Mechanistic decoupling of exonuclease III multifunctionality into AP endonuclease and exonuclease activities at the single-residue level.

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3'→5' exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3' terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key determinants of functional selection between the two activities are poorly understood. Here, we use a series of mutational analyses and single-molecule imaging to unravel the pivotal rules governing these endo- and exonuclease activities at the single amino acid level. An aromatic residue, either W212 or F213, recognizes AP sites to allow for the AP endonuclease activity, and the F213 residue also participates in the stabilization of the melted state of the 3' terminal nucleobases, leading to the catalytically competent state that activates the 3'→5' exonuclease activity. During exonucleolytic cleavage, the DNA substrate must be maintained as a B-form helix through a series of phosphate-stabilizing residues (R90, Y109, K121 and N153). Our work decouples the AP endonuclease and exonuclease activities of ExoIII and provides insights into how this multifunctional enzyme controls each function at the amino acid level.

RNase H is an exo- and endoribonuclease with asymmetric directionality, depending on the binding mode to the structural variants of RNA:DNA hybrids.

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3' DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5' DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.

Allosteric ring assembly and chemo-mechanical melting by the interaction between 5'-phosphate and λ exonuclease.

Phosphates along the DNA function as chemical energy frequently used by nucleases to drive their enzymatic reactions. Exonuclease functions as a machine that converts chemical energy of the phosphodiester-chain into mechanical work. However, the roles of phosphates during exonuclease activities are unknown. We employed λ exonuclease as a model system and investigated the roles of phosphates during degradation via single-molecule fluorescence resonance energy transfer (FRET). We found that 5' phosphates, generated at each cleavage step of the reaction, chemo-mechanically facilitate the subsequent post-cleavage melting of the terminal base pairs. Degradation of DNA with a nick requires backtracking and thermal fraying at the cleavage site for re-initiation via the formation of a catalytically active complex. Unexpectedly, we discovered that a phosphate of a 5' recessed DNA acts as a hotspot for an allosteric trimeric-ring assembly without passing through the central channel. Our study provides new insight into the versatile roles of phosphates during the processive enzymatic reaction.

Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding.

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.

Tumor Growth Kinetics Based on Initial Tumor Volume Doubling Time in Active Surveillance of Low-Risk Papillary Thyroid Carcinoma.

BACKGROUND: During active surveillance (AS) of low-risk papillary thyroid carcinomas (PTCs), the majority remain stable, while some exhibit either increase or decrease in tumor diameter or tumor volume (TV). We aimed to evaluate the clinical outcomes and relevant parameters influencing tumor growth kinetics of low-risk PTCs. METHODS: This retrospective cohort study evaluated clinical parameters of 402 patients with low-risk PTC sized <2 cm, with a follow-up duration over 3 years. Changes in maximum tumor diameter, TV, and initial TV doubling time (i-TVDT) calculated within 3-year were assessed. A significant change in TV was defined as a change of 75% or more. RESULTS: Of the 402 patients with low-risk PTC, 93.3% (375/402) were diagnosed with papillary thyroid microcarcinoma. During a median follow-up of 5 years, 3.4% (14/402) of patients developed new cervical lymph node (LN) metastasis, and 8.2% (33/402) experienced maximal diameter increase of ≥3 mm. The i-TVDT of <5 years emerged as an independent risk factor for both maximal diameter growth and new LN metastasis (p<0.001 and p=0.04, respectively). Based on TV changes and i-TVDT during AS, we identified four statistically significant tumor kinetic patterns (p<0.001): Stable (±75% change in TV), Rapid growth (TV increase >75% and i- TVDT <5 years), Slow growth (TV increase >75% and i-TVDT ≥5 years), and Shrinkage (TV decrease >75%). Most of the PTCs remained stable (67.7%), but 17.2% were rapidly growing, with a median onset of growth of 2.0 years. Slowly growing PTCs, comprising 10.9%, grew at a median of 4.3 years. A minority, 4.2%, exhibited shrinkage. In total, 115 (28.6%) patients underwent delayed surgery >12 months after initiating AS. The reasons for delayed surgery included patient preference (51/115, 44.3%), disease progression (31/115, 27.0%), and suspected disease progression, which was referred to as tumor growth not meeting the criteria of an increase of ≥3 mm in maximal tumor diameter (17/115, 14.8%). CONCLUSION: An i-TVDT of <5 years serve as an important prognostic indicator for disease progression, including tumor growth and new LN metastasis. The four tumor kinetic patterns based on TV changes and i-TVDT assist in guiding personalized decisions early in AS.

