A precise and sustainable doxycycline-inducible cell line development platform for reliable mammalian cell engineering with gain-of-function mutations.

For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a Robust Overexpression via Site-specific integration of Effector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells.

Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 µM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (q mAb ) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a q mAb of 9.1-11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.

Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells.

Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1α, SV40, and TK promoters, into the well-known genomic safe harbor site, AAVS1. EGFP expression driven by the CMV promoter increased up to 1.5-fold at 32 °C versus 37 °C under stable expression, while others showed no hypothermic response. Integration of short CMV variants revealed that the CMV-enhancer region is responsible for the positive hypothermic response. CMV-enhancer-specific transcription factors (TFs) were then predicted through in silico analysis and RNA-sequencing analysis, resulting in the selection of one TF, NKX3-1. At 37 °C, overexpression of NKX3-1 in recombinant HEK293 cells expressing EGFP through the CMV promoter (CMV-EGFP) increased EGFP expression up to 1.6-fold, compared with that in CMV-EGFP, the expression level of which was comparable to that of CMV-EGFP at 32 °C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences.

Universal three-dimensional crosslinker for all-photopatterned electronics.

All-solution processing of large-area organic electronics requires multiple steps of patterning and stacking of various device components. Here, we report the fabrication of highly integrated arrays of polymer thin-film transistors and logic gates entirely through a series of solution processes. The fabrication is done using a three-dimensional crosslinker in tetrahedral geometry containing four photocrosslinkable azide moieties, referred to as 4Bx. 4Bx can be mixed with a variety of solution-processable electronic materials (polymer semiconductors, polymer insulators, and metal nanoparticles) and generate crosslinked network under exposure to UV. Fully crosslinked network film can be formed even at an unprecedentedly small loading, which enables preserving the inherent electrical and structural characteristics of host material. Because the crosslinked electronic component layers are strongly resistant to chemical solvents, micropatterning the layers at high resolution as well as stacking the layers on top of each other by series of solution processing steps is possible.

Transparent and Colorless Polyimides Containing Multiple Trifluoromethyl Groups as Gate Insulators for Flexible Organic Transistors with Superior Electrical Stability.

A suitable insulating polymer material that is compatible with the fabrication process of organic transistors and has excellent electrical properties is critically required for the next-generation flexible organic electronics. In this study, using a one-step polymerization method, we synthesized two different solution-processable polyimides (PIs) incorporated with abundant trifluoromethyl groups. Not only were the two resulting PIs-termed 6FDA-6FDAM-PI and 6FDA-TFMB-PI-well soluble in organic solvents, but also they showed transparent and colorless optical properties. The fluorinated PI films showed smooth surface topographies and surface energy values that were appropriate for their use in bottom-gate organic transistors. Organic transistors separately fabricated with 6FDA-6FDAM-PI and 6FDA-TFMB-PI as the gate insulators showed excellent device performance and electrical stability under various testing conditions, especially for pentacene-based devices. The excellent performance of the devices with fluorinated PIs was attributed to the enhanced microstructure of the organic semiconductor and the fluorine-rich characteristic of the underlying gate insulator. Furthermore, organic complementary circuits including the basic logic gates of NOT, NOR, and NAND were demonstrated using these devices.

Comparative Study on Osseointegration of Implants After Flap and Flapless Surgery in the Mandible of Dogs.

PURPOSE: The objective of this study was to compare the implant stability and osseointegration of implants using a flap or flapless technique. MATERIAL AND METHODS: Mandibular premolars and molars were extracted from both sides in 6 dogs. After 8 weeks, 4 fixtures were implanted using either a flap or flapless technique. Implant stability quotient was measured on insertion and at 2, 4, and 8 weeks later. The animals were killed while the tissues were histologically analyzed. RESULTS: Implant stability increased for 8 weeks, and no statistically significant differences were observed between the surgical protocols. Bone-implant contact showed 60.27% ± 30.99% for flapless surgery and 59.73% ± 17.12% for flap surgery. And the results of new bone formation area from total area showed 56.07% ± 27.78% for flapless surgery and 57.00% ± 14.66% for flap surgery. There were no statistically significant differences. CONCLUSION: This study showed no significant difference in implant stability as well as osseointegration regardless of flap or flapless technique.

Gate-Bias Stability Behavior Tailored by Dielectric Polymer Stereostructure in Organic Transistors.

