A versatile and convenient tool for regulation of DNA strand displacement and post-modification on pre-fabricated DNA nanodevices.

Toehold-mediated strand displacement and its regulatory tools are fundamental for DNA nanotechnology. However, current regulatory tools all need to change the original sequence of reactants, making the regulation inconvenient and cumbersome. More importantly, the booming development of DNA nanotechnology will soon promote the production of packaged and batched devices or circuits with specified functions. Regarding standardized, packaged DNA nanodevices, access to personalized post-modification will greatly help users, whereas none of the current regulatory tools can provide such access, which has greatly constrained DNA nanodevices from becoming more powerful and practical. Herein, we developed a novel regulation tool named Cap which has two basic functions of subtle regulation of the reaction rate and erasability. Based on these functions, we further developed three advanced functions. Through integration of all functions of Cap and its distinct advantage of working independently, we finally realized personalized tailor-made post-modification on pre-fabricated DNA circuits. A pre-fabricated dual-output DNA circuit was successfully transformed into an equal-output circuit, a signal-antagonist circuit and a covariant circuit according to our requirements. Taken together, Cap is easy to design and generalizable for all strand displacement-based DNA nanodevices. We believe the Cap tool will be widely used in regulating reaction networks and personalized tailor-made post-modification of DNA nanodevices.

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA.

Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combining pre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM-SE+ dsDNA). We discovered that CRISPR-Cas12a had special enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.

Self-Internal-Reference Probe System for Control-Free Quantification of Mutation Abundance.

Gene mutations are important biomarkers for the diagnosis, classification, monitoring, and prognosis evaluation of cancers and genetic diseases. Both personalized cancer treatment and noninvasive prenatal testing require methods to accurately determine the abundance of mutation. At present, the widely adopted and convenient methods for measuring mutation abundance are mainly based on relative quantification, which requires negative samples and strict control of the analyte amounts. The development of DNA-probe-based methods that can determine the mutation abundance without negative samples nor control of analyte amount is highly preferred. The key to solving this bottleneck lies in whether the probe's response to mutation abundance can be completely independent of the number of targeted DNA strands. Herein, we propose the design of a self-internal-reference probe system. We established a theoretical model of this system and used the model to guide the design of probes. In this model, we provided quantitative corrections to the test results from the internal reference, thereby eliminating the influence of substrate amount. Therefore, the purification and quantification processes toward polymerase chain reaction (PCR) amplicons can be omitted. We applied this system to analyze unquantified PCR products aimed at cancer mutation detection and noninvasive prenatal testing.

Long-term quality of life between duodenum-preserving pancreatic head resection and pancreatoduodenectomy: a systematic review and meta-analysis.

BACKGROUND: The authors aimed to compare the differences in quality of life (QOL) and overall survival (OS) between duodenum-preserving pancreatic head resection (DPPHR) and pancreatoduodenectomy (PD) during long-term follow-up. DPPHR and PD have been shown to be effective in alleviating symptoms and controlling malignancies, but there is ongoing debate over whether DPPHR has an advantage over PD in terms of long-term benefits. METHOD: The authors searched the PubMed, Cochrane, Embase, and Web of Science databases for relevant studies comparing DPPHR and PD published before 1 May 2023. This study was registered with PROSPERO. Randomised controlled trials and non-randomised studies were included. The Mantel-Haenszel model and inverse variance method were used as statistical approaches for data synthesis. Subgroup analyses were conducted to evaluate the heterogeneity of the results. The primary outcome was the global QOL score, measured using the QLQ-C30 system. RESULTS: The authors analysed ten studies involving 976 patients (456 DPPHR and 520 PD). The global QOL score did not differ significantly between the DPPHR and PD groups [standard mean difference (SMD) 0.21, 95% CI (-0.05, 0.46), P =0.109, I2 =70%]; however, the OS time of patients with DPPHR was significantly improved [hazard ratio 0.59, 95% CI (0.44, 0.77), P <0.001, I2 =0%]. The follow-up length may be an important source of heterogeneity. Studies with follow-up length between two to seven years showed better global QOL for DPPHR than for PD [SMD 0.43, 95% CI (0.23, 0.64), P <0.001, I2 =0%]. There were no significant differences between the two groups in any of the functional scales of the QLQ-C30 system (all P >0.05). On the symptom scale, patients in the DPPHR group had lower scores for fatigue, nausea and vomiting, loss of appetite, insomnia, and diarrhoea than those in the PD group (all P <0.05). CONCLUSIONS: There were no significant differences in global QOL scores between the two surgeries; however, DPPHR had advantages over PD in terms of safer perioperative outcomes, lower long-term symptom scores, and longer OS times. Therefore, DPPHR should be recommended over PD for the treatment of benign pancreatic diseases and low-grade malignant tumours.