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease.

λ exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.

The effects of augmented reality on consumer responses in mobile shopping: The moderating role of task complexity.

Augmented reality (AR) solutions help facilitate consumers' direct examinations, enhancing their shopping experiences in the digital commerce context. This study examines consumer responses to AR in mobile shopping. It investigates the relationships among perceived media richness, interactivity, telepresence, utilitarian and hedonic values, and behavioral intentions. Furthermore, it explores whether these relationships differ depending on consumers' perceived task complexity. A total of 279 mobile application users participated in the online survey. The participants were guided to answer an online questionnaire after utilizing an AR mobile application to purchase a jewelry product. The findings reveal that media richness and interactivity positively influence telepresence, and telepresence increases behavioral intentions through perceived utilitarian and hedonic values. The effect of interactivity on telepresence and the impact of telepresence on utilitarian value are higher for consumers with low task complexity perception. By contrast, the impact of telepresence on hedonic value is higher for consumers with high task complexity perception. The results suggest practical implications for mobile retailers that apply advanced AR technology in retailing.

Morphometric Analysis of Lateral Cervical Lymph Node Metastasis in Papillary Thyroid Carcinoma Using Digital Pathology.

Digital pathology uses digitized images for cancer research. We aimed to assess morphometric parameters using digital pathology for predicting recurrence in patients with papillary thyroid carcinoma (PTC) and lateral cervical lymph node (LN) metastasis. We analyzed 316 PTC patients and assessed the longest diameter and largest area of metastatic focus in LNs using a whole slide imaging scanner. In digital pathology assessment, the longest diameters and largest areas of metastatic foci in LNs were positively correlated with traditional optically measured diameters (R = 0.928 and R2 = 0.727, p < 0.001 and p < 0.001, respectively). The optimal cutoff diameter was 8.0 mm in both traditional microscopic (p = 0.009) and digital pathology (p = 0.016) evaluations, with significant differences in progression-free survival (PFS) observed at this cutoff (p = 0.006 and p = 0.002, respectively). The predictive area's cutoff was 35.6 mm2 (p = 0.005), which significantly affected PFS (p = 0.015). Using an 8.0-mm cutoff in traditional microscopic evaluation and a 35.6-mm2 cutoff in digital pathology showed comparable predictive results using the proportion of variation explained (PVE) methods (2.6% vs. 2.4%). Excluding cases with predominant cystic changes in LNs, the largest metastatic areas by digital pathology had the highest PVE at 3.9%. Furthermore, high volume of LN metastasis (p = 0.001), extranodal extension (p = 0.047), and high ratio of metastatic LNs (p = 0.006) were associated with poor prognosis. Both traditional microscopic and digital pathology evaluations effectively measured the longest diameter of metastatic foci in LNs. Moreover, digital pathology offers limited advantages in predicting PFS of patients with lateral cervical LN metastasis of PTC, especially those without predominant cystic changes in LNs.

Undercover active surveillance of small highly suspicious thyroid nodules without fine needle aspiration.

