CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Contrasting mixed scaling patterns and mechanisms of nanofiltration and membrane distillation.

Oriented towards the pressing needs for hypersaline wastewater desalination and zero liquid discharge (ZLD), the contrasting mixed scaling of thermal-driven vacuum membrane distillation (VMD) and pressure-driven nanofiltration (NF) were investigated in this work. Bulk crystallization was the main mechanism in VMD due to the high salinity and temperature, but the time-independent resistance by the adsorption of silicate and organic matter dominated the initial scaling process. Surface crystallization and the consequent pore-blocking were the main scaling mechanisms in NF, with the high permeate drag force, hydraulic pressure, and cross-flow rate resulting in the dense scaling layer mainly composed of magnesium-silica hydrate (MSH). Silicate enhanced NF scaling with a 75% higher initial flux decline rate attributed to the MSH formation and compression, but delayed bulk crystallization in VMD. Organic matter presented an anti-scaling effect by delaying bulk crystallization in both VMD and NF, but specifically promoted CaCO3 scaling in NF. Furthermore, the incipient scaling was intensified as silicate and organic matter coexisted. The scaling mechanism shifted from surface to bulk crystallization due to the membrane concentration in both VMD and NF. This work fills the research gaps on mixed scaling mechanisms in different membrane processes, which offers insights for scaling mitigation and thereby supports the application of ZLD.

Mixed scaling patterns and mechanisms of high-pressure nanofiltration in hypersaline wastewater desalination.

Nanofiltration (NF) will play a crucial role in salt fractionation and recovery, but the complicated and severe mixed scaling is not yet fully understood. In this work, the mixed scaling patterns and mechanisms of high-pressure NF in zero-liquid discharge (ZLD) scenarios were investigated by disclosing the role of key foulants. The bulk crystallization of CaSO4 and Mg-Si complexes and the resultant pore blocking and cake formation under high pressure were the main scaling mechanisms in hypersaline desalination. The incipient scalants were Mg-Si hydrates, CaF2, CaCO3, and CaMg(CO3)2. Si deposited by adsorption and polymerization prior to and impeded Ca scaling when Mg was not added, thus pore blocking was the main mechanism. The amorphous Mg-Si hydrates contribute to dense cake formation under high hydraulic pressure and permeate drag force, causing rapid flux decline as Mg was added. Humic acid has a high affinity to Ca2+by complexation, which enhances incipient scaling by adsorption or lowers the energy barrier of nucleation but improves the interconnectivity of the foulants layer and inhibits bulk crystallization due to the chelation and directional adsorption. Bovine serum albumin promotes cake formation due to the low electrostatic repulsion and acts as a cement to particles by adsorption and bridging in bulk. This work fills the research gaps in mixed scaling of NF, which is believed to support the application of ZLD and shed light on scaling in hypersaline/ultra-hypersaline wastewater desalination applications.

Deep screening of proximal and distal splicing-regulatory elements in a native sequence context.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is impeded by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identified not only known SREs, but also a novel distal intronic splicing enhancer, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Hypoxia and TNF-α Synergistically Induce Expression of IL-6 and IL-8 in Human Fibroblast-like Synoviocytes via Enhancing TAK1/NF-κB/HIF-1α Signaling.

Hypoxia and increased levels of inflammatory cytokines in the joints are characteristics of rheumatoid arthritis (RA). However, the effects of hypoxia and tumor necrosis factor-α (TNF-α) on interleukin (IL)-6 and IL-8 production on fibroblast-like synoviocytes (FLSs) remain to be clarified. This study aimed to explore how hypoxia and TNF-α affect the expression of IL-6 and IL-8 in human FLSs isolated from RA patients. Hypoxia or TNF-α treatment alone significantly increased the expression and promoter activity of IL-6, IL-8, and hypoxia-inducible factor-1α (HIF-1α). Treatment of hypoxic FLSs with TNF-α further significantly elevated the expression of these cytokines and enhanced promoter activity of HIF-1α, which was abrogated by treatment with the HIF-1α inhibitor YC-1. Similarly, TNF-α alone elevated the phosphorylation and promoter activity of nuclear factor-κBp65 (NF-κBp65) in the FLSs. These effects were further enhanced by the combined treatment of hypoxia and TNFα but were attenuated by the NF-κB inhibitor BAY11-7082. NF-κB-p65 inhibition decreased the effect of TNF-α on HIF-1α upregulation in the FLSs in response to hypoxia. The combination of hypoxia and TNF-α also significantly upregulated transforming growth factor-ß-activated kinase 1 (TAK1) expression, and silencing TAK1 dramatically decreased NF-κB-p65, HIF-1α, IL-6, and IL-8 expression under the same conditions. Our results indicate that hypoxia and TNF-α synergistically increase IL-6 and IL-8 expression in human FLSs via enhancing TAK1/NF-κB/HIF-1α signaling.

