Recent advances in DNAzymes for bioimaging, biosensing and cancer therapy.

DNAzymes, a class of single-stranded catalytic DNA with good stability, high catalytic activity, and easy synthesis, functionalization and modification properties, have garnered significant interest in the realm of biosensing and bioimaging. Their integration with fluorescent dyes or chemiluminescent moieties has led to remarkable bioimaging outcomes, while DNAzyme-based biosensors have demonstrated robust sensitivity and selectivity in detecting metal ions, nucleic acids, proteins, enzyme activities, exosomes, bacteria and microorganisms. In addition, by delivering DNAzymes into tumor cells, the mRNA therein can be cleaved to regulate the expression of corresponding proteins, which has further propelled the application of DNAzymes in cancer gene therapy and synergistic therapy. This paper reviews the strategies for screening attractive DNAzymes such as SELEX and high-throughput sequencing, and briefly describes the amplification strategies of DNAzymes, which mainly include catalytic hairpin assembly (CHA), DNA walker, hybridization chain reaction (HCR), DNA origami, CRISPR-Cas12a, rolling circle amplification (RCA), and aptamers. In addition, applications of DNAzymes in bioimaging, biosensing, and cancer therapy are also highlighted. Subsequently, the possible challenges of these DNAzymes in practical applications are further pointed out, and future research directions are suggested.

Androgen Receptor Inhibitors in Patients With Nonmetastatic Castration-Resistant Prostate Cancer.

Importance: Novel androgen receptor inhibitors (ARIs; darolutamide, enzalutamide, and apalutamide) are standard-of-care treatments for nonmetastatic castration-resistant prostate cancer (nmCRPC). However, there are sparse data comparing their clinical use and tolerability. Objective: To compare clinical use and outcomes for darolutamide, enzalutamide, and apalutamide in patients with nmCRPC. Design, Setting, and Participants: This retrospective cohort study reviewed electronic medical records from the Precision Point Specialty network of US urology practices. Eligible patients had nmCRPC and no prior novel hormonal therapy and initiated novel ARI treatment between August 1, 2019, and March 31, 2022. Data were analyzed from February 1, 2019, to December 31, 2022. Exposures: Patients were prescribed darolutamide, enzalutamide, or apalutamide as their first novel ARI for nmCRPC. Main Outcomes and Measures: The main outcome was a composite of 2 end points, treatment discontinuation and progression to metastatic CRPC (mCRPC), whichever occurred first. Both end points were also assessed separately. Results: All 870 patients meeting eligibility criteria were included (362 receiving darolutamide [41.6%]; 382, enzalutamide [43.9%]; 126, apalutamide [14.5%]); mean (SD) age was 78.8 (8.7) years. Self-reported race was Black or African American in 187 patients (21.5%), White in 585 (67.2%), and other or unknown in 98 (11.3%). The darolutamide cohort had lower proportions of patients with a composite end point event (134 [37.0%] vs 201 [52.6%] for enzalutamide and 66 [52.4%] for apalutamide), discontinuation (110 [30.4%] for darolutamide vs 156 [40.8%] for enzalutamide and 58 [46.0%] for apalutamide), and progression to mCRPC (64 [17.7%] for darolutamide vs 108 [28.3%] for enzalutamide and 35 [27.8%] for apalutamide) during the study period. After adjusting for baseline covariates, patients receiving darolutamide had a lower risk of a composite end point event compared with enzalutamide (risk reduction, 33.8%; hazard ratio [HR], 0.66 [95% CI, 0.53-0.84]) and apalutamide (risk reduction, 35.1%; HR, 0.65 [95% CI, 0.48-0.88]). Similarly, patients receiving darolutamide had a lower risk of discontinuation compared with enzalutamide (risk reduction, 27.4%; HR, 0.73 [95% CI, 0.56-0.94]) and apalutamide (risk reduction, 39.1%; HR, 0.61 [95% CI, 0.44-0.85]) and a lower risk of progression to mCRPC compared with enzalutamide (risk reduction, 40.6%; HR, 0.59 [95% CI, 0.43-0.82]) and apalutamide (risk reduction, 35.3%; HR, 0.65 [95% CI, 0.42-0.99]). There was no difference between enzalutamide and apalutamide treatment across outcomes. Conclusions and Relevance: In this large cohort study of patients with nmCRPC treated with novel ARIs, results suggest better tolerability for darolutamide compared with enzalutamide and apalutamide, which may be associated with a clinical effectiveness advantage. Comparative clinical studies are needed to guide treatment decisions in the absence of head-to-head clinical trials.