Understanding charge trapping in a polymer dielectric is critical to the design of high-performance organic field-effect transistors (OFETs). We investigated the OFET stability as a function of the dielectric polymer stereostructure under a gate bias stress and during long-term operation. To this end, iso-, syn-, and atactic poly(methyl methacrylate) (PMMA) polymers with identical molecular weights and polydispersity indices were selected. The PMMA stereostructure was found to significantly influence the charge trapping behavior and trap formation in the polymer dielectrics. This influence was especially strong in the bulk region rather than in the surface region. The regular configurational arrangements (isotactic > syntactic > atactic) of the pendant groups on the PMMA backbone chain facilitated closer packing between the polymer interchains and led to a higher crystallinity of the polymer dielectric, which caused a reduction in the free volumes that act as sites for charge trapping and air molecule absorption. The PMMA dielectrics with regular stereostructures (iso- and syn-stereoisomers) exhibited more stable OFET operation under bias stress compared to devices prepared using irregular a-PMMA in both vacuum and air.

Identification of Domain Boundary Defects in Crystalline Self-Assembled Monolayers.

One of the major challenges to the fabrication of functionalized templates using self-assembled monolayers (SAMs) is the characterization of nanoscale defects, particularly SAM domain boundaries (DBs). In this study, an etchant was used to chemically amplify the DBs in a SAM by forming microscale pits in the underlying SiO2 layer. This approach revealed that the naturally occurring DBs acted as structural defects in the SAMs. The DB structures were characterized by systematically varying the octadecyltrichlorosilane (ODTS) monolayer domain size from the nanoscale to the microscale by varying the preparation temperature. These approaches showed that the SAM DBs, which were visualized as having round- and line-shaped nanoscale structures, provided potentially chemical and mechanical surface defect sites. Our principal findings open up exciting new possibilities for understanding the structural defects in SAMs on the molecular level and suggest an approach for optimizing the conditions used to generate defect-free SAM templates.

The use of lung ultrasonography to confirm lung isolation in an infant who underwent emergent video-assisted thoracoscopic surgery: a case report.

Video-assisted thoracoscopic surgery for pediatric patients has gained popularity due to better outcomes than open surgery. For this procedure, one-lung ventilation may be necessary to provide an adequate surgical field. Confirming lung isolation is crucial when one-lung ventilation is required. Recently, we experienced a case in which one-lung ventilation was confirmed by ultrasonography using the lung sliding sign and the lung pulse in an infant. Since lung ultrasonography can be performed easily and quickly, it may be a useful method to confirm lung isolation, particularly in emergency surgeries with limited time, devices, and experienced anesthesiologists.

Sclerotherapy using 1% sodium tetradecyl sulfate to treat a vascular malformation: a report of two cases.

Vascular malformations are the most common congenital and neonatal vascular anomalies in the head and neck region. The demand for simple and esthetic vascular malformation treatments have increased more recently. In this study, two patients that were diagnosed with venous malformations were treated with sodium tetradecyl sulfate as a sclerosing agent. Recurrence was not found one year after the surgery. This article gives a brief case report of sclerotherapy as an effective approach to treat vascular malformations in the oral cavity.

Dynamic optic nerve sheath diameter responses to short-term hyperventilation measured with sonography in patients under general anesthesia.

BACKGROUND: Rapid evaluation and management of intracranial pressure (ICP) can help to early detection of increased ICP and improve postoperative outcomes in neurocritically-ill patients. Sonographic measurement of optic nerve sheath diameter (ONSD) is a non-invasive method of evaluating increased intracranial pressure at the bedside. In the present study, we hypothesized that sonographic ONSD, as a surrogate of ICP change, can be dynamically changed in response to carbon dioxide change using short-term hyperventilation. METHODS: Fourteen patients were enrolled. During general anesthesia, end-tidal carbon dioxide concentration (ETCO2) was decreased from 40 mmHg to 30 mmHg within 10 minutes. ONSD, which was monitored continuously in the single sonographic plane, was repeatedly measured at 1 and 5 minutes with ETCO2 40 mmHg (time-point 1 and 2) and measured again at 1 and 5 minutes with ETCO2 30 mmHg (time-point 3 and 4). RESULTS: The mean ± standard deviation of ONSD sequentially measured at four time-points were 5.0 ± 0.5, 5.0 ± 0.4, 3.8 ± 0.6, and 4.0 ± 0.4 mm, respectively. ONSD was significantly decreased at time-point 3 and 4, compared with 1 and 2 (P < 0.001). CONCLUSIONS: The ONSD was rapidly changed in response to ETCO2. This finding may support that ONSD may be beneficial to close ICP monitoring in response to CO2 change.

The accuracy of the new landmark using respiratory jugular venodilation and direct palpation in right internal jugular vein access.

