Supramolecular complexation between cucurbit[7]uril and folate and analytical applications

Folate (FA) plays a key role in the biosynthesis of amino acids, purines, and pyrimidines in the human body, and intracellular folate metabolism has become an attractive target of tumor chemotherapy. In this work, an inclusion interaction was found between FA and cucurbit[7]uril (CB[7]), and the formation of a CB[7]-FA 2:1 supramolecular inclusion complex was confirmed by fluorescence spectra, UV-Vis absorption spectroscopy, 1H NMR, and molecular modeling calculations. In addition, FA is generally determined through the indirect fluorescent method because it shows weak fluorescence in aqueous solution. Therefore, a simple, direct fluorescence probe method for rapidly measuring FA was investigated, and the linear equation of FA was ΔF = 14.691C + 37.366 within the concentration ranges of 0.82 ~ 18.31 µg mL–1. The proposed direct fluorescence method was applied to the determination of spiked plasma. We demonstrated that this method could provide an experimental basis for the targeted administration of the CB[7]-FA complex, and it could be extended as a promising fluorescence detection method for drugs in vivo.

Direct fluorescence antibody testing augments syphilis diagnosis, compared to serology alone

In Ottawa, Canada, we initiated protocols to include non-serologic syphilis testing, as direct fluorescence antibody (DFA) for patients with syphilis symptoms. The purpose was to assess the ability of DFA to detect syphilis during acute infection and to determine if non-serologic testing could yield an increased number of syphilis diagnoses. We reviewed charts of patients of our local sexual health clinic for whom syphilis was suspected. A total of 69 clinical encounters were recorded for 67 unique patients, most of whom were male. The most common symptom was a painless genital lesion. Of the 67 patients, 29 were found to have a new syphilis diagnosis, among whom, 52% had positive syphilis serology and positive DFA, 34% had a positive syphilis serology and negative DFA, and 14% had negative syphilis serology and positive DFA. While DFA testing did not yield an abundance of new cases, it was useful to support findings from syphilis serology or confirm diagnosis where serology was negative. Where available, alternate non-serologic tests, such as nucleic acid amplification tests, should be considered above DFA due to its higher sensitivity for detecting syphilis in primary lesions; however, in clinical situations, when new syphilis infection is suspected, empiric treatment should not be delayed.

Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Objective The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. Results We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

Europium-Doped Ceria Nanocrystals as Nanozyme Fluorescent Probes for Biosensing

Molecular nanoprobes with intrinsic enzyme-like activity represent a new wave of technology for rapid and sensitive detection of molecular targets. This work reports synthesis and characterization of novel and well-dispersed europium-doped ceria nanocrystals (EuCe NCs) with self-integrated catalytic and fluorescence sensing functions. The NCs have an average size of ∼5 nm and exhibit bright and stable fluorescence for more than 6 months in aqueous media. Their dual cooperative function as both a catalyst and fluorescent probe was explored to develop a universally applicable fluorescence-based biosensing method to monitor enzyme reactions and quantitatively measure clinically relevant molecules. Sensing capabilities are demonstrated for detection of H2O2, glucose/glucose oxidase, lactate/lactate oxidase, phosphatase activity, and the catecholamine neurotransmitter, dopamine. Results indicate that EuCe NCs not only provide high enzyme-mimetic activity, but also impart direct fluorescence sensing ability enabling all-in-one recognition, catalytic amplification, and the detection of biomolecular targets. The EuCe nanozyme offers a stable alternative to the more complex systems based on the combined use of natural enzymes and fluorescent dyes. The high stability and fluorescence detection capabilities demonstrate that EuCe NCs have the potential to be used as a generic platform in chemical and biological sensing and bioimaging applications.

Activity of the Mouse Notch ligand DLL1 is Sensitive to C-terminal Tagging in Vivo

ObjectiveThe mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. ResultsWe generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP protein fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

Onychomycosis and Tinea Pedis in the Feet of Patients With Diabetes

This study aimed to determine the prevalence of onychomycosis and interdigital tinea pedis in a cohort of Spanish patients with diabetes in whom onychomycosis was clinically suspected (n = 101). Samples from a first toenail scraping and the fourth toe clefts were subjected to potassium hydroxide direct vision and incubated in Sabouraud and dermatophyte test medium. Fifty-eight samples were also analyzed by a pathologist using periodic acid-Schiff staining and Calcofluor white direct fluorescence microscopy. Onychomycosis was only confirmed in 41 patients (40.6%). The most frequent aetiological agent was Trichophyton rubrum, isolated in 10 patients (36%), followed by Candida parapsilosis in 7 patients (25%). Tests on the fourth toe cleft samples were only positive in 11 patients (10.9%), and in all cases, onychomycosis was also diagnosed. Neuroischemic foot was the only significant variable associated with onychomycosis in the univariate analysis ( P < .01). A positive result for mycosis in the fourth toe cleft was found in 11 cases (10.9%) and was associated with a history of myocardial infarction ( P< .01; odds ratio [OR]: 84.2, confidence interval [CI]: 6.8-1036.4) and neuroischemic foot ( P< .01; OR: 13.7, CI: 12.6-71.6) in the multivariate model. In conclusion, the prevalence of onychomycosis and tinea pedis in patients with diabetes in whom onychomycosis was clinically suspected was 40.6% and 10.9%, respectively. In addition, onychomycosis was not always associated with tinea pedis. These results show that clinical diagnosis has low accuracy in people with diabetes mellitus, and that diagnosis should not be based on clinical toenail characteristics alone.

Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2

Despite the importance of nucleic acid testing in managing the COVID-19 pandemic, current detection approaches remain limited due to their high complexity and extensive processing. Here, we describe a molecular nanotechnology that enables direct and sensitive detection of viral RNA targets in native clinical samples. The technology, termed catalytic amplification by transition-state molecular switch (CATCH), leverages DNA-enzyme hybrid complexes to form a molecular switch. By ratiometric tuning of its constituents, the multicomponent molecular switch is prepared in a hyperresponsive state—the transition state—that can be readily activated upon the binding of sparse RNA targets to turn on substantial enzymatic activity. CATCH thus achieves superior performance (~8 RNA copies/μl), direct fluorescence detection that bypasses all steps of PCR (<1 hour at room temperature), and versatile implementation (high-throughput 96-well format and portable microfluidic assay). When applied for clinical COVID-19 diagnostics, CATCH demonstrated direct and accurate detection in minimally processed patient swab samples.

An undiagnosed patient with skin rash, polyarthritis, and edema responding to low-dose colchicine: A case report

A 54-year-old man was referred to our hospital with painful rashes on the extremities. He also developed polyarthritis and pitting pedal edema. Blood tests showed no specific autoantibodies and were negative for human leukocyte antigens B51, B15, and B27. Lower extremity venous ultrasonography and computed tomography angiography showed no vascular disorders. Skin biopsy showed no evidence of thrombosis or vasculitis. Direct fluorescence antibody analysis showed no antibody or complement deposition. Joint ultrasonography showed mild synovial thickening and/or synovial effusion in the extremities. Non-steroidal anti-inflammatory drugs and topical steroids were administered, followed by oral steroids. However, the signs and symptoms did not improve. Oral steroids were discontinued, and colchicine (0.5 mg/day) was administered. Thereafter, the symptoms of arthritis improved, and no skin rash developed. In potentially inflammatory conditions with skin rash, edema, and polyarthritis that are difficult to diagnose, low-dose colchicine administration may be considered for prompt relief of symptoms.