The pathogenesis and regulatory role of HIF-1 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent autoimmune disease that involves the overgrowth and inflammation of synovial tissue, leading to the degeneration and impairment of joints. In recent years, numerous studies have shown a close relationship between the hypoxic microenvironment in joints and the occurrence and progression of RA. The main cause of the pathological changes in RA is widely believed to be the abnormal expression of hypoxia-inducible factor-1 (HIF-1) in joints. This paper describes and illustrates the structure and primary functions of HIF-1 and explains the main regulatory methods of HIF-1, including the PHDs/HIF-1 α/pVHL pathway, factor-inhibiting HIF (FIH), regulation of inflammatory cytokines, and the NF-κB pathway. Furthermore, this paper discusses the mechanism of HIF-1 and its impact on inflammation, angiogenesis, and cartilage destruction in greater detail. We summarize previous research findings on the mechanism of HIF-1 and propose new potential treatments for RA based on the pathogenesis of HIF-1 in RA.

On the force field optimisation of [Formula: see text]-lactam cores using the force field Toolkit.

When employing molecular dynamics (MD) simulations for computer-aided drug design, the quality of the used force fields is highly important. Here we present reparametrisations of the force fields for the core molecules from 9 different [Formula: see text]-lactam classes, for which we utilized the force field Toolkit and Gaussian calculations. We focus on the parametrisation of the dihedral angles, with the goal of reproducing the optimised quantum geometry in MD simulations. Parameters taken from CGenFF turn out to be a good initial guess for the multiplicity of each dihedral angle, but the key to a successful parametrisation is found to lie in the phase shifts. Based on the optimised quantum geometry, we come up with a strategy for predicting the phase shifts prior to the dihedral potential fitting. This allows us to successfully parameterise 8 out of the 11 molecules studied here, while the remaining 3 molecules can also be parameterised with small adjustments. Our work highlights the importance of predicting the dihedral phase shifts in the ligand parametrisation protocol, and provides a simple yet valuable strategy for improving the process of parameterising force fields of drug-like molecules.

A portable analytical system for rapid on-site determination of total nitrogen in water.

Excessive total nitrogen (TN) in the aqueous environment causes a notable negative impact on agriculture, human health, and the economy on a global scale. Conventional analytical techniques for determining TN in water usually involve long and tedious procedures with extensive sample preparation for digestion and titration. In recent years, lab-on-a-chip platforms have enabled in-situ measurements of water pollutants such as nitrate, nitrite, and ammonium. However, the digestion of organic nitrogen compounds in aqueous solutions still remains to be a challenge for portable analytical systems. In this paper, a portable TN analytical system is developed for on-site measurement of TN in a short assay time. It contains a TN reaction chamber for nitrogen digestion and reduction, and an optical measurement chamber for colorimetric determination of total nitrite. The ultraviolet-C (UVC)-thermal digestion method and the United States Environmental Protection Agency (EPA)-standard nitrate-nitrite determination method are implemented on the TN analytical system. Thorough investigations are performed to explore the optimum reaction conditions and reagent volumes in the process of TN oxidation, nitrate reduction, and nitrite detection, including oxidation time, temperature and substrate, oxidizer concentrations, nitrate reduction time, nitrite colorimetric reaction time, and reagents stability over time. Our system can complete fast oxidation and colorimetric determination of TN within 36 min, with a measurement range of 1 µg/L to 10 g/L and a limit of detection of 1.2 mg/L (lower than the World Health Organization standard of 10 mg/L). This portable TN analytical system enables the digestion and measurement of TN in a quick, portable, and low-cost manner.

A thread-based wearable sweat nanobiosensor.

Non-invasive wearable biosensors provide an efficient way of continuously quantifying a person's biochemical parameters, and are highly valuable for predicting human physiological status and flagging risks and illness. Commercial wearable sensors are available for tracking a user's physical activities, but few could monitor user's health conditions through sweat analysis. Electronic textile (e-textile) biosensors enable new applications in this scenario because of its high flexibility/wearability, low cost, high level of electronic integration, and unobtrusiveness. However, challenges in developing e-textile sweat biosensors remain in the production of textile-based biosensing materials, skin interfacing design, and embedded data acquisition/transmission. Here, we propose a novel wearable electrochemical sweat biosensor based on conductive threads decorated with zinc-oxide nanowires (ZnO NWs) and apply it to detecting lactate and sodium in perspiration during physical exercise. The sweat biosensor is fully integrated with signal readout and data communication circuits in a wearable headband and is capable of monitoring human sweat accurately and wirelessly. We achieved the detection of lactate and sodium in linear ranges of 0-25 mM and 0.1-100 mM and limits of detection of 3.61 mM and 0.16 mM, respectively, which cover the clinically-relevant ranges of lactate and sodium in human sweat. We demonstrated accurate lactate and sodium measurements in human sweat from a healthy volunteer, and the results are in good agreement with standard test results. We also conducted on-body measurements on the same human volunteer during exercise and confirmed the robustness of the signal readout during body movements and the excellent accuracy of the testing results.