Rtt105 stimulates Rad51-ssDNA assembly and orchestrates Rad51 and RPA actions to promote homologous recombination repair.

Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.

Real-world dosing of regorafenib and outcomes among patients with metastatic colorectal cancer: a retrospective analysis using US claims data.

BACKGROUND: The randomized, dose-optimization, open-label ReDOS study in US patients with metastatic colorectal cancer (CRC) showed that, compared with a standard dosing approach, initiating regorafenib at 80 mg/day and escalating to 160 mg/day depending on tolerability increased the proportion of patients reaching their third treatment cycle and reduced the incidence of adverse events without compromising efficacy. Subsequently, the ReDOS dose-escalation strategy was included as an alternative regorafenib dosing option in the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines. A retrospective analysis was conducted using a US claims database to assess whether inclusion of this dose-escalation strategy in NCCN Guidelines has influenced the use of flexible dosing in routine US clinical practice, and to describe clinical outcomes pre- and post-inclusion in NCCN Guidelines. METHODS: Patients with CRC in the Optum's de-identified Clinformatics® Data Mart database initiating regorafenib for the first time between January 2016 and June 2020 were stratified based on whether they initiated regorafenib pre- or post-inclusion of ReDOS in NCCN Guidelines, and in two groups: flexible dosing (< 160 mg/day; < 84 tablets in the first treatment cycle) and standard dosing (160 mg/day; ≥ 84 tablets in the first treatment cycle). The primary endpoints were the proportion of patients who initiated their third treatment cycle and the mean number of treatment cycles per group. RESULTS: 703 patients initiated regorafenib during the study period, of whom 310 (44%) initiated before and 393 (56%) initiated after inclusion of ReDOS in NCCN Guidelines. After inclusion in the guidelines, the proportion of patients who received flexible dosing increased from 21% (n = 66/310) to 45% (n = 178/393), the proportion who received standard dosing decreased from 79% (n = 244/310) to 55% (n = 215/393), the proportion who initiated their third treatment cycle increased from 36% (n = 113/310) to 46% (n = 179/393), and the mean (standard deviation) number of treatment cycles increased from 2.6 (2.9) to 3.2 (3.1). CONCLUSIONS: Following inclusion of ReDOS in NCCN Guidelines, real-world data suggest that US clinicians have markedly increased use of flexible dosing in clinical practice, potentially maximizing clinical benefits and safety outcomes for patients with metastatic CRC receiving regorafenib.

Dynamic Addressing Molecular Robot (DAMR): An Effective and Efficient Trial-and-Error Approach for the Analysis of Single Nucleotide Polymorphisms.

Accurate and efficient molecular recognition plays a crucial role in the fields of molecular detection and diagnostics. Conventional trial-and-error-based molecular recognition approaches have always been challenged in distinguishing minimal differences between targets and non-targets, such as single nucleotide polymorphisms (SNPs) of oligonucleotides. To address these challenges, here, a novel concept of dynamic addressing analysis is proposed. In this concept, by dissecting the regions of the target and creating a corresponding recognizer, it is possible to eliminate the inaccuracy and inefficiency of recognition. To achieve this concept, a Dynamic Addressing Molecular Robot (DAMR), a DNA-based dynamic addressing device is developed which is capable of dynamically locating targets. DAMR is designed to first bind to the conserved region of the target while addressing the specific region dynamically until accurate recognition is achieved. DAMR has provided an approach for analyzing low-resolution targets and has been used for analyzing SNP of miR-196a2 in both cell and serum samples, which has opened new avenues for effective and efficient molecular recognition.

Liquid-colloid-solid modular assembly for three-dimensional electrochemical biosensing of small molecules.

Electrochemical biosensors hold promise for advanced analytical applications in modern life analysis due to their miniaturization and cost-effectiveness. Nevertheless, their implementation in complex biological systems necessitates overcoming challenges related to timeliness, sensitivity, and interference resistance. Here, we developed a novel DNA hydrogel three-dimensional electron transporter through liquid-colloid-solid assembly, integrating electronic mediators and employing porous electrode covers with 3D printing technology. Our approach facilitated the fabrication of a high-performance electrochemical sensor for small molecule detection, leveraging target-specific aptamers and catalytic hairpin assembly (CHA) elements within the DNA hydrogel, which exhibited outstanding selectivity, sensitivity, and universality, achieving detection limits of 0.047 nM for kanamycin and 2.67 pM for ATP. Furthermore, this sensor could detect kanamycin in real samples, demonstrating good accuracy and robust anti-interference capabilities in human serum. Our work not only possesses substantial application value in clinical sample analysis but also represents a breakthrough in traditional strategies, thereby contributing to advancements in the application of electrochemical biosensors for life analysis.

