Filter Membrane-Based Colorimetric Approach for Point-of-Care Detection of Biomarkers Using CRISPR-Cas12a.

CRISPR-Cas-based point-of-care testing (POCT) strategies have been widely explored for the detection of diverse biomarkers. However, these methods often require complicated operations, such as careful solution transfer steps, to achieve high sensitivity and accuracy. In this study, we combine a filter membrane-based POCT method with CRISPR-Cas12a for colorimetric detection of biomarkers. For the nucleic acid target, the trans-cleavage activity of CRISPR-Cas12a is directly triggered, cutting the single-stranded DNA linkers on glucose oxidase (GOx)-modified polymer nanoparticles. Due to the size difference between GOx and the polymer nanoparticles, GOx can be separated using a filter membrane. The filtrate containing GOx reacts with the substrate to generate a colorimetric signal. For the non-nucleic acid target, the non-nucleic acid signal is converted into a nucleic acid signal that activates CRISPR-Cas12a, resulting in a colorimetric signal. The entire operation is easy to perform, and the signal can be directly observed via the naked eye, which circumvents the use of costly instruments. The developed strategy holds great promise for accurate and accessible POCT detection of disease biomarkers in resource-limited settings.

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA.

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per µL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per µL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.

AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still challenging. In this study, we develop an AND logic-gate-based CRISPR-Cas12a biosensing platform to achieve the sensitive colorimetric detection of dual miRNAs. Specifically, the DNA probe was designed to recognize the binary input of miRNAs and to output trigger DNA, which activated the CRISPR-Cas12a system to cut single-stranded DNA (ssDNA). The ssDNA on magnetic beads (MBs) was cleaved by the activated CRISPR-Cas12a, causing the separation of glucose oxidase (GOx) from MB and the subsequent generation of a colorimetric signal. The color change induced by 1 pM of target miRNAs can be directly distinguished by the naked eye and the instrumental limit of detection reaches 36.4 fM. The overexpressed miR-205 and miR-944 in the real human serum can be detected, allowing us to differentiate between lung cancer patients and healthy people. Furthermore, the developed strategy achieves simultaneous detection of dual miRNAs using CRISPR-Cas12a with one kind of crRNA, avoiding sophisticated nucleic acid amplifications and the use of bulky instruments. The current method can broaden the CRISPR-Cas12a-based applications for multiple biomarkers detection and precise disease diagnosis.

The inhibitory effect of neurotropin on inflammation in rats with lumbar disc herniation based on the c-JNK/CXCL1 signaling pathway.

This study aimed to investigate the inhibitory effect and mechanism of neurotropin on inflammation in rats with lumbar disc herniation. For this purpose, forty-eight rats were randomly divided into sham group, autologous nucleus pulposus transplantation model group (NP group), neurotropin treatment group (NP+NT group), and solvent [normal saline (NS)] control group (NP+NS group). After 7 days of intervention, the mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) of the rats were measured, and the expression levels of Iba-1, c-JNK and CXCL1 in spinal cord tissues were measured by Western blot. The levels of tissue-associated inflammatory and anti-inflammatory factors in the spinal cord were detected by ELISA. Results showed that Neurotropin significantly alleviated mechanical and thermal hyperalgesia induced by NP transplantation and reduced levels of Iba-1, c-JNK, and CXCL1 proteins in the spinal cord tissue. In addition, neurotoxins also lowered concentrations of the inflammatory factors IL-1ß, IL-6 and TNF-α. It was concluded that Neurotropin has an inhibitory effect on lumbar disc herniation-induced spinal cord inflammation through inhibition of the c-JNK/CXCL signaling pathway.

Evidence-based practice implementation in healthcare in China: a living scoping review.