Automatic high-throughput and non-invasive selection of sperm at the biochemical level.

BACKGROUND: Sperm selection, a key step in assisted reproductive technology (ART), has long been restrained at the preliminary physical level (morphology or motility); however, subsequent fertilization and embryogenesis are complicated biochemical processes. Such an enormous "gap" poses tough problems for couples dealing with infertility, especially patients with severe/total asthenozoospermia . METHODS: We developed a biochemical-level, automatic-screening/separation, smart droplet-TO-hydrogel chip (BLASTO-chip) for sperm selection. The droplet can sense the pH change caused by sperm's respiration products and then transforms into a hydrogel to be selected out. FINDINGS: The BLASTO-chip system can select biochemically active sperm with an accuracy of over 90%, and its selection efficiency can be flexibly tuned by nearly 10-fold. All the substances in the system were proven to be biosafe via evaluating mice fertilization and offspring health. Live sperm down to 1% could be enriched by over 76-fold to 76%. For clinical application to patients with severe/total asthenozoospermia, the BLASTO-chip could select live sperm from human semen samples containing 10% live but 100% immotile sperm. The rates of fertilization, cleavage, early embryos, and blastocysts were drastically elevated from 15% to 70.83%, 10% to 62.5%, 5% to 37.5%, and 0% to 16.67%, respectively. CONCLUSIONS: The BLASTO-chip represents a real biochemical-level technology for sperm selection that is completely independent of sperm's motility. It can be a powerful tool in ART, especially for patients with severe/total asthenozoospermia. FUNDING: This work was funded by the Ministry of Science and Technology of China, the Ministry of Education of China, and the Shenzhen-Hong Kong Hetao Cooperation Zone.

Highly-sensitive and homogenous detection of 8-oxoguanine based DNA oxidative damage by a CRISPR-enhanced structure-switching aptamer assay.

8-oxoguanine (8-oxoG) based DNA damage is the most common type of DNA damage which greatly affect gene expression. Therefore, accurate quantification of 8-oxoG based DNA damage is of high clinical significance. However, current methods for 8-oxoG detection struggle to balance convenience, low cost, and sensitivity. Herein, we have proposed and investigated the shortened crRNA mode of CRISPR-Cas12a system and greatly enhanced its signal-to-noise ratio. Taking advantages of the shortened crRNA mode, we further developed a CRISPR-enhanced structure-switching aptamer assay (CESA) for 8-oxoG. The analytical performance of CESA was thoroughly investigated via detecting free 8-oxoG and 8-oxoG on gDNA. The CESA displayed impressive sensitivity for free 8-oxoG, with detection and quantification limits of 32.3 pM and 0.107 nM. These limits modestly rose to 64.5 pM and 0.215 nM when examining 8-oxoG on gDNA. To demonstrate the clinical practicability and significance of the CESA system, we further applied it to measuring 8-oxoG levels in 7 plasma samples (Cervical carcinoma, 11.87 ± 0.69 nM VS. Healthy control, 2.66 ± 0.42 nM), 24 seminal plasma samples (Asthenospermia, 22.29 ± 7.48 nM VS. Normal sperm, 9.75 ± 3.59 nM), 10 breast-tissue gDNA samples (Breast cancer, 2.77 ± 0.63 nM/µg VS. Healthy control, 0.41 ± 0.09 nM/µg), and 24 sperm gDNA samples (Asthenospermia, 28.62 ± 4.84 VS. Normal sperm, 16.67 ± 3.31). This work not only proposes a novel design paradigm of shortened crRNA for developing CRISPR-Cas12a based biosensors but also offers a powerful tool for detecting 8-oxoG based DNA damage.

TRIM21-Promoted FSP1 Plasma Membrane Translocation Confers Ferroptosis Resistance in Human Cancers.

Ferroptosis, an iron-dependent form of regulated cell death driven by excessive accumulation of lipid peroxides, has become a promising strategy in cancer treatment. Cancer cells exploit antioxidant proteins, including Ferroptosis Suppressor Protein 1 (FSP1), to prevent ferroptosis. In this study, it is found that the E3 ubiquitin ligase TRIM21 bound to FSP1 and mediated its ubiquitination on K322 and K366 residues via K63 linkage, which is essential for its membrane translocation and ferroptosis suppression ability. It is further verified the protective role of the TRIM21-FSP1 axis in RSL3-induced ferroptosis in cancer cells and a subcutaneous tumor model. Moreover, TRIM21 is highly expressed in multiple gastrointestinal (GI) tumors, and its expression is further stimulated upon ferroptosis induction in cancer cells and the KPC mouse model. In summary, This study identifies TRIM21 as a negative regulator of ferroptosis through K63 ubiquitination of FSP1, which can serve as a therapeutic target to enhance the chemosensitivity of tumors based on ferroptosis induction.