PURPOSE: Active surveillance (AS) is an alternative treatment approach for small, low-risk papillary thyroid microcarcinoma (PTMC). This study aimed to assess the clinical outcomes of small, highly suspicious nodules lacking initial cytological confirmation. METHODS: This study included 112 patients with highly suspicious nodules measuring ≤ 10 mm who underwent serial ultrasound at Asan Medical Center, Korea, between 2010 and 2023. RESULTS: The median participant age was 51.9 years, and 74.1% were female. The median maximal tumor diameter and tumor volume (TV) were 4.5 (interquartile range [IQR] 3.7-5.2, range 2.2-9.3) mm and 25.2 (IQR 13.1-49.2) mm3, respectively. During a median follow-up period of 4.8 years, four (3.6%) patients showed a ≥ 3 mm increase in maximal diameter, and two (1.8%) developed new lymph node (LN) metastasis. Disease progression was associated with a TV doubling time (TVDT) of < 5 years and a ≥ 75% increase in TV (p = 0.017 and p < 0.005, respectively). Furthermore, 34.8% of patients underwent fine needle aspiration (FNA), primarily at their own request, yielding 46.2%, 5.1%, 41.0%, and 12.8 % malignant, benign, indeterminate, and non-diagnostic results, respectively. Of 18 patients with PTMC, 8 (44.4%) underwent surgery and 10 continued AS, with no LN metastasis during AS and no postoperative recurrence. CONCLUSION: Small, highly suspicious nodules had a low disease progression rate during AS without FNA. Disease progression was associated with a TVDT of < 5 years and a ≥ 75% increase in TV. FNA can be performed more conservatively than it currently is in patients with highly suspicious nodules measuring ≤ 10 mm.

Inhibitory effect of eriodictyol on IgE/Ag-induced type I hypersensitivity.

Mast cells are the principal effector cells involved in the allergic response, through the release of histamine. We investigated the effect of eriodictyol, derived from the painted maple and yerba santa, on mast cell degranulation and on an allergic response in an animal model. We also investigated its effect on the expression of the ceramide kinase (CERK) involved in calcium-dependent degranulation, and on ceramide activation by multiple cytokines. Eriodictyol suppressed the release of beta-hexosaminidase, a marker of degranulation, and the expression of interleukin (IL)-4 mRNA. It inhibited the expression of CERK mRNA, reduced the ceramide concentration in antigen-stimulated mast cells, and suppressed the passive cutaneous anaphylaxis (PCA) reaction in mice in a dose-dependent manner. These results suggest that eriodictyol can inhibit mast cell degranulation through inhibition of ceramide kinase, and that it might potentially serve as an anti-allergic agent.

Bacterial etiology and pneumococcal urinary antigen in moderate exacerbation of chronic obstructive pulmonary disease.

Background: This study aimed to establish nationwide data for the distributions of typical and atypical bacterial pathogens in Korean patients with moderate acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and evaluate the clinical usefulness of a urinary antigen test (UAT) to detect Streptococcus pneumoniae. Methods: This study was a post hoc analysis of a randomized controlled trial designed to compare oral zabofloxacin with moxifloxacin for treating outpatients with moderate AECOPD. From clinics across South Korea, 342 subjects with AECOPD were enrolled, and their blood, sputum, and urine samples were collected at baseline. A serologic test, sputum culture and polymerase chain reaction (PCR), and UAT were performed to identify bacterial pathogens. Bacterial prevalence and regional distributions were analyzed. The patients' characteristics and clinical response between UAT-positive and UAT-negative groups were compared, as were the Streptococcus pneumoniae detection rates using conventional sputum culture and PCR versus UAT. Results: The most commonly isolated pathogen was Haemophilus influenzae (30.3%), followed by Streptococcus pneumoniae (24.7%) and Pseudomonas aeruginosa (14.0%), with no significant regional differences in bacterial distribution. Patients with positive UAT for Streptococcus pneumoniae showed no clinical failure when treated with respiratory quinolone (0.0%), whereas 11.8% of patients with negative UAT showed clinical failure (P=0.037). UAT showed moderate agreement with sputum culture by kappa coefficient (κ=0.476). Conclusions: The bacterial prevalence in patients with moderate AECOPD in South Korea showed correlations with the global prevalence, without significant regional differences. In outpatient settings, UAT has the potential to be used as a supplemental tool with sputum culture as a guide for determining the suspicion of bacterial exacerbation.