BACKGROUND: Evidence-based practice (EBP) implementation plays a crucial role in bridging the knowledge-action gaps and reducing health inequities. Little is known about its development in China. This study aims to provide an overview of the EBP implementation research progress in healthcare in China and identify gaps for future studies. METHODS: We conducted a scoping review following the Joanna Briggs Institute scoping review methodology and the Cochrane Collaboration's guidance on living reviews. We performed a literature search in four Chinese databases (i.e., China National Knowledge Infrastructure, Wan Fang Database, The VIP Database, and China Biology Medicine) and three English databases (i.e., Ovid MEDLINE, the Cumulative Index to Nursing and Allied Health Literature, and EMBASE), Google scholar, and Baidu scholar from 1996 to 2021. We included EBP implementation studies conducted in healthcare settings in China and were published in Chinese and English literature. The search will be run on a regular basis to monitor the development of new literature and determine when to update the review. FINDINGS: Of the 11,276 records identified, we finally included 309 papers. The publications were on a sharp rise since 2013 and were predominantly from the nursing field (292/309, 94.50%). The commonly researched areas were symptom management (75/309, 24.27%), tube care (46/309, 14.89%), perioperative care (43/309, 13.92%), and fundamental care (43/309, 13.92%). Joanna Briggs Institute model was the most frequently used model to guide the implementation process (92/159, 59.75%). A median number of 8 people often comprised an implementation team, with 113 studies (36.57%) taking a multidisciplinary approach. 204 studies reported utilizing audit criteria to assist evaluation of evidence implementation rate with diversified methods measuring the criteria. Lack of knowledge, skills, and resources, and incomplete procedures or pathways were top barriers impeding EBP implementation. Leadership support was considered the most common facilitator. Education and training were the most frequently described implementation strategies for healthcare professionals and patients. Optimizing workflows and developing evaluation tools were the primary strategies adopted by organizations. 291 studies measured patient outcomes and 174 studies measured healthcare professional outcomes. INTERPRETATION: To our knowledge, this scoping review is the first one to systematically examine the EBP implementation research progress in healthcare in China. Based on this review, we identified contributions that Chinese EBP implementation research made to the global community, and provided eight recommendations for Chinese researchers in conducting implementation studies in the future. FUNDING: None.

Strand Displacement Amplification Assisted CRISPR-Cas12a Strategy for Colorimetric Analysis of Viral Nucleic Acid.

The development of a sensitive, facile, and cost-effective colorimetric method is of great significance for the point-of-care testing of viral nucleic acid. Herein, we reported a strand displacement amplification assisted CRISPR-Cas12a (SDACC) method for the colorimetric analysis of viral nucleic acid. The hepatitis B virus (HBV) DNA was chosen as the target to trigger strand displacement amplification (SDA) and generate abundant single-strand DNA (ssDNA) products. The ssDNA amplicon hybridized with template DNA to activate the trans-cleavage activity of CRISPR-Cas12a, leading to the nonspecific cleavage of ssDNA on GOx-ssDNA-modified magnetic beads and the release of GOx. The released GOx was capable of catalyzing the substrate solution to generate a color change, which could be directly observed by naked eyes. The SDACC strategy could identify a single-base mismatch located in the DNA sequence and achieve a sensitive detection for HBV DNA with the limit of detection as low as 41.8 fM. Notably, the sophisticated primer design for target amplification and complicated detection process could be circumvented. The current approach realizes a simple, low-cost, and sensitive colorimetric detection for viral nucleic acid and holds great promise for the practical application of virus infection diagnosis.

CRISPR/Cas-Based In Vitro Diagnostic Platforms for Cancer Biomarker Detection.

Timely diagnosis is of great benefit to improve the survival rate of cancer patients. Body fluid cancer biomarker detection is a critical kind of noninvasive method for cancer diagnosis. Nevertheless, traditional methods for cancer biomarker detection always rely on a large-scale instrument and involve sophisticated operation. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, holding great promise as the next-generation molecular diagnostic technology. In this Feature, we introduce the working mechanisms of different kinds of CRISPR/Cas systems for biosensing and CRISPR/Cas-mediated detection strategies for different kinds of cancer biomarkers including nucleic acids, proteins, and extracellular vesicles. In addition, the perspective and challenges of CRISPR/Cas-based strategies for cancer biomarkers are discussed.

Duplex-Specific Nuclease-Assisted CRISPR-Cas12a Strategy for MicroRNA Detection Using a Personal Glucose Meter.

