Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions.

CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.

Low-concentration imiquimod treatment promotes enhanced skin barrier functions through epidermal melanization reaction regulation.

The primary function of the skin is to form a mechanical, permeability, antimicrobial, and ultraviolet radiation barrier, which is essential for maintaining physiological homeostasis. Our previous studies demonstrated that cutaneous pigmentation could promote skin barrier function in addition to providing anti-ultraviolet irradiation defense. The present study aimed to develop a new regimen that enhances skin barrier function by regulating skin pigmentation using low-concentration imiquimod. Results showed that topical application of low-concentration imiquimod effectively induced skin hyperpigmentation in the dorsal skin and external ear of mice without inducing inflammatory cell infiltration. An in vitro study also revealed that low-concentration imiquimod did not induce any cytotoxic effects on melanoma cells but triggered excessive melanin synthesis. In coculture systems, low-concentration imiquimod was noted to increase tyrosinase activity in a broader cellular context, revealing the potential role of neighboring cells in melanin production. The next-generation sequencing result indicated that PKCη and Dnm3 might regulate melanin synthesis and release during imiquimod treatment. Overall, our study presents new insights into the regulation of melanin production by low-concentration imiquimod, both in a mice model and cultured cells. Furthermore, our study highlights the potential benefits of imiquimod in promoting melanin synthesis without causing skin disruptions or inducing inflammation, validating its potential to serve as a method for enhancing skin barrier functions by regulating the epidermal melanization reaction.

Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions.

CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as 6-fold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.

Incidence and contributing factors of non-root canal treated teeth with chronic fatigue root fracture: A cross-sectional study.

BACKGROUND/PURPOSE: Chronic fatigue root fracture describes a root fracture in a non-root canal treated (non-RCT) tooth. This study aimed to report the incidence and contributing factors of non-RCT teeth with chronic fatigue root fracture in a Taiwanese population. METHODS: This cross-sectional study included teeth extracted at Taipei Veterans General Hospital in Taiwan between 2018 and 2019. The reasons for extractions were recorded and included vertical and horizontal root fractures (VRF and HRF). Comparisons of clinical factors between teeth with fatigue VRF and teeth with fatigue HRF were performed by chi-square or Fisher exact test, where appropriate. RESULTS: Of the 4207 extracted teeth examined, 263 (6.25%) had tooth fracture. Thirty-two non-RCT teeth had chronic fatigue root fracture, including 16 with VRF and 16 with HRF. The incidence was 0.76% (32/4207). The occurrence of chronic fatigue root fracture was higher in males (83.9%). The mean age of the 31 patients with chronic fatigue root fracture was 71.7 ± 13.1 years. More than half of these teeth had intact crowns with severe attrition. The fatigue VRF occurred more frequently in molars (P = 0.003), in roots with a long oval cross-section (P = 0.037), and in terminal teeth (P = 0.013) than the fatigue HRF. CONCLUSION: The incidence of chronic fatigue root fracture is 0.76%. Both VRF and HRF occur mainly in aged males, in posterior teeth with attrition, and in teeth without restoration. Tooth position, cross-section root morphology, and terminal tooth are contributing factors related to chronic fatigue root fracture.

Nonlinear compression toward high-energy single-cycle pulses by cascaded focus and compression.

The advancement of contemporary ultrafast science requires reliable sources to provide high-energy few-cycle light pulses. Through experiments and simulations, we demonstrate an arrangement of pulse postcompression, referred to as cascaded focus and compression (CASCADE), for generating millijoule-level, single-cycle pulses in a compact fashion. CASCADE is realized by a series of foci in matter, whereas pulse compression is provided immediately after each focus to maintain a high efficiency of spectral broadening. By implementing four stages of CASCADE in argon cells, we achieve 50-fold compression of millijoule-level pulses at 1030 nanometers from 157 to 3.1 femtoseconds, with an output pulse energy of 0.98 millijoules and a transmission efficiency of 73%. When driving high harmonic generation, these single-cycle pulses enable the creation of a carrier-envelope phase-dependent extreme ultraviolet continuum with energies extending up to 180 electron volts, providing isolated attosecond pulses at the output.

Evaluation of students' self-assessment performance in preclinical endodontic training by means of rubrics and a 3D printed model.