Tumor-associated antigen-specific cell imaging based on upconversion luminescence and nucleic acid rolling circle amplification.

Tumor-associated antigen (TAA)-based diagnosis has gained prominence for early tumor screening, treatment monitoring, prognostic assessment, and minimal residual disease detection. However, limitations such as low sensitivity and difficulty in extracting non-specific binding membrane proteins still exist in traditional detection methods. Upconversion luminescence (UCL) exhibits unique physical and chemical properties under wavelength near-infrared light excitation. Rolling circle amplification (RCA) is an efficient DNA amplification technique with amplification factors as high as 105. Therefore, the above two excellent techniques can be employed for highly accurate imaging analysis of tumor cells. Herein, we developed a novel nanoplatform for TAA-specific cell imaging based on UCL and RCA technology. An aptamer-primer complex selectively binds to Mucin 1 (MUC1), one of TAA on cell surface, to trigger RCA reaction, generating a large number of repetitive sequences. These sequences provide lots of binding sites for complementary signal probes, producing UCL from lanthanide-doped upconversion nanoparticles (UCNPs) after releasing quencher group. The experimental results demonstrate the specific attachment of upconversion nanomaterials to cancer cells which express a high level of MUC1, indicating the potential of UCNPs and RCA in tumor imaging.

Dynamic Interface-Assisted Rapid Self-Assembly of DNA Origami-Framed Anisotropic Nanoparticles.

The ordered arrangement of nanoparticles can generate unique physicochemical properties, rendering it a pivotal direction in the field of nanotechnology. DNA-based chemical encoding has emerged as an unparalleled strategy for orchestrating precise and controlled nanoparticle assemblies. Nonetheless, it is often time-consuming and has limited assembly efficiency. In this study, we developed a strategy for the rapid and ordered assembly of DNA origami-framed nanoparticles assisted by dynamic interfaces. By assembling Au nanoparticles (AuNPs) onto DNA origami with different sticky ends in various directions, we endowed them with anisotropic specific affinities. After assembling DNA origami-framed AuNPs onto supported lipid bilayers with freely diffusing single-stranded DNA via DNA hybridization, we found that DNA origami-framed AuNPs could form larger ordered assemblies than those in 3D solution within equivalent time frames. Furthermore, we also achieved rapid and ordered assembly of liposome nanoparticles by employing the aforementioned strategy. Our work provides a novel avenue for efficient and rapid assembly of nanoparticles across two-dimensional interfaces, which is expected to promote the application of ordered nanoparticle assemblies in sensor and biomimetic system construction.

Urine-derived exosomes and their role in modulating uroepithelial cells to prevent hypospadias.

PURPOSE: Urethral hypospadias, a common congenital malformation in males, is closely linked with disruptions in uroepithelial cell (UEC) processes. Evidence exists reporting that urine-derived exosomes (Urine-Exos) enhance UEC proliferation and regeneration, suggesting a potential role in preventing hypospadias. However, the specific influence of Urine-Exos on urethral hypospadias and the molecular mechanisms involved are not fully understood. This study focuses on investigating the capability of Urine-Exos to mitigate urethral hypospadias and aims to uncover the underlying molecular mechanisms. METHODS: Bioinformatics analysis was performed to identify key gene targets in Urine-Exos potentially involved in hypospadias. Subsequent in vitro and in vivo experiments were conducted to validate the regulatory effects of Urine-Exos on hypospadias. RESULTS: Bioinformatics screening revealed syndecan-1 (SDC1) as a potential pivotal gene for the prevention of hypospadias. In vitro experiments demonstrated that Urine-Exos enhanced the proliferation and migration of UECs by transferring SDC1 and inhibiting cell apoptosis. Notably, Urine-Exos upregulated ß-catenin expression through SDC1 transfer, further promoting UEC proliferation and migration. These findings were confirmed in a congenital hypospadias rat model induced by di(2-ethylhexyl) phthalate (DEHP). CONCLUSION: This study reveals the therapeutic potential of Urine-Exos in hypospadias, mediated by the SDC1/ß-catenin axis. Urine-Exos promote UEC proliferation and migration, thereby inhibiting the progression of hypospadias. These findings offer new insights and potential therapeutic targets for the management of congenital malformations.