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. Nevertheless, the current CRISPR-Cas12a-based detection strategies always need bulky instruments or auxiliary devices to obtain a quantitative signal output, which restrains its point-of-care testing application. Herein, we proposed a duplex-specific nuclease-assisted CRISPR-Cas12a strategy to detect microRNA (miRNA) with a personal glucose meter. The target miRNA was first converted into an amplified initiator DNA via duplex-specific nuclease. Afterward, the initiator DNA activated the collateral cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA-modified magnetic beads, which led to the release of sucrase. The released sucrase was collected and then utilized to catalyze sucrose to glucose, which could be quantitatively detected by a personal glucose meter. The change in the glucose signal directly reflected the concentration of miRNA, which avoided expensive equipment for signal quantification. Two different miRNAs (miRNA21 and miRNA205) could be detected by simply changing the sequence of the template strand (H strand). The developed strategy showed high sensitivity with a limit of detection (LOD) of 2.4 and 1.1 pM for miRNA21 and miRNA205, respectively. In addition, good selectivity and anti-interference ability were achieved using this method, which enabled it promising for miRNA detection at the point-of-care.

A hybridization-based dual-colorimetric kit for circulating cancer miRNA detection.

A dual-colorimetric miRNA detection kit that can simultaneously detect two miRNAs with high sensitivity and selectivity is developed, and the data can be read by the naked eye easily. The kit is able to distinguish the patients from healthy people and achieve lung cancer diagnosis using clinical serum samples in a short time.

A dual-catalytic nanoreactor for synergistic chemodynamic-starvation therapy toward tumor metastasis suppression.

Tumor metastasis is extremely deadly for cancer patients and developing effective treatments for deep metastatic tumors remains a major challenge. In this study, we demonstrated a dual-catalytic nanoreactor for tumor metastasis suppression by synergistic Fenton reaction activated chemodynamic therapy (CDT) and glucose oxidase (GOx) initiated starvation therapy. GOx on the surface of hollow mesoporous silica nanoparticles can catalyze the decomposition of intratumoral glucose to generate gluconic acid and H2O2, while Fe3O4 nanoparticles as a Fenton reaction catalyst can in situ catalyze H2O2 to produce highly toxic hydroxyl radicals (˙OH). The oxygen-carrying perfluorohexane (PFC) in the hole of the hollow structures can alleviate the hypoxic environment and promote dual-catalytic reactions. After being disguised by the cancer cell membrane, the delivery efficiency and biological safety of the nanoreactor were effectively improved. The nanoreactor can realize sequential glucose depletion and ˙OH aggregation, which effectively suppress tumor metastasis with negligible side effects.

Avoiding False Positive Signals: A Powerful and Reliable Au-Se Dual-Color Probe.

Nucleic acids as the important tumor markers play a crucial role in the identification of cancer. Various kinds of probes such as gold nanoparticles and graphene oxide have been explored to detect different nucleic acid markers. However, the existing probes are mostly used to detect a single tumor marker and susceptible to harsh conditions in the complex and dynamic physiological environment, which may lead to false positive results and greatly limit the sensing performance of the probe. Herein, a powerful and reliable Au-Se probe was developed for high-fidelity imaging of two cancer markers simultaneously in living cells. Compared with the traditional nucleic acid probe based on the Au-S bond, this probe was more stable against biological thiols and could effectively distinguish normal cells and cancer cells to avoid false positive results, which is more suitable for imaging in a complex physiological environment. This strategy will provide more valuable insights into designing and exploring novel biosensors in the future.

Umbelliferone Ameliorates Complete Freund Adjuvant-Induced Arthritis via Reduction of NF-κB Signaling Pathway in Osteoclast Differentiation.

Osteoclasts, bone-resorbing somatic cells, are directly responsible for bone destruction during rheumatoid arthritis. Complete Freund adjuvant (CFA) is a widely used animal model using rodents for studying rheumatoid arthritis (RA), which effectively manifests serious cartilage destruction and progressive bone erosion, affecting synovial joints and serious joint dysfunction. It was considered that joint injury in RA is induced through systemic inflammation pathway. Umbelliferone (UF), a coumarin derivative of Agele marmilosa, possesses anti-inflammatory activity. In the current study, we scrutinize the effect of umbelliferone on CFA-induced arthritis model and explore the possible mechanism on bone destruction. Intradermal administration of CFA (0.05 mL) was to induce RA manifestations in the experimental rats and the same oral administration of UF was received. The anti-arthritic activity of UF was determined by its inhibitory activity on various biochemical markers, viz., pro-inflammatory, inflammatory, antioxidant enzymes, and hematological parameters elevated during RA condition. We also estimated the mRNA expression of osteoclast parameters. Obtained result disclosed significant reduction in the paw edema and increment of the body weight after UF administration. UF reduce the inflammatory mediatory such as COX-2, PGE2, NF-kB, and VEGF; pro-inflammatory cytokines include TNF-α, IL-1ß, IL-6, IL-10, and IL-17 significantly. Moreover, UF treatment significantly reduced the osteoclast number via modulating the RANKL/RANK/OPG ratio. Furthermore, administration of umbelliferone significantly (P < 0.001) suppressed the NF-κB and VEGF. Collectively, our results indicated the novel role of umbelliferone in osteoclastogenesis and proved that umbelliferone is a modern therapeutic tool as a natural agent for treating arthritis and other autoimmune disorders with bone degradation.

