Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis.

Quantitative polymerase chain reaction (qPCR) offers the capabilities of real-time monitoring of amplified products, fast detection, and quantitation of infectious units, but poses technical hurdles for point-of-care miniaturization compared with end-point polymerase chain reaction. Here we demonstrate plasmonic thermocycling, in which rapid heating of the solution is achieved via infrared excitation of nanoparticles, successfully performing reverse-transcriptase qPCR (RT-qPCR) in a reaction vessel containing polymerase chain reaction chemistry, fluorescent probes and plasmonic nanoparticles. The method could rapidly detect SARS-CoV-2 RNA from human saliva and nasal specimens with 100% sensitivity and 100% specificity, as well as two distinct SARS-CoV-2 variants. The use of small optical components for both thermocycling and multiplexed fluorescence monitoring renders the instrument amenable to point-of-care use. Overall, this study demonstrates that plasmonic nanoparticles with compact optics can be used to achieve real-time and multiplexed RT-qPCR on clinical specimens, towards the goal of rapid and accurate molecular clinical diagnostics in decentralized settings.

Prognostic relevance of the hexosamine biosynthesis pathway activation in leiomyosarcoma.

Metabolic reprogramming of tumor cells and the increase of glucose uptake is one of the hallmarks of cancer. In order to identify metabolic pathways activated in leiomyosarcoma (LMS), we analyzed transcriptomic profiles of distinct subtypes of LMS in several datasets. Primary, recurrent and metastatic tumors in the subtype 2 of LMS showed consistent enrichment of genes involved in hexosamine biosynthesis pathway (HBP). We demonstrated that glutamine-fructose-6-phosphate transaminase 2 (GFPT2), the rate-limiting enzyme in HBP, is expressed on protein level in a subset of LMS and the expression of this enzyme is frequently retained in patient-matched primary and metastatic tumors. In a new independent cohort of 327 patients, we showed that GFPT2 is associated with poor outcome of uterine LMS but not extra-uterine LMS. Based on the analysis of a small group of patients studied by 18F-FDG-PET imaging, we propose that strong expression of GFPT2 in primary LMS may be associated with high metabolic activity. Our data suggest that HBP is a potential new therapeutic target in one of the subtypes of LMS.

Efficacy and safety of 5 anesthetics in adult zebrafish (Danio rerio).

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 °C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthe- sia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures.