CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.

Peroxiredoxin II regulates exosome secretion from dermal mesenchymal stem cells through the ISGylation signaling pathway.

BACKGROUND: Exosomes are small extracellular vesicles that play important roles in intercellular communication and have potential therapeutic applications in regenerative medicine. Dermal mesenchymal stem cells (DMSCs) are a promising source of exosomes due to their regenerative and immunomodulatory properties. However, the molecular mechanisms regulating exosome secretion from DMSCs are not fully understood. RESULTS: In this study, the role of peroxiredoxin II (Prx II) in regulating exosome secretion from DMSCs and the underlying molecular mechanisms were investigated. It was discovered that depletion of Prx II led to a significant reduction in exosome secretion from DMSCs and an increase in the number of intracellular multivesicular bodies (MVBs), which serve as precursors of exosomes. Mechanistically, Prx II regulates the ISGylation switch that controls MVB degradation and impairs exosome secretion. Specifically, Prx II depletion decreased JNK activity, reduced the expression of the transcription inhibitor Foxo1, and promoted miR-221 expression. Increased miR-221 expression inhibited the STAT signaling pathway, thus downregulating the expression of ISGylation-related genes involved in MVB degradation. Together, these results identify Prx II as a critical regulator of exosome secretion from DMSCs through the ISGylation signaling pathway. CONCLUSIONS: Our findings provide important insights into the molecular mechanisms regulating exosome secretion from DMSCs and highlight the critical role of Prx II in controlling the ISGylation switch that regulates DMSC-exosome secretion. This study has significant implications for developing new therapeutic strategies in regenerative medicine. Video Abstract.

Phosphorylation of ß-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis.

ß-Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that ß-catenin contributes to cytokinesis. Here, we identify a novel p-S60 epitope on ß-catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of ß-catenin leads to cytokinesis-defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of ß-catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between ß-catenin, Plk1, and Ect2. Time-lapse image analysis confirmed the importance of ß-catenin phospho-Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of ß-catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure-derived human diseases.

Clinical Application of Intravitreal Aflibercept Injection for Diabetic Macular Edema Comparing Two Loading Regimens.

Background and Objectives: We investigated and compared the efficacy of three and five monthly loading regimens of an intravitreal aflibercept injection (IVA) in patients with diabetic macular edema (DME). Materials and Methods: This was a retrospective study that included patients diagnosed with DME and treated with an either three or five monthly aflibercept loading regimen from July 2018 to March 2022. Information on clinical characteristics and changes in the central retinal thickness (CRT) were obtained from medical records. Results: In total, 44 eyes of 44 patients with DME treated with IVA were included in this study, with 30 eyes treated with 3-monthly loadings (three-loading group) and 14 eyes with 5-monthly loadings (five-loading group). The mean CRT significantly decreased from the baseline one month after loading in both the three-loading and five-loading groups (p < 0.001). Four cases were refractory to treatment in the three-loading group, while there were no cases of refractory DME in the five-loading group. The stability rate was significantly higher in the five-loading group at three months after loading (p = 0.033). Conclusions: Five-monthly loading regimens of IVA might be favorable for DME considering the rate of refractory cases, stable duration, and the importance of early responsiveness to IVA in DME.

Synergistic effect of anticancer drug resistance and Wnt3a on primary ciliogenesis in A549 cell-derived anticancer drug-resistant subcell lines.

Primary cilia, antenna-like cellular sensor structures, are generated from the mother centriole in the G0/G1 cell-cycle phase under control by cellular signaling pathways involving Wnt, hedgehog, and platelet-derived growth factor. Although primary ciliary dynamics have been reported to be closely related to ciliopathy and tumorigenesis, the molecular basis for the role of primary cilia in human disease is lacking. To clarify how Wnt3a affects primary ciliogenesis in anticancer drug-resistant cells, we derived specific drug-resistant subcell lines from A549 human lung cancer cells using anticancer drugs doxorubicin, dasatinib, and paclitaxel (A549/Dox, A549/Das, and A549/Pac, respectively). The primary cilia-containing cell population and primary cilia length increased in the A549/Dox and A549/Pac subcell lines under increased MDR1 expression, when compared to those in the parental A549 cells. In the A549/Das subcell line, primary cilia length increased but the cell population was not affected. In addition, Wnt3a increased primary cilia-containing cell population and primary cilia length in A549/Dox, A549/Das, and A549/Pac cells, without change of cell growth. Abnormal shapes of primary cilia were frequently observed by anticancer drug resistance and Wnt3a stimulation. Taken together, our results indicate that anticancer drug resistance and Wnt3a affect primary ciliogenesis synergistically, suggesting a potential new strategy for overcoming anticancer drug resistance.