Robot-assisted technique vs conventional freehand technique in spine surgery: A meta-analysis.

BACKGROUND: The impact of robot-assisted techniques versus conventional freehand techniques in terms of the accuracy of pedicle screw placement remains conflicting. This meta-analysis was performed to evaluate this relationship. METHODS: A systematic literature search up to July 2020 was performed and 15 studies were detected with 6041 pedicle screw placements with 2748 of them were using robot-assisted techniques and 3293 were conventional freehand techniques. They reported relationships between robot-assisted techniques and conventional freehand techniques in pedicle screw placement. Odds ratio (OR) or Mean differences (MD) with 95% confidence intervals (CIs) was calculated comparing the robot-assisted techniques to conventional freehand techniques in pedicle screw placement risks using the dichotomous and continuous method with a random or fixed-effect model. RESULTS: Robot-assisted techniques had a significantly higher screw position grade A in Gertzbein-Robbins classification of the screw placement accuracy (OR, 2.43; 95% CI, 1.66-3.54, P < .001); shorter postoperative stay (MD, -0.67; 95% CI, -1.16 to -0.19, P < .001); lower intraoperative blood loss (MD, -91.64; 95% CI, -152.44 to -30.83, P = .003); fewer intraoperative radiation dose (MD, -23.52; 95% CI, -40.12 to -6.0.93, P = .005); and low proximal facet violations (MD, 0.08; 95% CI, 0.03-0.20, P < .001) compared with conventional freehand techniques. However, no significant difference was found between robot-assisted techniques and conventional freehand techniques in surgical time (OR, 11.71; 95% CI, 03.27-26.70, P = .13); visual analogue scale scores (MD, -0.15; 95% CI, -0.54 to 0.23, P = .44); and Oswestry disability index scores (MD, 0.21; 95% CI, -5.09-5.51, P = .94). CONCLUSIONS: The extent of the improvement with robot-assisted techniques in screw position grade A in Gertzbein-Robbins classification of the screw placement accuracy, postoperative stay, intraoperative blood loss, intraoperative radiation dose, and proximal facet violations was significantly better than conventional freehand techniques. This relationship forces us to recommend robot-assisted techniques for pedicle screw placement to avoid any possible negative postoperative results.

An in vitro site-specific cleavage assay of CRISPR-Cas9 using a personal glucose meter.

A sensitive and cost-effective assay based on a personal glucose meter was developed to detect the in vitro site-specific cleavage efficiency of CRISPR-Cas9.

A tumor acidity activatable and Ca2+-assisted immuno-nanoagent enhances breast cancer therapy and suppresses cancer recurrence.

Breast cancer recurrence is the greatest contributor to patient death. As the immune system has a long-term immune memory effect, immunotherapy has great potential for preventing cancer recurrence. However, cancer immunotherapy is often limited due to T cell activation being blocked by insufficient tumor immunogenicity and the complex immunosuppressive tumor microenvironment. Here we show a tumor acidity activatable and Ca2+-assisted immuno-nanoagent to synergistically promote T cell activation and enhance cancer immunotherapy. When the immuno-nanoagent reaches the acidic tumor microenvironment, the CaCO3 matrix disintegrates to release immune stimulants (CpG ODNs and IDOi) and Ca2+. CpG ODNs are responsible for triggering dendritic cell maturation to increase the immunogenicity for activation of T cells. And IDOi can inhibit the oxidative catabolism of tryptophan to kynurenine for preventing T-cell anergy and apoptosis. Due to the complexity of the immunosuppressive microenvironment, it is difficult to restore T cell activation by inhibiting only one pathway. Fortunately, the released Ca2+ can promote the activation and proliferation of T cells with the support of the immune stimulants. In vivo experiments demonstrate that our Ca2+-assisted immuno-nanoagent can significantly suppress tumor progression and protect mice from tumor rechallenge due to the long-term memory effect. This immunotherapeutic strategy may provide more possibilities for clinical applications such as treating cancer and preventing relapse.

A nuclear-targeted titanium dioxide radiosensitizer for cell cycle regulation and enhanced radiotherapy.

A nuclear-targeted titanium dioxide radiosensitizer was developed to regulate the cell cycle and enhance the radiation effect.

The role of HD-Zip class I transcription factors in plant response to abiotic stresses.

Abiotic stresses usually affect plant growth and development, indirectly or directly causing crop production reduction and even plant death. To survive, plants utilize different mechanisms to adapt themselves to continuously changing surrounding environmental stresses. Homeodomain-leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom and divided into four different subfamilies (HD-Zip I∼IV). Many HD-Zip I members have been shown to play critical roles in the regulation of plant developmental processes, signaling networks and responses to environmental stresses. This review focuses on the role of HD-Zip I transcription factors in plant responses to various abiotic stresses, including abscisic acid-mediated stress, drought and cold stress, oxidative stress, helping to identify the potential regulatory mechanisms that alleviate abiotic stress in plants.

A DR4 capturer with AKT siRNA for the synergetic enhancement of death receptor-mediated apoptosis.

A graphene-based nanocarrier modified with death receptor 4 (DR4) antibody and AKT siRNA has been developed, which can synergistically strengthen death receptor-mediated apoptosis and enhance the cancer therapeutic effect in vivo.

Advances in DNA Barcoding of Toxic Marine Organisms.

There are more than 200,000 marine species worldwide. These include many important economic species, such as large yellow croaker, ribbonfish, tuna, and salmon, but also many potentially toxic species, such as blue-green algae, diatoms, cnidarians, ctenophores, Nassarius spp., and pufferfish. However, some edible and toxic species may look similar, and the correct identification of marine species is thus a major issue. The failure of traditional classification methods in certain species has promoted the use of DNA barcoding, which uses short, standard DNA fragments to assist with species identification. In this review, we summarize recent advances in DNA barcoding of toxic marine species such as jellyfish and pufferfish, using genes including cytochrome oxidase I gene (COI), cytochrome b gene (cytb), 16S rDNA, internal transcribed spacer (ITS), and Ribulose-1,5-bisphosphate carboxylase oxygenase gene (rbcL). We also discuss the application of this technique for improving the identification of marine species. The use of DNA barcoding can benefit the studies of biological diversity, biogeography, food safety, and the detection of both invasive and new species. However, the technique has limitations, particularly for the analysis of complex objects and the selection of standard DNA barcodes. The development of high-throughput methods may offer solutions to some of these issues.

MicroRNA166 Modulates Cadmium Tolerance and Accumulation in Rice.

MicroRNAs (miRNAs) are 20- to 24-nucleotide small noncoding RNAs that regulate gene expression in eukaryotic organisms. Several plant miRNAs, such as miR166, have vital roles in plant growth, development and responses to environmental stresses. One such environmental stress encountered by crop plants is exposure to cadmium (Cd), an element highly toxic to most organisms, including humans and plants. In this study, we analyzed the role of miR166 in Cd accumulation and tolerance in rice (Oryza sativa). The expression levels of miR166 in both root and leaf tissues were significantly higher in the reproductive stage than in the seedling stage in rice. The expression of miR166 in the roots of rice seedlings was reduced after Cd treatment. Overexpression of miR166 in rice improved Cd tolerance, a result associated with the reduction of Cd-induced oxidative stress in transgenic rice plants. Furthermore, overexpression of miR166 reduced both Cd translocation from roots to shoots and Cd accumulation in the grains. miR166 targets genes encoding the class-III homeodomain-Leu zipper (HD-Zip) family proteins in plants. In rice, HOMEODOMAIN CONTAINING PROTEIN4 (OsHB4) gene (Os03g43930), which encodes an HD-Zip protein, was up-regulated by Cd treatment but down-regulated by overexpression of miR166 in transgenic rice plants. Overexpression of OsHB4 increased Cd sensitivity and Cd accumulation in the leaves and grains of transgenic rice plants. By contrast, silencing OsHB4 by RNA interference enhanced Cd tolerance in transgenic rice plants. These results indicate a critical role for miR166 in Cd accumulation and tolerance through regulation of its target gene, OsHB4, in rice.