Upregulation of P2X7 Exacerbates Myocardial Ischemia-Reperfusion Injury through Enhancing Inflammation and Apoptosis in Diabetic Mice.

Diabetes-aggravated myocardial ischemia-reperfusion (MI/R) injury remains an urgent medical issue, and the molecular mechanisms involved with diabetes and MI/R injury remain largely unknown. Previous studies have shown that inflammation and P2X7 signaling participate in the pathogenesis of the heart under individual conditions. It remains to be explored if P2X7 signaling is exacerbated or alleviated under double insults. We established a high-fat diet and streptozotocin-induced diabetic mouse model, and we compared the differences in immune cell infiltration and P2X7 expression between diabetic and nondiabetic mice after 24 h of reperfusion. The antagonist and agonist of P2X7 were administered before and after MI/R. Our study showed that the MI/R injury of diabetic mice was characterized by increased infarct area, impaired ventricular contractility, more apoptosis, aggravated immune cell infiltration, and overactive P2X7 signaling compared with nondiabetic mice. The major trigger of increased P2X7 was the MI/R-induced recruitment of monocytes and macrophages, and diabetes can be a synergistic factor in this process. Administration of P2X7 agonist eliminated the differences in MI/R injury between nondiabetic mice and diabetic mice. Both 2 wk of brilliant blue G injection before MI/R and acutely administered A438079 at the time of MI/R injury attenuated the role of diabetes in exacerbating MI/R injury, as evidenced by decreased infarct size, improved cardiac function, and inhibition of apoptosis. Additionally, brilliant blue G blockade decreased the heart rate after MI/R, which was accompanied by downregulation of tyrosine hydroxylase expression and nerve growth factor transcription. In conclusion, targeting P2X7 may be a promising strategy for reducing the risk of MI/R injury in diabetes.

Diverse Chiral Nanotubes Assembled from Identical DNA Strands.

DNA nanotubes with controllable geometries hold a wide range of interdisciplinary applications. When preparing DNA nanotubes of varying widths or distinct chirality, existing methods require repeatedly designing and synthesizing specific DNA sequences, which can be costly and laborious. Here, we proposed an intercalator-assisted DNA tile assembly method which enables the production of DNA nanotubes of diverse widths and chirality using identical DNA strands. Through adjusting the concentration of intercalators during assembly, the twisting direction and extent of DNA tiles could be modulated, leading to the formation of DNA nanotubes featuring controllable widths and chirality. Moreover, through introducing additional intercalators and secondary annealing, right-handed nanotubes could be reconfigured into distinct left-handed nanotubes. We expect that this method could be universally applied to modulating the self-assembly pathways of various DNA tiles and other chiral materials, advancing the landscape of DNA tile assembly.

A Chemo-Mechanically Coupled DNA Origami Clamp Capable of Generating Robust Compression Forces.

DNA nanostructures have been utilized to study biological mechanical processes and construct artificial nanosystems. Many application scenarios necessitate nanodevices able to robustly generate large single molecular forces. However, most existing dynamic DNA nanostructures are triggered by probabilistic hybridization reactions between spatially separated DNA strands, which only non-deterministically generate relatively small compression forces (≈0.4 piconewtons (pN)). Here, an intercalator-triggered dynamic DNA origami nanostructure is developed, where large amounts of local binding reactions between intercalators and the nanostructure collectively lead to the robust generation of relatively large compression forces (≈11.2 pN). Biomolecular loads with different stiffnesses, 3, 4, and 6-helix DNA bundles are efficiently bent by the compression forces. This work provides a robust and powerful force-generation tool for building highly chemo-mechanically coupled molecular machines in synthetic nanosystems.

DNA Kirigami Driven by Polymerase-Triggered Strand Displacement.

The precursors of functional biomolecules in living cells are synthesized in a bottom-up manner and subsequently activated by modification into a delicate structure with near-atomic precision. DNA origami technology provides a promising way to mimic the synthesis of precursors, although mimicking the modification process is a challenge. Herein, a DNA paper-cutting (DNA kirigami) method to trim origami into designer nanostructures is proposed, where the modification is implemented by a polymerase-triggered DNA strand displacement reaction. Six geometric shapes are created by cutting rectangular DNA origami. Gel electrophoresis and atomic force microscopy results demonstrate the feasibility and capability of the DNA paper-cutting method. The proposed DNA paper-cutting strategy can enrich the toolbox for dynamically transforming DNA origami and has potential applications in biomimetics. .

Cryopreservation of stool samples altered the microbial viability quantitively and compositionally.

Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at - 80 °C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at - 80 °C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.

Tuning curved DNA origami structures through mechanical design and chemical adducts.

The bending and twisting of DNA origami structures are important features for controlling the physical properties of DNA nanodevices. It has not been fully explored yet how to finely tune the bending and twisting of curved DNA structures. Traditional tuning of the curved DNA structures was limited to controlling the in-plane-bending angle through varying the numbers of base pairs of deletions and insertions. Here, we developed two tuning strategies of curved DNA origami structures fromin silicoandin vitroaspects.In silico, the out-of-plane bending and twisting angles of curved structures were introduced, and were tuned through varying the patterns of base pair deletions and insertions.In vitro, a chemical adduct (ethidium bromide) was applied to dynamically tune a curved spiral. The 3D structural conformations, like chirality, of the curved DNA structures were finely tuned through these two strategies. The simulation and TEM results demonstrated that the patterns of base pair insertions and deletions and chemical adducts could effectively tune the bending and twisting of curved DNA origami structures. These strategies expand the programmable accuracy of curved DNA origami structures and have potential in building efficient dynamic functional nanodevices.

Switching the activity of Taq polymerase using clamp-like triplex aptamer structure.

In nature, allostery is the principal approach for regulating cellular processes and pathways. Inspired by nature, structure-switching aptamer-based nanodevices are widely used in artificial biotechnologies. However, the canonical aptamer structures in the nanodevices usually adopt a duplex form, which limits the flexibility and controllability. Here, a new regulating strategy based on a clamp-like triplex aptamer structure (CLTAS) was proposed for switching DNA polymerase activity via conformational changes. It was demonstrated that the polymerase activity could be regulated by either adjusting structure parameters or dynamic reactions including strand displacement or enzymatic digestion. Compared with the duplex aptamer structure, the CLTAS possesses programmability, excellent affinity and high discrimination efficiency. The CLTAS was successfully applied to distinguish single-base mismatches. The strategy expands the application scope of triplex structures and shows potential in biosensing and programmable nanomachines.

Fuzzy DNA Strand Displacement: A Strategy to Decrease the Complexity of DNA Network Design.

Toehold-mediated DNA strand displacement endows DNA nanostructures with dynamic response capability. However, the complexity of sequence design dramatically increases as the size of the DNA network increases. We attribute this problem to the mechanism of toehold-mediated strand displacement, termed exact strand displacement (ESD), in which one input strand corresponds to one specific substrate. In this work, we propose an alternative to toehold-mediated DNA strand displacement, termed fuzzy strand displacement (FSD), in which one-to-many and many-to-one relationships are established between the input strand and the substrate, to reduce the complexity. We have constructed four modules, termed converter, reporter, fuzzy detector, and fuzzy trigger, and demonstrated that a sequence pattern recognition network composed of these modules requires less complex sequence design than an equivalent network based on toehold-mediated DNA strand displacement.

Multimode adaptive logic gates based on temperature-responsive DNA strand displacement.

Living organisms switch their intrinsic biological states to survive environmental turbulence, in which temperature changes are prevalent in nature. Most artificial temperature-responsive DNA nanosystems work as switch modules that transit between "ON-OFF" states, making it difficult to construct nanosystems with diverse functions. In this study, we present a general strategy to build multimode nanosystems based on a temperature-responsive DNA strand displacement reaction. The temperature-responsive DNA strand displacement was controlled by tuning the sequence of the substrate hairpin strands and the invading strands. The nanosystems were demonstrated as logic gates that performed a set of Boolean logical functions at specific temperatures. In addition, an adaptive logic gate was fabricated that could exhibit different logic functions when placed in different temperatures. Specifically, upon the same input strands, the logic gate worked as an XOR gate at 10 °C, an OR gate at 35 °C, an AND gate at 46 °C, and was reset at 55 °C. The design and fabrication of the multifunctional nanosystems would help construct advanced temperature-responsive systems that may be used for temperature-controlled multi-stage drug delivery and thermally-controlled multi-step assembly of nanostructures.

Controllable DNA nanodevices regulated by logic gates for multi-stimulus recognition.

DNA biosensors have attracted considerable attention due to their great potential in environmental monitoring and medical diagnosis. Despite the great achievements, the single function and uncontrollability of the sensors restrict their further application. Therefore, it is necessary to construct controllable nanodevices with both sensing and responding capabilities to external stimuli. Herein, we develop a strategy to engineer structure-switching biosensors which can respond to external stimuli while preserving the sensing capability. The engineered nanodevice consists of an actuation module and a sensing module. Initially, the sensing module is disabled by a blocker strand which acts as an allosteric switch. Once the stimuli-responsive actuation module displaces the blocker DNA, the sensing module is activated. Based on the strategy, the engineered nanodevice could recognize both the target and external stimuli. As a demonstration of this strategy, a controllable Hg2+ sensor was designed, in which a 'YES', 'AND', and 'OR' logic gate is employed as the actuation module respectively to facilitate recognition of oligonucleotide inputs. The modular nature of the proposed strategy makes it easily generalizable to other structure-switching sensors. As a demonstration of this, we successfully apply it to the ATP sensor. The proposed strategy has potential in the fields of programmable biosensing, disease diagnosis, DNA computing, and intelligent nanodevices.