BACKGROUND: Although ultrasonography is recommended in internal jugular vein (IJV) catheterization, the landmark-guided technique should still be considered. The central landmark using the two heads of the sternocleidomastoid muscle is widely used, but it is inaccurate for IJV access. As an alternative landmark, we investigated the accuracy of the new landmark determined by inspection of the respiratory jugular venodilation and direct IJV palpation in right IJV access by ultrasonography. METHODS AND FINDINGS: Thirty patients were enrolled. After induction of anesthesia, the central landmark was marked at the cricoid cartilage level (M1) and the alternative landmark determined by inspection of the respiratory jugular venodilation and direct palpation of IJV was also marked at the same level (M2). Using ultrasonography, the location of IJV was identified (M3) and the distance between M1 and M3 as well as between M2 and M3 were measured. The median (interquartile range) distance between the M2 and M3 was 3.5 (2.0-6.0) mm, compared to 17.5 (12.8-21.3) mm between M1 and M3. (P<0.001) The dispersion of distances between M2 and M3 was significantly smaller than between M1 and M3. (P<0.001) The visibility of respiratory jugular venodilation was associated with CVP more than 4 mmHg. Limitations of the present study are that the inter-observer variability was not investigated and that the visibility of the alternative landmark can be limited to right IJV in adults. CONCLUSION: The alternative landmark may allow shorter distance for the right side IJV access than the central landmark and can offer advantages in right IJV catheterization when ultrasound device is unavailable. TRIAL REGISTRATION: Clinical Research Informational Service KCT0000812.

Deep Neck Space Infection Caused by Keratocystic Odontogenic Tumor.

Keratocystic odontogenic tumor (KCOT) is a benign cystic intraosseous tumor of odontogenic origin. An infection of a KCOT is not common because KCOT is a benign developmental neoplasm. Moreover, a severe deep neck space infection with compromised airway caused by infected KCOT is rare. This report presents a 60-year-old male patient with a severe deep neck space infection related to an infected KCOT due to cortical bone perforation and rupture of the exudate. Treatment of the deep neck space infection and KCOT are reported.

Microstructural control over soluble pentacene deposited by capillary pen printing for organic electronics.

Research into printing techniques has received special attention for the commercialization of cost-efficient organic electronics. Here, we have developed a capillary pen printing technique to realize a large-area pattern array of organic transistors and systematically investigated self-organization behavior of printed soluble organic semiconductor ink. The capillary pen-printed deposits of organic semiconductor, 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS_PEN), was well-optimized in terms of morphological and microstructural properties by using ink with mixed solvents of chlorobenzene (CB) and 1,2-dichlorobenzene (DCB). Especially, a 1:1 solvent ratio results in the best transistor performances. This result is attributed to the unique evaporation characteristics of the TIPS_PEN deposits where fast evaporation of CB induces a morphological evolution at the initial printed position, and the remaining DCB with slow evaporation rate offers a favorable crystal evolution at the pinned position. Finally, a large-area transistor array was facilely fabricated by drawing organic electrodes and active layers with a versatile capillary pen. Our approach provides an efficient printing technique for fabricating large-area arrays of organic electronics and further suggests a methodology to enhance their performances by microstructural control of the printed organic semiconducting deposits.

Directly drawn organic transistors by capillary pen: a new facile patterning method using capillary action for soluble organic materials.

A capillary pen drawing technique, developed as a new patterning methodology for the large-area patterning and fabrication of organic electronics, provides several advantages over conventional approaches: the method is simple and versatile, there are no restrictions on the patterning shapes that could be produced, and the method can be tailored to a variety of substrates.

Comparison of the spinal neuropathic pain induced by intraspinal injection of N-methyl-d-aspartate and quisquate in rats.

OBJECTIVE: Excitatory amino acids play important roles in the development of secondary pathology following spinal cord injury (SCI). This study was designed to evaluate morphological changes in the dorsal horn of the spinal cord and assess profiles of pain behaviors following intraspinal injection of N-methyl-D-aspartate (NMDA) or quisqualate (QUIS) in rats. METHODS: Forty male Sprague-Dawley rats were randomized into three groups : a sham, and two experimental groups receiving injections of 125 mM NMDA or QUIS into their spinal dorsal horn. Following injection, hypersensitivity to cold and mechanical stimuli, and excessive grooming behaviors were assessed serially for four weeks. At the end of survival periods, morphological changes in the spinal cord were evaluated. RESULTS: Cold allodynia was developed in both the NMDA and QUIS groups, which was significantly higher in the QUIS group than in the NMDA group. The mechanical threshold for the ipsilateral hind paw in both QUIS and NMDA groups was significantly lower than that in the control group. The number of groomers was significantly higher in the NMDA group than in the QUIS group. The size of the neck region of the spinal dorsal horn, but not the superficial layer, was significantly smaller in the NMDA and QUIS groups than in the control group. CONCLUSION: Intraspinal injection of NMDA or QUIS can be used as an excitotoxic model of SCI for further research on spinal neuropathic pain.