Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.

Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.

Enhanced gelling property and freeze-thaw stability of potato, tapioca and corn starches modified by mild heating in aqueous ethanol solution.

BACKGROUND: Physically modified starches can be classified as natural ingredients on food labels and clean label products. Thus, the market demand for physically modified starch is increasing. Potato, tapioca and corn starches were physically modified by mild heat treatment in an alcoholic solution to enhance their gelling property and freeze-thaw stability. RESULTS: During mild heating of starch suspension (40% w/w) in 10% ethanol solution at the onset gelatinization temperature, granular swelling of starch occurred, followed by amylose leaching with medication of the surface structure of the starch granules. All treated starches exhibited increased gelatinization and pasting temperatures and decreased breakdown for pasting as a result of improved stability against shear and heat. The treated starches had higher hardness, cohesiveness and springiness of gel than the respective native starches, and these gel properties were more pronounced in potato starch than in tapioca and corn starches. The treated starches showed substantially reduced gel syneresis during freeze-thawing. CONCLUSION: Physical modification of starch by mild heat treatment in an alcoholic solution substantially improved its gelation ability and freeze-thaw stability. © 2024 Society of Chemical Industry.

Effective mRNA Delivery by Condensation with Cationic Nanogels Incorporated into Liposomes.

The challenge in effective delivery of mRNA has been a major hurdle in their development as therapeutics. Herein, we present that the incorporation of cationic nanogels as the condensing material for mRNA into liposomes enables stable and enhanced mRNA delivery to cells in vitro. We prepared dextran-based nanogel particles, which were surface functionalized with oligoarginine peptide (DNPR9) and complexed with mRNA for incorporation into liposomes (LipoDNPR9). The use of DNPR9 with the liposomes resulted in enhanced internalization, as well as a 4-fold increase in transfection of luciferase mRNA when treated with A549 cells in vitro, compared to control liposomes. The enhancement in transfection efficiency was also observed in various cell lines while causing low cytotoxicity. The versatility of the strategy was also investigated by applying DNPR9 for mRNA condensation to ionizable lipid particles, which resulted in an ∼55% increase in transfection. The current development based on nanogel-incorporated liposomes introduces an effective platform for mRNA delivery, while the condensation strategy using DNPR9 can be widely applied for various lipid-based formulations to enhance their efficacy.

Factors contributing to uncertainty in paediatric abdominal ultrasound reports in the paediatric emergency department.

BACKGROUND: Abdominal pain, which is a common cause of children presenting to the paediatric emergency department (PED), is often evaluated by ultrasonography (US). However, uncertainty in US reports may necessitate additional imaging. OBJECTIVE: In this study, we evaluated factors contributing to uncertainty in paediatric abdominal US reports in the PED. MATERIALS AND METHODS: This retrospective cohort study included children younger than 18 years of age who underwent abdominal US in the PED of the study hospital between January 2017 and December 2019. After exclusion, the researchers manually reviewed and classified all US reports as 'certain' or 'uncertain'. Univariate and multivariate logistic regression analyses were performed to identify the factors contributing to uncertain reports. RESULTS: In total, 1006 patients were included in the final analysis., 796 patients were tagged as having certain reports, and 210 as having uncertain reports. Children with uncertain reports had a significantly higher rate of undergoing an additional computed tomography (CT) scan (31.0% vs. 2.5%, p < 0.001) and a longer PED median length of stay (321.0 (Interquartile range (IQR); 211.3-441.5) minutes vs. 284.5 (IQR; 191.8-439.5) minutes, p = 0.042). After logistic regression, US performed by a radiology resident (odds ratio, 5.01; 95% confidence interval, 3.63-7.15) was the most significant factor contributing to uncertainty in paediatric abdominal US reports followed by obesity and age. CONCLUSION: Several factors contribute to uncertainty in paediatric abdominal US reports. Uncertain radiological reports increase the likelihood of additional CT scans. Measures to improve the clarity of radiological reports must be considered to improve the quality of care for children visiting the PED.

Recyclable Periodic Nanostructure Formed by Sublimable Liquid Crystals for Robust Cell Alignment.