BACKGROUND/PURPOSE: Effective evaluation of students' self-assessment ability is crucial. This study was to develop a standardized 3D printed teaching model accompanied by structured scoring rubrics for preclinical endodontic training, to appraise students' self-assessment ability, and to evaluate their perceptions of the training system. METHODS: The 3D printing model was designed to house a standardized central incisor. Forty-four undergraduate dental students were enrolled and their endodontic performance was self-assessed and also rated by a teaching assistant and a tutor using 3 structured rubrics including access cavity, mechanical preparation, and obturation assessments. In total, 21 rubric evaluation items of preclinical tasks were assessed, and the assessment results from the student, teaching assistant, and tutor were compared. Furthermore, questionnaires were used to evaluate students' experiences with the new system. RESULTS: The tutor, teaching assistant, and students all had similar scorings and high intraclass correlation coefficients (ICC) for mechanical preparation and obturation rubrics, while the students had underestimated scores on the access cavity rubric with a rather low ICC of 0.387, which also lead to the underestimated overall scoring (P < 0.05). Among the 21 evaluation criteria, 9 items were rated statistically different (P < 0.05), which denoted the major deficiencies of students' self-assessments. More than 80% of students rated satisfied for most of the questions regarding the new training system, except the tactile sensation, hardness, and radiopacity. CONCLUSION: The proposed standardized 3D printed model and structured scoring rubrics is feasible for preclinical endodontic training, and standardized evaluation of students' self-assessment ability.

The role of fibroblasts in the modulation of dental pulp inflammation.

BACKGROUND/PURPOSE: Dental pulp fibroblasts can protect dental pulp from microbial invasion. However, little is known about the interaction between pulp fibroblasts and the immune cells. In this study, the production of proinflammatory cytokines related to inflammatory cell recruitment was evaluated in tumor necrosis factor (TNF)-α-stimulated human dental pulp fibroblasts (HDPFs). The role of TNF-α-stimulated HDPFs in the cell fusion under inflammatory process was determined with the cell co-culture with peripheral blood mononuclear cells (PBMCs). METHODS: HDPFs were stimulated with various concentrations of TNF-α, and the secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 was analyzed by the enzyme-linked immunosorbent assay. The mRNA expression levels of intercellular adhesion molecule-1 (ICAM-1), macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) were determined by real-time quantitative polymerase chain reaction. TNF-α-treated HDPFs were co-cultured with PBMCs for 21 days, and characteristics of cell differentiation were assessed. RESULTS: TNF-α induced IL-6, IL-8 and MCP-1 production in HDPFs. Moreover, mRNA expression levels of ICAM-1, M-CSF and OPG were significantly increased in TNF-α-treated HDPFs. Co-culture of TNF-α-treated HDPFs and PBMCs stimulated formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and the F-actin rings were observed in these multinucleated cells. CONCLUSION: Our results indicate that under the stimulation of TNF-α, HDPFs may amplify inflammatory response by cytokines production, which in turn can modulate the differentiation of immune cells.

Application of Intelligent Modeling Method to Optimize the Multiple Quality Characteristics of the Injection Molding Process of Automobile Lock Parts.

This study focuses on applying intelligent modeling methods to different injection molding process parameters, to analyze the influence of temperature distribution and warpage on the actual development of auto locks. It explores the auto locks using computer-aided engineering (CAE) simulation performance analysis and the optimization of process parameters by combining multiple quality characteristics (warpage and average temperature). In this experimental design, combinations were explored for each single objective optimization process parameter, using the Taguchi robust design process, with the L18 (21 × 37) orthogonal table. The control factors were injection time, material temperature, mold temperature, injection pressure, packing pressure, packing time, cooling liquid, and cooling temperature. The warpage and temperature distribution were analysed as performance indices. Then, signal-to-noise ratios (S/N ratios) were calculated. Gray correlation analysis, with normalization of the S/N ratio, was used to obtain the gray correlation coefficient, which was substituted into the fuzzy theory to obtain the multiple performance characteristic index. The maximum multiple performance characteristic index was used to find multiple quality characteristic-optimized process parameters. The optimal injection molding process parameters with single objective are a warpage of 0.783 mm and an average temperature of 235.23 °C. The optimal parameters with multi-objective are a warpage of 0.753 mm and an average temperature of 238.71 °C. The optimal parameters were then used to explore the different cooling designs (original cooling, square cooling, and conformal cooling), considering the effect of the plastics temperature distribution and warpage. The results showed that, based on the design of the different cooling systems, conformal cooling obtained an optimal warpage of 0.661 mm and a temperature of 237.62 °C. Furthermore, the conformal cooling system is smaller than the original cooling system; it reduces the warpage by 12.2%, and the average temperature by 0.46%.

Efficient nonlinear compression of a mode-locked thin-disk oscillator to 27 fs at 98 W average power.

We demonstrate efficient pulse compression of a 13.4 MHz, 534 fs, 123 W, Yb:YAG thin-disk oscillator down to 27 fs at 98 W average power, resulting in a record-high 166 MW peak power from an amplifier-free oscillator-driven setup. Our compressor is based on two stages: one multipass cell allowing us to reduce the pulse duration to sub-90 fs and, subsequently, a multiple-plate compressor, allowing us to reach 27 fs. The overall average power compression efficiency is 80%, and the beam has excellent beam quality and homogeneity. In addition, we demonstrate further spectral broadening that supports a transform limit of 5 fs in a second multiple-plate stage, demonstrating the potential for reaching a 100 W class, amplifier-free, few-cycle source in the near future. The performance of this unique source is very promising for applications previously restricted to amplified sources, such as efficient generation of extreme ultraviolet light at high repetition rate, and the generation of high-power broadband THz radiation.

