Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway.

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

A Novel Oral Drugs Delivery System for Borneol Based on HiCap®100 and Maltodextrin: Preparation, Characterization, and the Investigation as an Intestinal Absorption Enhancer.

The objective of this study was to create a new method for delivering oral borneol (BN) drug that would improve stability. This was accomplished through microencapsulation using HiCap®100 and maltodextrin (MD), resulting in HiCap®100/MD/BN microcapsules (MCs). The HiCap®100/MD/BN MCs were evaluated in terms of encapsulation efficiency (EE%), drug loading (DL%), morphological observations, particle size distribution, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), thermal analysis, drug degradation rate studies, and in vitro release behavior. The effect of MCs on intestinal permeability in a rat model was assessed using the model drug "florfenicol" (FF) in single-pass intestinal perfusion (SPIP) study. The relationship between MCs and P-glycoprotein (P-gp) was further investigated in comparison with verapamil (Ver). The irritation of MCs was assessed by histological analysis. The MCs in a spherical structure with micron-scale dimensions were obtained. The EE% and DL% were (86.71 ± 0.96)% and (6.03 ± 0.32)%, respectively. MCs played a significantly protective role in drug degradation rate studies. In vitro release studies indicated that the release behavior of MCs was significantly better than BN at the three-release media, and the cumulative release rate exceeded 90% in 15 min. The SPIP studies showed that MCs significantly enhanced the absorption of FF in rats. Compared with Ver, MCs were not promoted by a single inhibition of P-gp. Hematoxylin-eosin (HE)-stained images showed that MCs had no obvious irritation and toxic effects on the intestines of rats. Thus, the preparation of HiCap®100/MD/BN MCs improves the stability of BN, which has certain scientific value for the development and application of BN, and provides unique perspectives for future BN-related researches.

Solid-State Nanopore/Nanochannel Sensing of Single Entities.

Solid-state nanopores/nanochannels, with their high stability, tunable geometry, and controllable surface chemistry, have recently become an important tool for constructing biosensors. Compared with traditional biosensors, biosensors constructed with solid-state nanopores/nanochannels exhibit significant advantages of high sensitivity, high specificity, and high spatiotemporal resolution in the detection single entities (such as single molecules, single particles, and single cells) due to their unique nanoconfined space-induced target enrichment effect. Generally, the solid-state nanopore/nanochannel modification method is the inner wall modification, and the detection principles are the resistive pulse method and the steady-state ion current method. During the detection process, solid-state nanopore/nanochannel is easily blocked by single entities, and interfering substances easily enter the solid-state nanopore/nanochannel to generate interference signals, resulting in inaccurate measurement results. In addition, the problem of low flux in the detection process of solid-state nanopore/nanochannel, these defects limit the application of solid-state nanopore/nanochannel. In this review, we introduce the preparation and functionalization of solid-state nanopore/nanochannel, the research progress in the field of single entities sensing, and the novel sensing strategies on solving the above problems in solid-state nanopore/nanochannel single-entity sensing. At the same time, the challenges and prospects of solid-state nanopore/nanochannel for single-entity electrochemical sensing are also discussed.

Characterization, Properties and Mixing Mechanism of Rubber Asphalt Colloid for Sustainable Infrastructure.

Rubber asphalt has always been considered to have the most potential for the disposal of waste tires as sustainable infrastructure. However, the covalently cross-linked tire rubber presents an extreme challenge in reusing waste rubbers in roads. Rubberized asphalt with finely dispersed or colloidal structure has been regarded as a potential binder used as road material because of the improved properties in terms of storage stability, easy processing and high content of incorporation. However, the mixing mechanism between the finely dispersed rubber on micro-nano scale with asphalt is still not clear, which restricts its further development as value-added material. Devulcanized rubber (DR) was introduced to improve the compatibility between asphalt and rubber. The basic chemicals of DR and asphalt were introduced based on their structures. Furthermore, the interactions between DR and asphalt were discussed according to the functional elements at different levels, and the concept of DR as "the fifth component" of asphalt was put forward. Finally, high performance, environmental and economic effects and applications of devulcanized-rubber-modified asphalt (DRMA) were discussed. The review is expected to provide a guide for the wide application of DRMA, which is still restricted by poor compatibility and bad stability during processing, storage and recycling.

Efficient targeted insertion of large DNA fragments without DNA donors.

Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.

A ratiometric electrochemical sensor for selectively monitoring monoamine oxidase A in the live brain.

Herein, an electrochemical method for selectively sensing and accurately quantifying monoamine oxidase A (MAO-A) in the cortex and thalamus of a live mouse brain was reported. Using this tool, it was found that MAO-A increased Ca2+ entry into neurons via the TPRM2 channel in the live mouse brain of an AD model.

A Robust Au-C≡C Functionalized Surface: Toward Real-Time Mapping and Accurate Quantification of Fe2+ in the Brains of Live AD Mouse Models.