We demonstrate a facile method to fabricate a recyclable cell-alignment scaffold using nanogrooves based on sublimable liquid crystal (LC) material. Randomly and uniaxially arranged smectic LC structures are obtained, followed by sublimation and recondensation processes, which directly produce periodic nanogrooves with dimensions of a couple of hundreds of nanometers. After treatment with osmium tetroxide (OsO4), the nanogroove can serve as a scaffold to efficiently induce directed cell growth without causing cytotoxicity, and it can be used repeatedly. Together, various cell types are applied to the nanogroove, proving the scaffold's broad applicability. Depending on the nanotopography of the LC structures, cells exhibit different morphologies and gene expression patterns, compared to cells on standard glass substrates, according to microscopic observation and qPCR. Furthermore, cell sheets can be formed, which consist of oriented cells that can be repeatedly formed and transferred to other substrates, while maintaining its organization. We believe that our cell-aligning scaffold may pave the way for the soft material field to bioengineering, which can involve fundamentals in cell behavior and function, as well as applications for regenerative medicine.

Enhancing Resistant Starch Content of High Amylose Rice Starch through Heat-Moisture Treatment for Industrial Application.

The objective of the present study is to enhance the resistant starch (RS) content of high amylose rice starch with heat-moisture treatment (HMT) for industrial application. The optimized HMT condition for achieving the highest RS content established using response surface methodology (RSM) was a temperature of 100 °C, moisture content of 24.2%, and a time of 11.5 h. Upon HMT, the RS content increased from 32.1% for native starch to 46.4% in HMT starch with optimized condition. HMT of the starches reduced the solubility and swelling power. The surface of HMT starch granules was more irregular than native starch. The X-ray diffraction (XRD) peak intensity at 2θ = 5° was greatly reduced by HMT, and the peaks at 22.7° and 24.2° were merged. HMT increased the gelatinization temperature and reduced the gelatinization enthalpy. HMT provides a method for the production of high-yield RS2 with high amylose rice starch in industrial application.

Analysis of Targeted Metabolites and Molecular Structure of Starch to Understand the Effect of Glutinous Rice Paste on Kimchi Fermentation.

Bachu (Chinese cabbage) kimchi, a Korean traditional fermented dish, were prepared with or without the addition of glutinous (waxy) rice paste and their characteristics including pH, total bacteria count, total starch content, sugar metabolites, and molecular structure of starch were examined periodically for 20 days to investigate the effect of adding glutinous rice paste to kimchi during fermentation. The pH and total bacteria count showed that the fermentation of kimchi added with glutinous rice paste (GRP kimchi) progressed a little more quickly than that of control kimchi without glutinous rice paste. The GRP kimchi had higher glucose content but lower fructose content than control kimchi. Interestingly, maltose was only detected in GRP kimchi during fermentation. The GRP kimchi contained much greater amount of mannitol throughout fermentation than control kimchi. Total starch content in GRP kimchi gradually decreased during fermentation, which might have contributed to its greater glucose content and the larger amount of maltose production. In GRP kimchi, peak height and area for all degrees of polymerization (DP) of starch decreased during fermentation and its average chain length decreased while the proportion of short chains increased as fermentation processed, indicating degradation of starch chains by enzymes presented in the kimchi.

Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

Childhood immunizations in China: disparities in health care access in children born to North Korean refugees.

BACKGROUND: Childhood immunization rates are at an all-time high globally, and national data for China suggests close to universal coverage. Refugees from North Korea and their children may have more limited health care access in China due to their legal status. However, there is no data on immunization rates or barriers to coverage in this population. METHODS: This study was conducted to determine the rates and correlates of immunizations in children (≥1 year) born to North Korean refugees in Yanbien, China. Child immunization data was obtained from vaccination cards and caregiver self-report for 7 vaccines and 1:3:3:3:1 series. Age-appropriate vaccination rates of refugee children were compared to Chinese and migrant children using a goodness-of-fit test. Logistic regression was used to determine correlates of immunization coverage for each vaccine and the 1:3:3:3:1 series. RESULTS: Age-appropriate immunization coverage rates were significantly lower in children born to North Korean refugees (12.1-97.8 %), compared to Chinese (99 %) and migrant (95 %) children. Increased father's age and having a sibling predicted significantly lower vaccination rates. CONCLUSIONS: Children born to North Korean refugees had significantly lower immunization rates, compared to Chinese or migrant children. Further research is needed to examine barriers of health care access in this high-risk population.