R-catcher, a potent molecular tool to unveil the arginylome.

Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.

Is gas/air tamponade essential for eyes with small peripheral retinal breaks without detachment during vitrectomy?

BACKGROUND: To investigate the safety of vitrectomy with laser photocoagulation in eyes with small peripheral retinal breaks without air or gas tamponade. METHODS: Among patients who underwent vitrectomy for various retinal disorders, those with small peripheral retinal breaks treated by laser photocoagulation without air or gas tamponade were included in this study. Their medical records were assessed retrospectively, and we investigated the characteristics of small peripheral retinal breaks and the incidence of postoperative retinal detachment (RD). RESULTS: Thirty-one eyes of 31 patients who presented with small peripheral retinal breaks requiring endolaser photocoagulation during vitrectomy were included in this analysis. There were two cases of iatrogenic retinal breaks that occurred during vitrectomy, while others were preexisting lesions, including retinal tears, atrophic retinal holes, and retinal holes with lattice degeneration. There were no cases of RD during the follow-up period of at least 6 months. CONCLUSIONS: Adequate laser treatment without gas or air tamponade may be sufficient during vitrectomy in cases with small peripheral retinal breaks without concurrent RD, along with complete removal of vitreoretinal traction.

Prediction of Heart Function and Volume Status in End-Stage Kidney Disease Patients through N-Terminal Pro-Brain Natriuretic Peptide.

Background and Objectives: N-terminal pro-brain natriuretic peptide (NT-proBNP) is a biomarker used to predict heart failure and evaluate volume status in hemodialysis (HD) patients. However, it is difficult to determine the cutoff value for NT-proBNP in HD patients. In this study, we analyzed whether NT-proBNP helps predict heart function and volume status in HD patients. Materials and Methods: This prospective observational study enrolled 96 end-stage kidney disease patients with HD. All patients underwent echocardiography and bioimpedance spectroscopy (BIS) after an HD session. Overhydration (OH) was measured by BIS. Laboratory data were obtained preHD, while serum NT-proBNP was measured after HD. Interventions for blood pressure control and dry weight control were performed, and NT-proBNP was re-assessed after a month. Results: There was an inverse correlation between NT-proBNP and ejection fraction (EF) (ß = -0.34, p = 0.001). OH (ß = 0.331, p = 0.001) and diastolic dysfunction (ß = 0.226, p = 0.027) were associated with elevated NT-proBNP. In a subgroup analysis of diastolic dysfunction grade, NT-proBNP increased according to dysfunction grade (normal, 4177 pg/mL [2637-10,391]; grade 1, 9736 pg/mL [5471-21,110]; and grades 2-3, 26,237 pg/mL [16,975-49,465]). NT-proBNP showed a tendency toward a decrease in the 'reduced dry weight' group and toward an increase in the 'increased dry weight' group compared to the control group (ΔNT-proBNP, -210 pg/mL [-12,899 to 3142], p = 0.104; 1575 pg/mL [-113 to 6439], p = 0.118). Conclusions: We confirmed that NT-proBNP is associated with volume status as well as heart function in HD patients.

Cullin 1 (CUL1) Promotes Primary Ciliogenesis through the Induction of Ubiquitin-Proteasome-Dependent Dvl2 Degradation.

Primary cilia are nonmotile cellular signal-sensing antenna-like structures composed of microtubule-based structures that distinguish them from motile cilia in structure and function. Primary ciliogenesis is regulated by various cellular signals, such as Wnt, hedgehog (Hh), and platelet-derived growth factor (PDGF). The abnormal regulation of ciliogenesis is closely related to developing various human diseases, including ciliopathies and cancer. This study identified a novel primary ciliogenesis factor Cullin 1 (CUL1), a core component of Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, which regulates the proteolysis of dishevelled 2 (Dvl2) through the ubiquitin-proteasome system. Through immunoprecipitation-tandem mass spectrometry analysis, 176 Dvl2 interacting candidates were identified, of which CUL1 is a novel Dvl2 modulator that induces Dvl2 ubiquitination-dependent degradation. Neddylation-dependent CUL1 activity at the centrosomes was essential for centrosomal Dvl2 degradation and primary ciliogenesis. Therefore, this study provides a new mechanism of Dvl2 degradation by CUL1, which ultimately leads to primary ciliogenesis, and suggest a novel target for primary cilia-related human diseases.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Lymphatic endothelial cells promote T lymphocyte migration into lymph nodes under hyperlipidemic conditions.

