No abstract

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

Induction and secretion of acid phosphatases (APases) is thought to be an adaptive mechanism that helps plants survive and grow under phosphate (Pi) deprivation. In Arabidopsis, there are 29 purple acid phosphatase (AtPAP) genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAP10 is mainly a secreted APase. On Pi-deficient (PÀ) medium or PÀ medium supplemented with the organophosphates ADP and fructose-6-phosphate (Fru-6-P), growth of atpap10 was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type (WT). Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on PÀ or PÀ medium supplemented with ADP or Fru-6-P. Interestingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.

Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

Some acoustically active substances can combine with specific molecules/cells and enable imaging of complex disease processes at the molecular level. [8,9] In parallel, accumulating evidence demonstrates that US is an appealing regimen for managing various diseases including Alzheimer's diseases, cardiovascular diseases, and cancers. [10][11][12][13] According to the frequency, US is usually categorized into high intensity focused US (HIFU; >200 kHz) and low intensity focused US (LIFU; 20-200 kHz). [14] HIFU enables truly non-invasive tumor ablation in clinical applications. [15] There have been tens of thousands of cases reporting HIFU guided by US or magnetic resonance imaging to treat solid organ tumors globally. [16,17] However, recent investigations indicate that the administration of HIFU can lead to adverse events such as skin burn and nerve injury. [18] Theoretically, LIFU with lower intensity can eliminate US-induced overheating and make the treatment safer. LIFU radiation combined with sonosensitizer administration (sonodynamic therapy, SDT), was developed as a suitable therapy alternative by Yumita et al. in 1989. [19] When US activates the preloaded sonosensitizers, they can trigger sensitive reactions to produce reactive oxygen species (ROS), which play a therapeutic role in specific sites through different mechanisms. [20,21] More importantly, due to the excellent regional focusing characteristics and strong tissue penetrating ability of US, SDT has higher therapeutic efficiency and fewer side effects. [22,23] Recent intriguing investigations had been carried out using SDT to treat cancer, cardiovascular disease, and multidrug-resistant (MDR) bacterial infection. [24] Notably, SDT of cancer and atherosclerotic peripheral artery disease (PAD) has been applied in clinical trials, and the results showed high efficacy and favorable safety on patients. [25,26] The sonosensitization efficiency, ROS quantum yield, and accumulation of the sonosensitizers in the target cells are essential prerequisites for SDT. [27,28] Therefore, designing and developing excellent sonosensitizers is an important topic in the development of SDT. Generally, sonosensitizers mainly include organic and inorganic sonosensitizers. Many inorganic sonosensitizers have superior physiochemical properties, but the clinical translation remains unresolved because of nonbiodegradation and potential biosafety issues. However, organic sonosensitizers have the advantages of clear structure and easy metabolism, which is conducive to clinical applications. [24] Sonodynamic therapy (SDT) is a novel noninvasive therapeutic modality that combines low-intensity ultrasound and sonosensitizers. Versus photomediated therapy, SDT has the advantages of deeper tissue penetration, high accuracy, and less side effects. Sonosensitizers are critical for therapeutic efficacy during SDT and organic sonosensitizers are important because of their clear structure, easy monitoring, evaluation of drug metabolism, and clinical transformation. Notably, nanotechnolo...