Impact of postoperative hepatitis B virus reactivation in hepatocellular carcinoma patients who formerly had naturally suppressed virus.

BACKGROUND AND AIM: Hepatitis B virus (HBV) replication detected before the resection of hepatocellular carcinoma (HCC) is to be controlled by antiviral agents. However, management strategy for patients with preoperatively undetectable HBV DNA without antiviral therapy is not clearly delineated. This study investigated viral reactivation after the liver resection in non-replicating HBV DNA-related HCC patients and its impact on the surgical outcome. METHODS: From 198 patients that underwent liver resection due to HBV-related HCC, 101 patients who had serially checked serum HBV DNA were analyzed. RESULTS: From 101 patients, 33 patients had baseline undetectable HBV DNA. Eleven patients (11/33, 33.3%) had viral replication after the liver resection. The postoperative viral reactivation (HR: 2.144; 95% CI: 1.122-4.097; P = 0.021), along with the existence of satellite nodules (HR: 3.034; 95% CI: 1.1.376-6.689; P = 0.006), existence of microvascular invasion (HR: 2.479; 95% CI: 1.303-4.718; P = 0.006), and HBeAg positivity (HR: 2.059; 95% CI: 1.155-3.670; P = 0.014) predicted recurrence after the surgery. Quantification of intrahepatic total and covalently closed circular DNA (cccDNA) was done in 14 patients whose baseline serum HBV DNA was undetectable without the use of antiviral agent. Amount of intrahepatic cccDNA expressed as copies/hepatocyte in patients with postoperative viral reactivation showed significantly higher than those in patients with sustained negative serum HBV DNA (P = 0.010). CONCLUSIONS: This study shows that naturally suppressed preoperative HBV without application of antiviral agent does not ensure undetectable serum HBV after the surgery, and postoperative viral reactivation might be associated with HCC recurrence.

Transarterial chemoembolization versus surgery/radiofrequency ablation for recurrent hepatocellular carcinoma with or without microvascular invasion.

BACKGROUND/AIMS: Microvascular invasion (MVI) is a well-known prognostic factor of postoperative recurrence and of overall survival (OS) in patients with hepatocellular carcinoma (HCC). We compared the treatment outcomes of transarterial chemoembolization (TACE) and surgery/radiofrequency ablation (RFA) according to the presence of MVI in patients with early or late recurrent HCC that presented as Barcelona Clinical Liver Cancer (BCLC) stage 0 or A after curative resection for HCC. METHODS: A consecutive 68 patients with recurrent HCC of BCLC stage 0 or A at our institution between 1998 and 2012 were retrospectively enrolled. We compared the outcomes of patients treated by TACE or surgery/RFA. Tumor recurrence after curative resection was classified as early (≤ 12 months) or late (> 12 months) recurrence. RESULTS: Median tumor size was 1.5 cm (range, 1-10 cm), and 67 (98.5%) had HCCs within the Milan criteria. Median post-retreatment follow-up duration was 27 months (range, 1-109 months). Of the 68 patients, 19 (27.9%) underwent surgery/RFA, 47 (69.1%) TACE, and 2 (2.9%) were lost to follow-up. After retreatment, TACE showed significantly higher OS and recurrence-free survival rates than surgery/RFA in MVI-positive patients (P = 0.03 and P = 0.05, respectively), but not in MVI-negative patients (P = 0.95 and P = 0.98, respectively). In particular, in early recurred MVI-positive patients, TACE had a significantly higher OS rate than surgery/RFA (P = 0.01). CONCLUSIONS: TACE may be the more effective treatment option for recurrent HCC of BCLC stage 0 or A than surgery/RFA in MVI-positive patients, especially in those that recur early after curative resection.

Relation between textural attributes and surface leachate structural and compositional characteristics of cooked rice.

The objective of this study was to evaluate the leachate and textural characteristics of cooked rice, and the correlations between the leachate properties and texture attributes were also investigated. Cooked waxy rice had much higher total solids and amylopectin amount in leachate than the normal and high-amylose rice. For all varieties, the amylopectin chain length of the leachate was similar, excluding Dodam cultivar. The rheological characteristics of the leachate solutions were highly dependent on the amylopectin amount of the leachate. Regarding the textural characteristics, Dodam had the highest hardness and the lowest adhesiveness. The principal component analysis showed substantial differences in leachate and textural characteristics of Korean cooked rice according to its amylose content. The adhesiveness was positively and negatively correlated with amylopectin amount of leachate and the proportion of long amylopectin chains, respectively. These results indicated that the leachate characteristics of cooked rice significantly influenced its textural attributes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01446-3.