Spectroscopic sensing and quantification of AP-endonucleases using fluorescence-enhancement by cis-trans isomerization of cyanine dyes.

Apurinic/apyrimidinic (AP) endonucleases are vital DNA repair enzymes, and proposed to be a prognostic biomarker for various types of cancer in humans. Numerous DNA sensors have been developed to evaluate the extent of nuclease activity but their DNA termini are not protected against other nucleases, hampering accurate quantification. Here we developed a new fluorescence enhancement (FE)-based method as an enzyme-specific DNA biosensor with nuclease-protection by three functional units (an AP-site, Cy3 and termini that are protected from exonucleolytic cleavage). A robust FE signal arises from the fluorescent cis-trans isomerization of a cyanine dye (e.g., Cy3) upon the enzyme-triggered structural change from double-stranded (ds)DNA to single-stranded (ss)DNA that carries Cy3. The FE-based assay reveals a linear dependency on sub-nanomolar concentrations as low as 10-11 M for the target enzyme and can be also utilized as a sensitive readout of other nuclease activities.

The mechanism of gap creation by a multifunctional nuclease during base excision repair.

During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3' end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.

Idesolide, an isolate of Idesia polycarpa, inhibits apoptosis through induction of intracellular heat shock protein 70 in C2C12 muscle cells.

Muscle disorders, such as muscular dystrophy, are associated with an increase in oxidative stress. Proposed treatments for muscular dystrophy, some in clinical trials, include gene therapy and muscle cell transplantation. In this study, we investigated the effects of idesolide, isolated from the fruits of Idesia polycarpa, on changes that occur in muscle disuse atrophy. We noted protective effects on oxidative stress response and HSP70 regulation. Pre-treatment with idesolide for 24 h maintained cell viability and decreased apoptosis in H(2)O(2)-treated C(2)C(12) muscle cells. The idesolide pretreatment also increased intracellular HSP70 protein. Our results suggest that idesolide inhibits cell death through induction of HSP70 in C(2)C(12) muscle cells. This work is the first to report that idesolide can regulate the decrease in HSP70 that occurs during skeletal muscle atrophy.

Applying an eMASS Customization Program as a Research Tool to Evaluate Consumer Benefits.

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization through marketing technology. The purpose of this study is to examine how to conduct consumer research using an online survey and analysis of data. This study examines consumers' perceived benefits while customizing a product as well as emotional product attachment, attitudes toward a customization program, and loyalty intentions in the context of online retailing. In addition, this study investigates how consumer responses are different based on individual characteristics such as fashion innovativeness. An online survey company in South Korea recruited 290 female apparel shoppers who purchased apparel online. To enhance external validity, this study used an existing retail website with a well-established mass customization program. After completing the customization program, participants complete the online questionnaire. Structural equation modeling (SEM) and latent mean analyses (LMAs) are then performed for analyses. This study stresses the importance of testing measurement invariance for mean comparisons. Before the SEM and LMA, this study follows the hierarchy of invariance tests (configural invariance test, metric invariance test, and scalar invariance test), which are not considered by traditional approaches such as ANOVA. These statistical analyses provide applicability of the invariance test procedures and LMA to consumer behaviors. The conclusions of mean differences have integrity and validity because they are guided by a sophisticated statistical procedure to ensure measurement invariance.

Benefits of mass customized products: moderating role of product involvement and fashion innovativeness.

The objective of this study was to explore impacts and benefits of mass customized products on emotional product attachment, favorable attitudes toward a mass customization program, and the ongoing effect on loyalty intentions. This study further investigated how benefits, attachment, attitudes, and loyalty intentions differed as a function of involvement and fashion innovativeness. 290 female online shoppers in South Korea participated in an online survey. Results of this study revealed that perceived benefits positively influenced emotional product attachment and attitudes toward a mass customization program. In addition, attachment positively influenced attitudes, which in turn affected loyalty intentions. This study also found that benefits, attachment, attitudes, and loyalty intentions were all higher in highly involved consumers (high fashion innovators) than those in less involved consumers (low fashion innovators). This study concludes with theoretical and practical implications for mass customization programs.

Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis.

Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg2+ binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while MgA2+ and MgB2+ have similar binding constants, the dissociation rate of MgA2+ is two order of magnitude lower than that of MgB2+ due to a kinetic-barrier-difference. At physiological Mg2+ concentration, the MgB2+ ion near the 5'-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of MgA2+ result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.

Periodontal and endodontic pathology delays extraction socket healing in a canine model.

PURPOSE: The aim of the present exploratory study was to evaluate extraction socket healing at sites with a history of periodontal and endodontic pathology. METHODS: The mandibular 4th premolar teeth in 5 adult beagle dogs served as experimental units. Periodontal and endodontic lesions were induced in 1 premolar site in each animal using wire ligatures and pulpal exposure over 3 months (diseased sites). The contralateral premolar sites served as healthy controls. The mandibular 4th premolar teeth were then extracted with minimal trauma, followed by careful wound debridement. The animals were sacrificed at days 1, 7, 30, 60, and 90 post-extraction for analysis, and the healing patterns at the healthy and diseased extraction sites were compared using radiography, scanning electron microscopy, histology, and histometry. RESULTS: During the first 7 days of healing, a significant presence of inflammatory granulation tissue was noted at the diseased sites (day 1), along with a slightly accelerated rate of fibrin clot resolution on day 7. On day 30, the diseased extraction sites showed a greater percentage of persistent fibrous connective tissue, and an absence of bone marrow formation. In contrast, healthy sites showed initial signs of bone marrow formation on day 30, and subsequently a significantly greater proportion of mature bone marrow formation on both days 60 and 90. Radiographs exhibited sclerotic changes adjoining apical endodontic lesions, with scanning electron microscopy showing collapsed Volkmann canals protruding from these regions in the diseased sites. Furthermore, periodontal ligament fibers exhibited a parallel orientation to the alveolar walls of the diseased sites, in contrast to a perpendicular arrangement in the healthy sites. CONCLUSIONS: Within the limitations of this study, it appears that a history of periodontal and endodontic pathology may critically affect bone formation and maturation, leading to delayed and compromised extraction socket healing.

Genetic interactions of TGA transcription factors in the regulation of pathogenesis-related genes and disease resistance in Arabidopsis.

TGA transcription factors are implicated as regulators of pathogenesis-related (PR) genes because of their physical interaction with the known positive regulator, nonexpresser of PR gene1 (NPR1). A triple-knockout mutant tga2-1 tga5-1 tga6-1 was shown previously to be defective in the induction of PR genes and systemic acquired resistance, confirming their role in disease resistance. However, the contributions of individual TGA factors have been difficult to discern because of functional redundancy among these factors, as well as possible dual functions for some single factors. In this study, we characterized six TGA factors by reverse genetics. We show that TGA3 is required for both basal and 2,6-dichloroisonicotinic acid-induced transcription of PR genes. The tga3-1 mutants were found to be defective in basal pathogen resistance, whereas induced resistance was unaffected. TGA1 and TGA4 play partially redundant roles in regulation of basal resistance, having only moderate effects on PR gene expression. Additionally, an activation-tagged mutant of TGA6 was able to increase basal as well as induced expression of PR1, demonstrating a positive role for TGA6 on PR gene expression. In contrast, TGA2 has repressor activity on PR gene expression even though it can act as a positive regulator in the tga5-1 tga6-1 null mutant background. Finally, we examined the genetic interaction between tga2-2 and suppressor of npr1 inducible1 (sni1-1). TGA2's repressor activity overlaps with SNI1 because the tga2-2 sni1-1 double mutant shows a synergistic effect on PR gene expression.