Pluripotency state transition of embryonic stem cells requires the turnover of histone chaperone FACT on chromatin.

The differentiation of embryonic stem cells (ESCs) begins with the transition from the naive to the primed state. The formative state was recently established as a critical intermediate between the two states. Here, we demonstrate the role of the histone chaperone FACT in regulating the naive-to-formative transition. We found that the Q265K mutation in the FACT subunit SSRP1 increased the binding of FACT to histone H3-H4, impaired nucleosome disassembly in vitro, and reduced the turnover of FACT on chromatin in vivo. Strikingly, mouse ESCs harboring this mutation showed elevated naive-to-formative transition. Mechanistically, the SSRP1-Q265K mutation enriched FACT at the enhancers of formative-specific genes to increase targeted gene expression. Together, these findings suggest that the turnover of FACT on chromatin is crucial for regulating the enhancers of formative-specific genes, thereby mediating the naive-to-formative transition. This study highlights the significance of FACT in fine-tuning cell fate transition during early development.

Inhibitory effects of catalpol on DNCB-induced atopic dermatitis and IgE-mediated mast cells reaction.

Atopic dermatitis (AD) is a chronic, inflammatory cutaneous disease driven by immune dysregulation. Catalpol is an iridoids, possessing anti-inflammatory, antioxidant, and neuroprotective activities. It can be added to food as a dietary supplement. To evaluate the effects and mechanisms of catalpol on AD, both in vitro and in vivo studies were conducted. It was found that catalpol downregulated the phosphorylation of Lyn and Syk to inhibit various downstream pathways, including intracellular Ca2+ elevation, cytokines generation, and histamine release, which ultimately controlled mast cell (MCs) degranulation. The results showed that catalpol alleviated AD-like skin lesions and MC infiltration via regulation of pro-Th2 and Th2 cytokines in vivo. Furthermore, this compound reduced the levels of IgE in AD mice and improved allergic reactions in PCA mice. The results provided that catalpol was potentially developed as a dietary supplement to improve AD and other atopic diseases.

Metabolic Glycoengineering-Programmed Nondestructive Capture of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are the "seeds" for malignant tumor metastasis, and they serve as an ideal target for minimally invasive tumor diagnosis. Abnormal glycolysis in tumor cells, characterized by glycometabolism disorder, has been reported as a universal phenomenon observed in various types of tumors. This provides a potential powerful tool for universal CTC capture. However, to the best of our knowledge, no metabolic glycoengineering-based CTC capture strategies have been reported. Here, we proposed a nondestructive CTC capture method based on metabolic glycoengineering and a nanotechnology-based proximity effect, allowing for highly specific, sensitive, and universal CTC capture. To achieve this goal, cells are first labeled with DNA tags through metabolic glycoengineering and then captured through a DNA tetrahedra-functionalized dual-tentacle magnetic nanodevice. Due to the difference in metabolic performance, only tumor cells are labeled with more densely packed DNA tags and captured through enhanced intermolecular interaction mediated by the proximity effect. In summary, we have constructed a versatile platform for nondestructive CTC capture, offering a novel perspective for the application of CTC liquid biopsy in tumor diagnosis and treatment.

Establishment of Digital PCR Method and Reference Material for Adenoviruses 40 and 41.

Coinfection with human adenovirus (HAdV) and SARS-CoV-2 has been associated with acute hepatitis in children with unknown etiology. Similar cases have been reported in many countries, and HAdV 40 and HAdV 41 have been identified. The quantification method is established based on digital PCR (dPCR) for HAdV 40/41, which is more convenient for low-concentration virus detection. The limit of detections of HAdV 40/41 dPCR were 4 and 5 copies/µL. Pseudovirus reference material (RM) that contains the highly conserved HEXON gene was developed and quantified with the dPCR method. The assigned values with expanded uncertainty were (1.43 ± 0.35) × 103 copies/µL for HAdV 40 RM and (1.21 ± 0.28) × 103 copies/µL for HAdV 41 RM. The values could be reproduced on multiple platforms. The dPCR method and pseudovirus RMs contribute to the improved accuracy of HAdV 40/41 detection, which is crucial for clinical diagnosis.