Lymphatic vessels serve as conduits through which immune cells traffic. Because lymphatic vessels are also involved in lipid transport, their function is vulnerable to abnormal metabolic conditions such as obesity and hyperlipidemia. Exactly how these conditions impact immune cell trafficking, however, is not well understood. Here, we found higher numbers of LYVE-1-positive lymphatic endothelial cells and CD3-positive T cells in the lymph nodes of mice fed high-cholesterol or high-fat diets compared with those of mice fed a normal chow diet. To confirm the effect of fat content on immune cell trafficking, the lymphatic endothelial SVEC4-10 cell line was treated with palmitic acid at a 100 µM concentration. After 24 h, palmitic acid-treated cells exhibited increased expression of podoplanin and vascular growth-associated molecules (VEGFC, VEGFD, VEGFR3, and NRP2) and enhanced tube formation. Microarray analysis showed an increase in pro-inflammatory cytokine and chemokine transcription after palmitic acid treatment. Finally, transwell migration assay confirmed that T cell line moved toward medium previously cultured with palmitic acid-treated SVEC4-10 cells. Together, our results suggest that hyperlipidemia drives lymphatic vessel remodeling and T cell migration toward lymphatic endothelial cells.

CPPF, A Novel Microtubule Targeting Anticancer Agent, Inhibits the Growth of a Wide Variety of Cancers.

In the past, several microtubule targeting agents (MTAs) have been developed into successful anticancer drugs. However, the usage of these drugs has been limited by the acquisition of drug resistance in many cancers. Therefore, there is a constant demand for the development of new therapeutic drugs. Here we report the discovery of 5-5 (3-cchlorophenyl)-N-(3-pyridinyl)-2-furamide (CPPF), a novel microtubule targeting anticancer agent. Using both 2D and 3D culture systems, we showed that CPPF was able to suppress the proliferation of diverse cancer cell lines. In addition, CPPF was able to inhibit the growth of multidrug-resistant cell lines that are resistant to other MTAs, such as paclitaxel and colchicine. Our results showed that CPPF inhibited growth by depolymerizing microtubules leading to mitotic arrest and apoptosis. We also confirmed CPPF anticancer effects in vivo using both a mouse xenograft and a two-step skin cancer mouse model. Using established zebrafish models, we showed that CPPF has low toxicity in vivo. Overall, our study proves that CPPF has the potential to become a successful anticancer chemotherapeutic drug.

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions.

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1-Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1-Plk1 complex-dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.

Interfacing a Personal Glucose Meter with Cell-Free Protein Synthesis for Rapid Analysis of Amino Acids.

We developed a method to analyze amino acids using a personal glucose meter (PGM). In this method, the principles of protein biosynthesis were interfaced with the sensing mechanism of a PGM to enable simple and ubiquitous measurement of amino acids. A reaction mixture for cell-free protein synthesis was designed to synthesize a bacterial invertase in response to exogenous addition of a specific amino acid. The invertase synthesized upon addition of an assay sample containing the amino acid of interest was used to convert sucrose into glucose, which was detected using a PGM. The titers of the amino acid in assay samples were precisely represented by the readouts of a PGM. In addition to the convenience provided by use of a PGM, the accuracy and reproducibility of this method were comparable to those of standard high-performance liquid chromatography based methods.

A novel tubulin inhibitor STK899704 induces tumor regression in DMBA/TPA-induced skin carcinogenesis model.

Skin cancer is the most common type of cancer. The incidence rate of skin cancer has continuously increased over the past decades. In an effort to discover novel anticancer agents, we identified a novel tubulin inhibitor STK899704, which is structurally distinct from other microtubule-binding agents such as colchicine, vinca alkaloids and taxanes. STK899704 inhibited microtubule polymerization leading to mitotic arrest and suppressed the proliferation of various cancer cell lines as well as multidrug resistance cancer cell lines. In this study, our investigation is further extended into animal model to evaluate the effect of STK899704 on skin carcinogenesis in vivo. Surprisingly, almost 80% of the tumors treated with STK899704 were regressed with a one-fifth reduction in tumor volume. Furthermore, the efficacy of STK899704 was nearly 2 times higher than that of 5-fluorouracil, a widely used skin cancer therapeutic. Overall, our results suggest that STK899704 is a promising anticancer chemotherapeutic that may replace existing therapies, particularly for skin cancer.