Comparative analysis of malt quality and starch characteristics of three South Korean barley cultivars.

In this study, malt was produced in pilot-scale facilities and conditioned using three barley (Hordeum vulgare L.) cultivars in South Korea (Heugho, Hopum, and Kwangmaeg). Quality and starch characteristics were compared. The starch content was considerably reduced in all malts. Coleoptile elongation was higher in Heugho (HHM; 85.7% ± 12.6%) and Hopum (HPM; 83.9% ± 10.7%) than in Kwangmaeg (KMM; 78.1% ± 9.9%) malt. Malt yield ranged from 81.8 to 84.9%, with no significant difference. All samples presented type A crystallinity, and granules showed discoid shapes. After malting, the mono- and di-saccharide contents (not including sucrose) were increased. The fermentable sugar level was the highest in HHM, whereas non-fermentable sugar was the highest in KMM. These results suggest that HPM enables efficient scarification based on the rapid degradation of starch, while Heugho barley and HHM have a high potential for beer and malt production, respectively. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01419-6.

Potential Migration and Health Risks of Heavy Metals and Metalloids in Take-Out Food Containers in South Korea.

The consumption of take-out food has increased worldwide; consequently, people are increasingly being exposed to chemicals from food containers. However, research on the migration of metals from containers to food is limited, and therefore, information required to determine the health risks is lacking. Herein, the amount of transfer of nine metals and metalloids (Pb, Sb, Cd, Ge, Co, Mn, Sn, As, and Hg) from food containers to food in South Korea was assessed from take-out food containers classified into paper and plastic container groups. The sample containers were eluted over time by either warming with 4% acetic acid at 70 °C or cooling with 4% acetic acid at 100 °C /deionized water at 25 °C. It was analyzed using an inductively coupled plasma mass spectrometer and a direct mercury analyzer. The reliability of the quantitative results was verified by calculating the linearity, limit of detection, and limit of quantification. We found that the amount of metals and metalloids (Pb, Sb, Cd, and Co) eluting over time was highly significant in the plastic group. Regardless of the food simulant and elution time, the amount of Sb transferred from the food containers to food was substantially higher in the plastic (average concentration: 0.488-1.194 µg/L) than in the paper group (average concentration: 0.001-0.03 µg/L). Fortunately, all food containers were distributed at levels safe for human health (hazard index: 0.000-64.756%). However, caution is needed when warm food is added to food containers. Overall, our results provide baseline data for the management and use of take-out containers.

A Wireless, CRISPR-Polymer Dot Electrochemical Sensor for the Diagnosis of Bacterial Pneumonia and Multi-Drug Resistance.

Rapid and accurate diagnosis is crucial for managing the global health threat posed by multidrug-resistant bacterial infections; however, current methods have limitations in either being time-consuming, labor-intensive, or requiring instruments with high costs. Addressing these challenges, we introduce a wireless electrochemical sensor integrating the CRISPR/Cas system with electroconductive polymer dot (PD) nanoparticles to rapidly detect bacterial pathogens from human sputum. To enhance the electroconductive properties, we synthesized copper-ion-immobilized PD (PD-Cu), followed by conjugation of the deactivated Cas9 protein (dCas9) onto PD-Cu-coated Si electrodes to generate the dCas9-PD-Cu sensor. The dCas9-PD-Cu sensor integrated with isothermal amplification can specifically detect target nucleic acids of multidrug-resistant bacteria, such as the antibiotic resistance genes kpc-2 and mecA. The dCas9-PD-Cu sensor exhibits high sensitivity, allowing for the detection of ∼54 femtograms of target nucleic acids, based on measuring the changes in resistivity of the Si electrodes through target capture by dCas9. Furthermore, a wireless sensing platform of the dCas9-PD-Cu sensor was established using a Bluetooth module and a microcontroller unit for detection using a smartphone. We demonstrate the feasibility of the platform in diagnosing multidrug-resistant bacterial pneumonia in patients' sputum samples, achieving 92% accuracy. The current study presents a versatile biosensor platform that can overcome the limitations of conventional diagnostics in the clinic.