Risk of hyperglycemic crisis episode in diabetic patients with bipolar disorder: A nationwide population-based cohort study.

BACKGROUND: Suboptimal management of diabetes can lead to a hyperglycemic crisis episode (HCE), which could be further enhanced in the presence of bipolar disorder (BD) and the prescription of antipsychotics. This study aims to investigate the risk of HCE in diabetic patients with BD. Additionally, the duration of antipsychotic prescription on HCE risk is examined. METHODS: Using the Taiwan National Health Insurance Research Database, 6099 diabetic patients with BD and 24,378 diabetic patients without BD matched by gender, age, index year, and Charlson Comorbidity Index score were enrolled between 1999 and 2010 and followed to the end of 2013. Participants who developed HCE during the follow-up period were identified. Cox regression analysis was used to calculate the hazard ratio (HR) with 95% confidence interval (CI) of the HCE incidence rate between two groups studied. RESULTS: Diabetic patients with BD were associated with an increased risk of HCE compared with unaffected controls after adjusted for baseline demographics and duration of antipsychotic prescription (3.84 versus 2.71 per 1,000 person-years, HR: 1.41, 95% CI: 1.15-1.71). Also, this study revealed that male gender, more comorbidities, and a longer duration of antipsychotic prescription were potential risk factors for developing HCE. LIMITATIONS: This study only deals with data on the duration of antipsychotic prescription, without showing the effects of different antipsychotics on HCE risk. CONCLUSION: This study highlights the need to pay attention to the risk of HCE in diabetic patients with BD and the importance of careful prescription of antipsychotics to reduce the HCE incident.

A cone-beam computed tomography study of C-shaped root canal systems in mandibular second premolars in a Taiwan Chinese subpopulation.

BACKGROUND/PURPOSE: The purpose of this study is to investigate the prevalence and the morphologic characteristics of the radicular groove and root canal system in human mandibular second premolars with C-shaped root in a Taiwan Chinese subpopulation using cone-beam computed tomographic (CBCT) imaging. METHODS: CBCT images of 580 mandibular second premolars were collected from 317 patients. All of the mandibular second premolars were examined in serial axial sections to identify the presence of any C-shaped root and C-shaped canal systems. The morphologic characteristics of mandibular second premolars with C-shaped roots were studied by performing measurements of serial axial sections. RESULTS: The prevalence rate of mandibular second premolars with a C-shaped root was 3.45% (20/580 teeth) and the rate of those with a C-shaped canal system was 2.24% (13/580 teeth). It was found that 69% of the radicular grooves were located on the lingual half of the root (9/13 teeth) in mandibular second premolars with a C-shaped canal system. In those teeth with a lingual radicular groove, the main canal was toward the buccal side. Frequently, the first C-shaped canal image was found at the mid-root level. The deepest part of the radicular groove was located at about 2.5 mm apical to the first C-shaped canal image. CONCLUSION: There was a 2-3% morphologic variation of the mandibular second premolar with a C-shaped root canal system among the Taiwan Chinese subpopulation investigated in this study. Detailed knowledge of the morphological characteristics of teeth may be valuable when choosing clinical treatments.

Broadband mid-infrared polarization rotator based on optically addressable LCs.

This work proposes a mid-infrared polarization rotator that incorporates a twisted nematic liquid crystal (TNLC) cell with a photo-controllable alignment layer. The TNLC device with a sufficient phase retardation can act as an achromic polarization rotation device over a wide wavelengths range and thus can rotate the polarization of a mid-IR laser beam. The photo-alignment technique enables TNLCs with arbitrary twisting angles to be generated by the use of visible polarized addressing light to control the directors of the photo-alignment layer. Therefore, arbitrary rotation angles of the polarization axis of a linearly polarized mid-IR laser beam can be realized. Moreover, the rewritable property and reliability of this polarization rotator are experimentally verified. The flexibility of polarization control for broadband mid-IR opens up a large range of potential mid-IR applications.

Non-invasive manipulation of Drosophila behavior by two-photon excited red-activatable channelrhodopsin.