Peptide nucleic acid (PNA) probe-based analysis to detect filaggrin mutations in atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose prevalence is increasing worldwide. Filaggrin (FLG) is essential for the development of the skin barrier, and its genetic mutations are major predisposing factors for AD. In this study, we developed a convenient and practical method to detect FLG mutations in AD patients using peptide nucleic acid (PNA) probes labelled with fluorescent markers for rapid analysis. Fluorescence melting curve analysis (FMCA) precisely identified FLG mutations based on the distinct difference in the melting temperatures of the wild-type and mutant allele. Moreover, PNA probe-based FMCA easily and accurately verified patient samples with both heterozygote and homozygote FLG mutations, providing a high-throughput method to reliable screen AD patients. Our method provides a convenient, rapid and accurate diagnostic tool to identify potential AD patients allowing for early preventive treatment, leading to lower incidence rates of AD, and reducing total healthcare expenses.

Complementary Cell-Free Translational Assay for Quantification of Amino Acids.

In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.

Assessing translational efficiency by a reporter protein co-expressed in a cell-free synthesis system.

We demonstrate the use of a cell-free protein synthesis system as a convenient tool for assessing the relative translational efficiencies of genes. When sfGFP was used as a common reporter gene and co-expressed with a series of target genes, the intensities of sfGFP fluorescence from the co-expression reactions were highly correlated with the individual expression levels of the co-expressed genes. The relative translational efficiencies of genes estimated by this method were reproducible when the same genes were expressed in transformed Escherichia coli, suggesting that this method could be used as a universal tool for prognostic assessment of translational efficiency.

Is MRI of the Liver Needed During Routine Preoperative Workup for Colorectal Cancer?

BACKGROUND: The clinical efficacy of gadoxetic acid-enhanced liver MRI as a routine preoperative procedure for all patients with colorectal cancer remains unclear. OBJECTIVE: The purpose of this study was to evaluate the efficacy of preoperative gadoxetic acid-enhanced liver MRI for the diagnosis of liver metastasis in patients with colorectal cancer. DESIGN: This was a retrospective analysis from a prospective cohort database. SETTINGS: All of the patients were from a subspecialty practice at a tertiary referral hospital. PATIENTS: Patients who received preoperative gadoxetic acid-enhanced liver MRI after CT and attempted curative surgery for colorectal cancer were included. MAIN OUTCOME MEASURES: The number of equivocal hepatic lesions based on CT and gadoxetic acid-enhanced liver MRI and diagnostic use of the gadoxetic acid-enhanced liver MRI were measured. RESULTS: We reviewed the records of 690 patients with colorectal cancer. Equivocal hepatic lesions were present in 17.2% of patients based on CT and in 4.5% based on gadoxetic acid-enhanced liver MRI. Among 496 patients with no liver metastasis based on CT, gadoxetic acid-enhanced liver MRI detected equivocal lesions in 15 patients and metastasis in 3 patients. Among 119 patients who had equivocal liver lesions on CT, gadoxetic acid-enhanced liver MRI indicated hepatic lesions in 103 patients (86.6%), including 90 with no metastasis and 13 with metastasis. Among 75 patients who had liver metastasis on CT, gadoxetic acid-enhanced liver MRI indicated that the hepatic lesions in 2 patients were benign, in contrast to CT findings. The initial surgical plans for hepatic lesions according to CT were changed in 17 patients (3%) after gadoxetic acid-enhanced liver MRI. LIMITATIONS: This study was limited by its retrospective design. CONCLUSIONS: The clinical efficacy of gadoxetic acid-enhanced liver MRI as a routine preoperative procedure for all patients with colorectal cancer is low, in spite of its high diagnostic value for detecting liver metastasis. However, this study showed gadoxetic acid-enhanced liver MRI was helpful in characterizing equivocal hepatic lesions identified in CT and could lead to change in treatment plans for some patients. See Video Abstract at http://links.lww.com/DCR/A420.