Regulating the Polymerization of DNA Structures via Allosteric Control of Monomers.

Regulation of self-assembly is crucial in constructing structural biomaterials, such as tunable DNA nanostructures. Traditional tuning of self-assembled DNA nanostructures was mainly conducted by introducing external stimuli after the assembly process. Here, we explored the allosteric assembly of DNA structures via introducing external stimuli during the assembly process to produce structurally heterogeneous polymerization products. We demonstrated that ethidium bromide (EB), a DNA intercalator, could increase the left-handed out-of-plane chirality of curved DNA structures. Then, EB and double strands were introduced as competing stimuli to transform monomers into allosteric conformations, leading to three different polymerization products. The steric trap between different polymerization products promoted the polymerized structures to keep their geometric properties, like chirality, under varying intensity of external stimuli. Our strategy harnesses allosteric effects for assembly of DNA-based materials and is expected to expand the design space for advanced control in synthetic materials.

Presketched DNA Origami Canvas for Polymerase-Driven DNA Kirigami.

Reconfigurable DNA origami provides a versatile tool to manipulate the conformation of matter on the nanometer scale. Typically, the DNA kirigami method enables the transformation of an origami structure from an initial shape to another predesigned shape by reconfiguring the staple strands. In a regular origami structure, since the perfectly matched and densely packed DNA duplexes block the removal of staple strands, the construction of finely trimmed "sub-origami" structures by the DNA kirigami method has remained challenging. Herein, we proposed a strategy to construct the presketched DNA origami canvas, where the offcut area in the canvas was sketched by loosely fixed staple strands with single-base insertion, to enhance the fineness of polymerase-driven DNA kirigami. We successfully trimmed presketched two-dimensional rectangular canvas, three-dimensional Möbius strip, and genie bottle canvases into complex letter patterns, supercoiled rings, and nanorods, respectively. Finally, we demonstrated a size-controlled DNA kirigami system: a presketched 6HB origami was trimmed into a set of shorter nanowires with predefined lengths, which quantitatively characterized the fineness of the improved DNA kirigami. The presketched origami design was a general method that applied to both 2D and 3D DNA origami structures in square and honeycomb lattices. Loosening DNA origami structures by introducing single-base insertions provides a practical approach to constructing dynamic components when designing DNA nanomachines. Furthermore, the delicate trimming of the DNA origami canvas driven by polymerase may inspire strategies for graphical information encryption and storage.

A microtubule stabilizer ameliorates protein pathogenesis and neurodegeneration in mouse models of repetitive traumatic brain injury.

Tau pathogenesis is a hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD). Although the events leading to initial tau misfolding and subsequent tau spreading in patient brains are largely unknown, traumatic brain injury (TBI) may be a risk factor for tau-mediated neurodegeneration. Using a repetitive TBI (rTBI) paradigm, we report that rTBI induced somatic accumulation of phosphorylated and misfolded tau, as well as neurodegeneration across multiple brain areas in 7-month-old tau transgenic PS19 mice but not wild-type (WT) mice. rTBI accelerated somatic tau pathology in younger PS19 mice and WT mice only after inoculation with tau preformed fibrils and AD brain-derived pathological tau (AD-tau), respectively, suggesting that tau seeds are needed for rTBI-induced somatic tau pathology. rTBI further disrupted axonal microtubules and induced punctate tau and TAR DNA binding protein 43 (TDP-43) pathology in the optic tracts of WT mice. These changes in the optic tract were associated with a decline of visual function. Treatment with a brain-penetrant microtubule-stabilizing molecule reduced rTBI-induced tau, TDP-43 pathogenesis, and neurodegeneration in the optic tract as well as visual dysfunction. Treatment with the microtubule stabilizer also alleviated rTBI-induced tau pathology in the cortices of AD-tau-inoculated WT mice. These results indicate that rTBI facilitates abnormal microtubule organization, pathological tau formation, and neurodegeneration and suggest microtubule stabilization as a potential therapeutic avenue for TBI-induced neurodegeneration.

Cofactor-assisted three-way DNA junction-driven strand displacement.

Toehold-mediated strand displacement is widely used to construct and operate DNA nanodevices. Cooperative regulation of strand displacement with diverse factors is pivotal in the design and construction of functional and dynamic devices. Herein, a cofactor-assisted three-way DNA junction-driven strand displacement strategy was reported, which could tune the reaction kinetics by the collaboration of DNA and other types of stimulus. This strategy is responsive to various inputs by incorporation of the specific sequence into the three-way junction structure. Specifically, the cooperation of multiple factors changes the conformation of the specific domain and promotes the reaction. To demonstrate the strategy, adenosine triphosphate (ATP), HG2+, and pH were used as cofactors to modulate the displacement reaction. The electrophoresis and fluorescence experiments showed that the cooperative regulation of the strand displacement reaction could be achieved by diverse factors using this strategy. The proposed strategy provides design flexibility for dynamic DNA devices and may have potential in biosensing and biocomputing.