A paper-based microfluidic platform with shape-memory-polymer-actuated fluid valves for automated multi-step immunoassays.

Smart fluid manipulation with automatically controlled paper valves will enable automated and multi-step immunoassays on paper-based microfluidic devices. In this work, we present an integrated paper-based microfluidic platform with shape-memory polymer (SMP)-actuated fluid valves capable of automated colorimetric enzyme-linked immunosorbent assays (ELISAs). A single-layer microfluidic paper-based analytical device (µPAD) was designed to store all the reagents on the chip, and sequentially transfer reagents to a paper test zone following a specific ELISA protocol through automatic fluidic flow control by the multiple SMP-actuated valves. The actuation of a paper valve was based on the thermally responsive, duel-state shape transformation of a SMP sheet attached to the root of a paper cantilever beam for driving a hydrophilic paper bridge to connect and disconnect two paper channels. A portable colorimetric reader was developed to control the on-chip valve operations, quantify the colorimetric signal output, display the assay result, and wirelessly transmit the data to a smart phone for the application of telemedicine. Reliable operations of the paper valve and the entire µPAD were demonstrated with success rates of 97% and 93%, respectively. A detection mechanism for valve malfunction was designed and confirmed effective to identify any mal-operation of individual valves, thus rendering our platform reliable in real assays. For device calibration, we conducted direct ELISAs of rabbit IgG in phosphate-buffered saline (PBS), and achieved a low limit of detection (LOD) of 27 pM (comparable to that of standard and paper-based ELISAs). In order to demonstrate the clinical application of our multi-step immunoassay platform, we also conducted sandwich ELISAs to quantify the protein level of an inflammatory cytokine, namely tumor necrosis factor (TNF)-α, in surgically injured laryngeal tissues of rats. The protein levels of TNF-α were shown similar between the conventional and µPAD ELISAs.

An assessment of the random-phase approximation functional and characteristics analysis for noncovalent cation-π interactions.

The binding energy is of great importance in understanding the formation and stability of noncovalent interactions. However, the determination of the binding energy with high precision and efficiency in medium- and long-range noncovalent interactions is still challenging for quantum chemistry. Here, we assess the performance of random-phase approximation (RPA), a fully non-local fifth-rung of the Jacob ladder functional, in determining the binding energy of cation-π systems (cation = Li+, Na+, Be2+, Mg2+, Al+, and NH4+; π = C6H6), which, to the best of our knowledge, has not been investigated. Using experimental results as the benchmark, we systematically compared the RPA method to the other ab initio methods (DFT/B3LYP, MP2, CCSD(T), and QCISD(T)) both in calculation accuracy and efficiency. From the perspective of accuracy, RPA is the best among these approaches, followed by the CCSD(T) and QCISD(T) methods. DFT/B3LYP and MP2 provide the worst accuracy. In addition, the computational efficiency of RPA is much faster than that of CCSD(T) and QCISD(T). We believe that RPA is a robust method for the precise description of medium- and long-range noncovalent interactions and is capable of providing benchmarking data. The interaction strength and interaction nature of cation-π systems are further analyzed by atoms in molecules (AIM) and the color-mapped reduced density gradient (RDG) isosurface, which are consistent with the characteristics of a typical cation-π interaction.

Large lateral shift near pseudo-Brewster angle on reflection from a weakly absorbing double negative medium.

We focus on the lateral shift for an electromagnetic wave reflected from a weakly absorbing double negative medium (DNM). A large lateral shift near the pseudo-Brewster angle is found, which may be negative or positive. We give an analytic expression for such a kind of lateral shift, from which the critical transition point for sign-changing of the lateral shift can be easily obtained. Theoretical analysis shows that the absorption of DNM, even though very weak, plays an important role in determining the lateral shift. As evidences, we calculate the lateral shift by means of the momentum method and perform the finite-difference time-domain (FDTD) simulations. We find that the results of our theoretical analysis are reliable.