Gold-Catalyzed C-O Cross-Coupling Reactions of Aryl Iodides with Silver Carboxylates.

We have developed a C-O cross-coupling reaction of (hetero)aryl iodides with silver carboxylates via a AuI/AuIII catalytic cycle. The transformation featured exclusive chemoselectivity and moisture/air insensitivity. Aromatic and aliphatic (including primary, secondary, and tertiary) silver carboxylates are all suitable substrates. Moreover, this protocol worked well intermolecularly and intramolecularly. Most importantly, good yields were obtained regardless of the substrates' electronic effect and steric hindrance.

Comprehensive analysis of the RSK gene family in acute myeloid leukemia determines a prognostic signature for the prediction of clinical prognosis and treatment responses.

OBJECTIVE: The prognosis of acute myeloid leukemia (AML) remains poor although the basic and translational research has been highly productive in understanding the genetics and pathopoiesis of AML and a plethora of targeted therapies have been developed. Consequently, it is crucial to deepen the knowledge of molecular pathogenesis underlying AML for the advancement of new treatment options. METHOD: A RSK gene family-related signature was constructed to investigate whether RSK gene family members were useful in predicting the prognosis of AML patients. The relationship between the RSK gene family-related signature and the infiltration of immune cells was further assessed using the CIBERSORT algorithm. The 'oncoPredict' package was used to analyze relationships between the RSK gene family-related signature and the sensitivity to drugs or small molecules. RESULTS: Patients were classified into two groups using the RSK gene family-related signature following the median risk score. Overall survival (OS) was significantly longer in patients with low-risk scores than that in patients with high-risk scores as showed by both training and validation datasets. Moreover, the signature was helpful in predicting 1-year, 3-year, and 5-year OS in training and validation datasets. In addition, it was identified that low-risk patients exhibited greater sensitivity to 20 drugs or small molecules and that high-risk patients had higher sensitivity to 38 drugs or small molecules. CONCLUSION: RSK gene family members, particularly RPS6KA1 and RPS6KA4, may help to predict prognosis for AML patients. Furthermore, RPS6KA1 may serve as a novel drug target for AML.

A portable and partitioned DNA hydrogel chip for multitarget detection.

A DNA hydrogel, owing to its dual properties of liquid and solid, is considered to be an ideal material for constructing biosensors that can integrate the advantages of both wet chemistry and dry chemistry. Nevertheless, it has struggled to cope with the demands of high-throughput analysis. A partitioned and chip-based DNA hydrogel is a potential avenue to achieve this, but currently remains a formidable challenge. Here, we developed a portable and partitioned DNA hydrogel chip that can be used for multitarget detection. The partitioned and surface-immobilized DNA hydrogel chip was formed by inter-crosslinking amplification by incorporating target-recognizing fluorescent aptamer hairpins into multiple rolling circle amplification products, which can achieve portable and simultaneous detection of multiple targets. This approach expands the application of semi-dry chemistry strategies, which can realize high throughput and point of care testing (POCT) of different targets, improving the development of hydrogel-based bioanalysis and providing new potential solutions for biomedical detection.

The Role of Nomogram Based on the Combination of Ultrasound Parameters and Clinical Indicators in the Degree of Pathological Remission of Breast Cancer.