Described here is that Au-C≡C bonds showed the highest stability under biological conditions, with abundant thiols, and the best electrochemical performance compared to Au-S and Au-Se bonds. The new finding was also confirmed by theorical calculations. Based on this finding, a specific molecule for recognition of Fe2+ was designed and synthesized, and used to create a selective and accurate electrochemical sensor for the quantification of Fe2+ . The present ratiometric strategy demonstrates high spatial resolution for real-time tracking of Fe2+ in a dynamic range of 0.2-120 µM. Finally, a microelectrode array with good biocompatibility was applied in imaging and biosensing of Fe2+ in the different regions of live mouse brains. Using this tool, it was discovered that the uptake of extracellular Fe2+ into the cortex and striatum was largely mediated by cyclic adenosine monophosphate (cAMP) through the CREB-related pathway in the brain of a mouse with Alzheimer's disease.

A SERS Optophysiological Probe for the Real-Time Mapping and Simultaneous Determination of the Carbonate Concentration and pH†Value in a Live Mouse Brain.

To have a profound understanding of the physiological and pathological processes in a brain, both chemical and electrical signals need to be recorded, but this is still very challenging. Herein, micrometer- to nanometer-sized SERS optophysiological probes were created to determine both the CO32- concentration and the pH in live brains and neurons because both species play important roles in regulating the acid-base balance in the brain. A ratiometric SERS microarray of eight microprobes with tip sizes of 5 µm was established and used for the first time for real-time mapping and simultaneous quantification of CO32- and pH in a live brain. We found that both the CO32- concentration and the pH value dramatically decreased under ischemic conditions. The present SERS technique can be combined with electrophysiology without cross-talk to record both electrical and chemical signals in brains. To deepen our understanding of the mechanism of ischemia on the single-cell level, a SERS nanoprobe with a tip size of 200 nm was developed for use in a single neuron.

Metabolites from carnivorous fungus Arthrobotrys entomopaga and their functional roles in fungal predatory ability.

The carnivorous fungus Arthrobotrys entomopaga (Drechsler) can develop adhesive knobs to capture nematodes. Chemical study on the culture medium of A. entomopaga producing adhesive knobs led to isolation of six trace amounts of metabolites, including two new metabolites, paganins A and B (1 and 2), blumenol A (3), talathermophilins A and B (4 and 5), and cyclo(glycyltryptophyl) (6). Compounds 3-6 were reported for the first time from carnivorous fungi. Compounds 1 and 2 promoted the formation of the predatory adhesive knobs with an increasing rate up to 118% at a concentration of 50 µM but showed moderate inhibitory activity at a concentration of 5 µM. Moreover, compounds 1 and 2 displayed strong inhibitory activities toward the formation of A. entomopaga conidiophores with inhibitory rates of 40-75%. Growth experiments suggested that compounds 1 and 2 could be involved in the regulation of the fungal predatory and reproductive abilities. Nematode chemotaxis bioassay indicated that compounds 1 and 3 displayed strong nematode-attracting abilities. These findings provided a new type of regulatory metabolite and support for the hypothesis that predators often evolve to respond to their metazoan prey.

Thermolides, potent nematocidal PKS-NRPS hybrid metabolites from thermophilic fungus Talaromyces thermophilus.

Macrocyclic PKS-NRPS hybrid metabolites represent a unique family of natural products mainly from bacteria with broad and outstanding biological activities. However, their distribution in fungi has rarely been reported, and little has been reported regarding their nematocidal activity. Here we describe an unprecedented class of PKS-NRPS hybrid metabolites possessing a 13-membered lactam-bearing macrolactone, thermolides A-F (1-6) from a thermophilic fungus Talaromyces thermophilus. We showed that 1 and 2 displayed potent inhibitory activity against three notorious nematodes with LC(50) values of 0.5-1 µg/mL, as active as commercial avermectins. This work provided a new class of promising lead compounds for nematocide discovery.

Morphology regulatory metabolites from Arthrobotrys oligospora.

Novel autoregulatory metabolites, arthrosporols A-C (1-3), involved in regulating the morphological switch in fungi, were purified and characterized from the carnivorous fungus Arthrobotrys oligospora. These compounds possess a novel hybrid carbon skeleton consisting of an epoxy-cyclohexenol combined with a rare monocyclic sesquiterpenol substructure. This is the first report of a monocyclic sesquiterpenol of this type of fungal origin. Compounds 1-3 displayed significant inhibitory activities toward the formation of conidiophores, while compounds 1 and 3 showed the opposite effects on the formation of a two-dimensional network with increasing rates of 40-90% and inhibiting rates of 30-90%, respectively.

Isolation of talathermophilins from the thermophilic fungus Talaromyces thermophilus YM3-4.

Six indole alkaloids with various levels of prenylation were isolated from the thermophilic fungus Talaromyces thermophilus strain YM3-4. Their structures were identified by NMR and MS spectroscopic analyses. Compounds 1 and 2 are new analogues of the key versatile precursor notoamide E. Compound 3 is a novel analogue of preechinulin, and compound 4 was reported as a natural occurring cyclo(glycyltryptophyl) for the first time. The metabolite profile of this thermophilic organism displayed a biosynthetic pathway for talathermophilins.