An Antibody-CRISPR/Cas Conjugate Platform for Target-Specific Delivery and Gene Editing in Cancer.

The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody-CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2-positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody-conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2-positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody-CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2-positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody-mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases.

Live-Cell Imaging of MicroRNA Expression via Photoinduced Electron Transfer Controlled by Catalytic Hairpin Assembly.

MicroRNAs (miRNAs) serve as emerging biomarkers for a range of diseases, and their quantitative analysis draws increasing attention. Yet, current invasive methods limit continuous tracking within living cells. To overcome this, a nonenzymatic DNA-based nanoprobe is developed for dynamic, noninvasive miRNA tracking via live-cell imaging. This probe features a unique hairpin DNA structure with five guanines that act as internal quenchers, suppressing fluorescence from an attached fluorophore via photoinduced electron transfer. Target miRNA initiates toehold-mediated strand displacement, restoring, and amplifying the fluorescence signal. Additionally, by introducing a single mismatch to the hairpin DNA, the nanoprobe's sensitivity is significantly enhanced, lowering the detection limit to about 60 pM without compromising specificity. To optimize intracellular delivery for prolonged monitoring, the nanoprobe is encapsulated within multilamellar lipid nanovesicles, fluorescently labeled for dual-wavelength ratiometric analysis. The proposed nanoprobe demonstrates a significant advance in live-cell miRNA detection, promising enhanced in situ analysis for a better understanding of miRNAs' pathophysiological function.

Prediction of sustained virologic response based on week 4 and week 12 response in hepatitis C virus genotype 1 patients treated with peginterferon and ribavirin: assessment in a favorable IL28B allele-prevalent area.

OBJECTIVES: The aim of this study was to evaluate rapid virologic response (RVR) rate after peginterferon (PegIFN) and ribavirin (RBV) dual combination therapy in Korean hepatitis C virus (HCV) genotype 1 patients whose IL28B polymorphism is generally favorable. This study also assessed the value of RVR in predicting sustained virologic response (SVR). METHODS: Treatment-naïve HCV genotype 1 patients who underwent initial treatment with either PegIFN-α-2a or α-2b and RBV were retrospectively evaluated. From 148 patients, 115 met inclusion criteria for the final analysis. RESULTS: Overall RVR rate was 47.8% and SVR rate was 67.8% (78/115). Positive RVR had the highest positive predictive value (PPV) for achieving SVR, whereas it had the lowest negative predictive value (NPV). Undetectable HCV RNA at treatment week 12, namely complete early virologic response (cEVR), had high PPV as well as high NPV. Factors predisposing SVR were absence of liver cirrhosis and achievement of RVR or cEVR. CONCLUSION: This study showed RVR rate close to 50% in HCV genotype 1 patients treated with dual combination therapy in the region where favorable IL28B polymorphism is reported to be as high as 90%. Even for the patients who failed to achieve RVR, positive cEVR demonstrated a fair chance of achieving SVR.

Starch Structure of Raw Materials with Different Amylose Contents and the Brewing Quality Characteristics of Korean Rice Beer.

This study aimed to explore suitable processing materials for rice beer (RB) production by analyzing the starch structure of the raw materials utilized for brewing beer and the quality characteristics of RB. We used malt, employing the Heugho cultivar as the main ingredient, and produced beer containing 30% rice. The regular amylose-containing cultivars Samgwang (SA) and Hangaru (HA) and the high-amylose-containing cultivar Dodamssal (DO) were used as adjuncts. Distribution of the short molecular chains of the starch amylopectin was the highest for SA and malt at 29.3% and 27.1%, respectively. Glucose content was the highest in the wort prepared with 100% malt and 30% SA + 70% malt. The alcohol content in SA RB and HA RB was higher than that in beer prepared with 100% malt. DO RB had the least bitterness and volatile components, such as acetaldehyde and ethyl acetate. The three rice cultivars tested in this study are suitable as starch adjuncts for RB production. The characteristics of RBs varied depending on the molecular structure of the ingredients, irrespective of their amylose contents. SA could be considered a craft beer with quality characteristics and rich flavor components, similar to 100% malt beer, compared to other RBs.