Scattering and absorption limit light penetration through inhomogeneous tissue. To reduce scattering, biochemists have shifted the wavelengths of excitation light for optogenetic actuators and fluorescent proteins to the orange-red range, while physicists have developed multiphoton technologies for deep tissue stimulation. We have built a rapid multiphoton spectroscopic screening system of genetically encoded red-activatable channelrhodopsin (ReaChR), and considered specific behaviors in transgenic Drosophila melanogaster as readouts to optimize the laser parameters for two-photon optogenetic activation. A wavelength-tunable optical parametric amplifier was adopted as the major light source for widefield two-photon excitation (TPE) of ReaChR. Our assays suggest that the optimized TPE wavelength of ReaChR is 1250 nm. Exploiting its capacity for optogenetic manipulation to induce macroscopic behavioral change, we realized rapid spectroscopic screening of genetically encoded effectors or indicators in vivo, and used modulation of ReaChR in the fly as a successful demonstration of such a system.

ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates.

BACKGROUND: Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase. RESULTS: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates. CONCLUSIONS: We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.

Phase-matching spectral phase measurement and domain period reconstruction of aperiodic quasi-phase matched gratings by nonlinear spectral interferometry.

We experimentally measured the phase-matching spectral phases of aperiodic quasi-phase matched gratings for the first time (to the best of our knowledge) by nonlinear spectral interferometry. The retrieved information is useful in determining the temporal shape of the nonlinearly converted ultrafast signal and reconstructing the slowly-varying domain period distribution. The method is nondestructive, fast, sensitive, accurate, and applicable to different nonlinear materials. Compared to taking microscopic images of the etched crystal surface, our method can directly measure the domain period distribution in the crystal interior and is free of the artificial random duty period error due to image concatenation.

Allergic colitis in infants related to cow's milk: clinical characteristics, pathologic changes, and immunologic findings.

BACKGROUND: Allergic colitis (AC) is an inflammatory condition characterized by eosinophils infiltrating the colonic wall. It can be a benign and/or severe illness among gastrointestinal diseases in infants. METHODS: We report five infants who, since January 2009, in whom AC under fibrotic endoscopic examinations has been diagnosed. The criterion for histopathologic diagnosis of AC in this study was five or more eosinophils per high-power field. Patients' clinical symptoms, pathologic findings, and immunologic studies, such as specific antibodies against component of cow's milk protein, were compared with those of allergic children without AC and those of nonatopic control children. RESULTS: Histopathologic examinations of biopsy specimens revealed acute inflammation with characteristic eosinophilic infiltration of lamina propria (5-15 eosinophils per high-power field) in all five patients. They all had strongly positive skin prick tests against milk protein, which were not correlated with in vitro allergen-specific immunoglobulin (Ig) E levels. In contrast, there were significantly higher levels of IgE antibodies, and lower specific IgG4 and IgA levels to components and whole milk proteins in AC, as compared to control children without AC. CONCLUSION: Endoscopic biopsy specimens of intestine confirm the diagnosis of AC. However, allergen skin prick test and IgE antibody to milk protein components also provide helpful diagnostic tools for this rare disease in children.

Higher order chromatin structure at the X-inactivation center via looping DNA.

In mammals, the silencing step of the X-chromosome inactivation (XCI) process is initiated by the non-coding Xist RNA. Xist is known to be controlled by the non-coding Xite and Tsix loci, but the mechanisms by which Tsix and Xite regulate Xist are yet to be fully elucidated. Here, we examine the role of higher order chromatin structure across the 100-kb region of the mouse X-inactivation center (Xic) and map domains of specialized chromatin in vivo. By hypersensitive site mapping and chromosome conformation capture (3C), we identify two domains of higher order chromatin structure. Xite makes looping interactions with Tsix, while Xist makes contacts with Jpx/Enox, another non-coding gene not previously implicated in XCI. These regions interact in a developmentally-specific and sex-specific manner that is consistent with a regulatory role in XCI. We propose that dynamic changes in three-dimensional architecture leads to formation of separate chromatin hubs in Tsix and Xist that together regulate the initiation of X-chromosome inactivation.

Mobilization of RAG-generated signal ends by transposition and insertion in vivo.

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.

Transient homologous chromosome pairing marks the onset of X inactivation.

Mammalian X inactivation turns off one female X chromosome to enact dosage compensation between XX and XY individuals. X inactivation is known to be regulated in cis by Xite, Tsix, and Xist, but in principle the two Xs must also be regulated in trans to ensure mutually exclusive silencing. Here, we demonstrate that interchromosomal pairing mediates this communication. Pairing occurs transiently at the onset of X inactivation and is specific to the X-inactivation center. Deleting Xite and Tsix perturbs pairing and counting/choice, whereas their autosomal insertion induces de novo X-autosome pairing. Ectopic X-autosome interactions inhibit endogenous X-X pairing and block the initiation of X-chromosome inactivation. Thus, Tsix and Xite function both in cis and in trans. We propose that Tsix and Xite regulate counting and mutually exclusive choice through X-X pairing.