Background: The mortality rate of breast cancer (BC) ranks first among female tumors worldwide and presents a trend of younger age, which poses a great threat to women's health and life. Neoadjuvant chemotherapy (NAC) for breast cancer is defined as the first step of treatment for breast cancer patients without distant metastasis before planned surgical treatment or local treatment with surgery and radiotherapy. According to the current NCCN guidelines, patients with different molecular types of BC should receive neoadjuvant chemotherapy (NAC), which can not only achieve tumor downstaging, increase the chance of surgery, and improve the breast-conserving rate. In addition, it can identify new genetic pathways and drugs related to cancer, improve patient survival rate, and make new progress in breast cancer management. Objective: To explore the role of the nomogram established by the combination of ultrasound parameters and clinical indicators in the degree of pathological remission of breast cancer. Methods: A total of 147 breast cancer patients who received neoadjuvant chemotherapy and elective surgery in the Department of Ultrasound, Nantong Cancer Hospital, from May 2014 to August 2021 were retrospectively included. Postoperative pathological remission was divided into two groups according to Miller-Payne classification: no significant remission group (NMHR group, n = 93) and significant remission group (MHR group, n = 54). Clinical characteristics of patients were recorded and collected. The multivariate logistic regression model was used to screen the information features related to the MHR group, and then, a nomogram model was constructed; ROC curve area, consistency index (C-index, CI), calibration curve, and H-L test were used to evaluate the model. And the decision curve is used to compare the net income of the single model and composite model. Results: Among 147 breast cancer patients, 54 (36.7%) had pathological remission. Multivariate logistic regression showed that ER, reduction/disappearance of strong echo halo, Adler classification after NAC, PR + CR, and morphological changes were independent risk factors for pathological remission (P < 0.05). Based on these factors, the nomogram was constructed and verified. The area under the curve (AUC) and CI were 0.966, the sensitivity and specificity were 96.15% and 92.31%, and the positive predictive value (PPV) and negative predictive value (NPV) were 87.72% and 97.15%, respectively. The mean absolute error of the agreement between the predicted value and the real value is 0.026, and the predicted risk is close to the actual risk. In the range of HRT of about 0.0∼0.9, the net benefit of the composite evaluation model is higher than that of the single model. H-L test results showed that χ 2 = 8.430, P=0.393 > 0.05. Conclusion: The nomogram model established by combining the changes of ultrasound parameters and clinical indicators is a practical and convenient prediction model, which has a certain value in predicting the degree of pathological remission after neoadjuvant chemotherapy.

Synthetic biology-based bioreactor and its application in biochemical analysis.

In the past few years, synthetic biologists have established some biological elements and bioreactors composed of nucleotides under the guidance of engineering methods. Following the concept of engineering, the common bioreactor components in recent years are introduced and compared. At present, biosensors based on synthetic biology have been applied to water pollution monitoring, disease diagnosis, epidemiological monitoring, biochemical analysis and other detection fields. In this paper, the biosensor components based on synthetic bioreactors and reporters are reviewed. In addition, the applications of biosensors based on cell system and cell-free system in the detection of heavy metal ions, nucleic acid, antibiotics and other substances are presented. Finally, the bottlenecks faced by biosensors and the direction of optimization are also discussed.

Development of highly accurate digital PCR method and reference material for monkeypox virus detection.

Human monkeypox has attracted attention recently. Monkeypox virus (MPXV) keeps evolving as it spreading around the world rapidly, which may threaten the health of more and more people. Here, we have developed a high order reference method based on digital PCR (dPCR) for MPXV detection, of which the limits of quantification (LoQ) and detection (LoD) are 38 and 6 copies/reaction, respectively. Pseudovirus reference materials (RM) containing the conserved F3L gene has been developed, and the homogeneity assessment showed that the RM was homogeneous. The reference value with its expanded uncertainty determined by the established dPCR is (2.74 ± 0.46) × 103 copies/µL. Six different MPXV test kits were accessed by the RM. Four out of six test kits cannot reach their claimed LoDs. The poor analytical sensitivity might cause false-negative results, which lead to incorrect diagnosis and treatment. The establishment of a high order reference method of dPCR and pseudovirus RM is very useful for improving the accuracy and reliability of MPXV detection.

Recent advances in tumor biomarker detection by lanthanide upconversion nanoparticles.

Early tumor diagnosis could reliably predict the behavior of tumors and significantly reduce their mortality. Due to the response to early cancerous changes at the molecular or cellular level, tumor biomarkers, including small molecules, proteins, nucleic acids, exosomes, and circulating tumor cells, have been employed as powerful tools for early cancer diagnosis. Therefore, exploring new approaches to detect tumor biomarkers has attracted a great deal of research interest. Lanthanide upconversion nanoparticles (UCNPs) provide numerous opportunities for bioanalytical applications. When excited by low-energy near-infrared light, UCNPs exhibit several unique properties, such as large anti-Stoke shifts, sharp emission lines, long luminescence lifetimes, resistance to photobleaching, and the absence of autofluorescence. Based on these excellent properties, UCNPs have demonstrated great sensitivity and selectivity in detecting tumor biomarkers. In this review, an overview of recent advances in tumor biomarker detection using UCNPs has been presented. The key aspects of this review include detection mechanisms, applications in vitro and in vivo, challenges, and perspectives of UCNP-based tumor biomarker detection.