characterization methods and compositions of in vivo PC are summarized firstly. Next, we highlight the impacts of the in vivo PC formation on absorption, blood circulation, biodistribution, metabolism, and toxicity of administered ENPs. We also introduce the applications of modulating in vivo PC formation in nanomedicine. We further discuss the challenges and future perspectives.

Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.