A strategy for programming the regulation of in vitro transcription with application in molecular circuits.

In vitro transcription is a convenient platform for fabricating nanodevices and has been used for assembling synthetic networks. However, it remains challenging to regulate synthetic cell-free in vitro transcription by multiple stimuli in a simple and programmable way. We proposed a strategy to regulate in vitro transcription by controlling the transcription templates' promoter domain via variable DNA inputs. To demonstrate the utility of this strategy, various logic circuits and cascading circuits were implemented. With the advantage of simplicity, modularity, programmability, and extensibility, the proposed strategy has potential in biocomputing, bioanalytical, and therapeutic applications.

Complete-noncontact photoacoustic microscopy by detection of initial pressures using a 3×3 coupler-based fiber-optic interferometer.

We demonstrate a 3×3 coupler-based fiber-optic interferometric system to detect the local initial photoacoustic pressure. In contrast with the existing interferometric photoacoustic microscopy (PAM) relying on the measurement of the phase change of the probe light caused by the sample surface vibration, the present method measures the intensity change of the probe light caused by the initial photoacoustic pressure. Compared with the conventional interferometric PAMs, this method has the advantages: (1) it is free from the influence of the rough tissue surface, achieving complete noncontact in vivo imaging; (2) the probe light and the excitation light are focused at a same point below the sample surface, and the confocal configuration makes it more convenient for in vivo imaging; and (3) there is no need for phase stabilization, allowing a high imaging speed. These advantages show that the method will be a promising technique for in vivo imaging. This method is verified by imaging of a resolution test target and in vivo imaging of the blood vessels in a mouse ear.

Aptamer-based regulation of transcription circuits.

We propose synthetic DNA/RNA transcription circuits based on specific aptamer recognition. By mimicking transcription factor regulation, combined with specific enzyme/DNA aptamer binding, multiple biomolecules including DNA, RNA, polymerase, restriction enzymes and methylase were used as regulators. In addition, multi-level cascading networks and methylation-switch circuits were also established. This regulation strategy has the potential to expand the toolkit of in vitro synthetic biology.

Initial gut microbiota structure affects sensitivity to DSS-induced colitis in a mouse model.

The dextran sulfate sodium (DSS)-induced colitis model is a widely applied mouse model, but controversial results have been obtained from experiments using the same mouse strain under the same conditions. Because the gut microbiota play an important role in DSS-induced colitis, it is essential to evaluate the influence of the initial gut microbiota in this model. Here, we identified significant variations in the initial gut microbiota of different batches of mice and found that the initial intestinal microbiota had a profound influence on DSS-induced colitis. We performed three independent trials using the same C57BL/6J mouse model with DSS treatment and used high-throughput 16S rRNA gene sequencing to analyze the gut microbiota. We found that the structure and composition of the gut microbiota in mice with severe colitis, as compared with mice with milder colon damage, had unique features, such as an increase in Akkermansia bacteria and a decrease in Barnesiella spp. Moreover, these varied gut bacteria in the different trials also showed different responses to DSS treatment. Our work suggests that, in studies using mouse models, the gut microbiota must be considered when examining mechanisms of diseases, to ensure that comparable results are obtained.

Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice.

BACKGROUND: Calorie restriction (CR), which has a potent anti-inflammaging effect, has been demonstrated to induce dramatic changes in the gut microbiota. Whether the modulated gut microbiota contributes to the attenuation of inflammation during CR is unknown, as are the members of the microbial community that may be key mediators of this process. RESULTS: Here, we report that a unique Lactobacillus-predominated microbial community was rapidly attained in mice within 2 weeks of CR, which decreased the levels of circulating microbial antigens and systemic inflammatory markers such as tumour necrosis factor alpha (TNF-α). Lactobacillus murinus CR147, an isolate in the most abundant operational taxonomic unit (OTU) enriched by CR, downregulated interleukin-8 production in TNF-α-stimulated Caco-2 cells and significantly increased the lifespan and the brood size of the nematode Caenorhabditis elegans. In gnotobiotic mice colonized with the gut microbiota from old mice, this strain decreased their intestinal permeability and serum endotoxin load, consequently attenuating the inflammation induced by the old microbiota. CONCLUSIONS: Our study demonstrated that a strain of Lactobacillus murinus was promoted in CR mice and causatively contributed to the attenuation of ageing-associated inflammation.