Small extracellular vesicle-mediated CRISPR-Cas9 RNP delivery for cardiac-specific genome editing.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Although clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing holds immense potential for genetic manipulation, its clinical application is hindered by the absence of an efficient heart-targeted drug delivery system. Herein, we developed CRISPR-Cas9 ribonucleoprotein (RNP)-loaded extracellular vesicles (EVs) conjugated with cardiac-targeting peptide (T) for precise cardiac-specific genome editing. RNP complexes containing Cas9 and single guide RNA targeting miR-34a, an MI-associated molecular target, were loaded into EVs (EV@RNP). Gene editing by EV@RNP attenuated hydrogen peroxide-induced apoptosis in cardiomyocytes via miR-34a inhibition, evidenced by increased B-cell lymphoma 2 levels, decreased Bcl-2-associated X protein levels, and the cleavage of caspase-3. Additionally, to improve cardiac targeting in vivo, we used click chemistry to form functional T-EV@RNP by conjugating T peptides to EV@RNP. Consequently, T-EV@RNP-mediated miR-34a genome editing might exert a protective effect against MI, reducing apoptosis, ameliorating MI injury, and facilitating the recovery of cardiac function. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting EVs is a powerful approach for precise and tissue-specific gene therapy for cardiovascular disease.

Inhibitory Effects of Fermented Sprouted Oat Extracts on Oxidative Stress and Melanin Overproduction.

Hyperpigmentation occurs due to irregular secretion of melanin pigment in the skin. This can affect quality of life depending on its severity, so prevention and management are essential. Oats (Avena sativa L.), a grain consumed worldwide, are known to offer improved health benefits upon germination and fermentation. This study is aimed to investigate the protective effects of lactobacilli-fermented sprouted oat extracts on oxidative stress and melanin overproduction in vitro. The anti-melanogenic effect was investigated using melanin content and tyrosinase activity assays in B16F10 cells, as well as a mushroom tyrosinase-based enzyme inhibition assay. The results showed that L. casei-fermented oat extracts were the most effective for reducing melanin formation by reducing the mRNA expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein 2. Furthermore, L. casei fermentation was effective in improving the total phenolic, flavonoid, and avenanthramide A contents of sprouted oat extracts. The results also demonstrated the antioxidant effects of L. casei-fermented sprouted oat extracts in promoting DPPH radical-scavenging activity, superoxide dismutase-like activity, and reduction in reactive oxygen species levels. Overall, the findings indicate that fermented sprouted oat extracts are promising candidates for antioxidant and anti-hyperpigmentation treatments.

Long-Term Health Effects of Work Trajectories Among Middle-Aged and Older Adults: The Mediating Role of Work, Material, and Social Environments.

Using data from 14 waves (2003-2016) of the Korean Labor and Income Panel Study (KLIPS) (N = 1,627 individuals aged 45-64; 22778 observations), in this study, we conducted sequence analysis and a multi-categorical variable mediation analysis (1) to examine to what extent long-term work histories exhibit varying degrees of de-standardization and precariousness using sequence analysis (2) to explore the potential mediating effects of work, material, and social environments in the association between multiple work sequences and self-rated health. We found the coexistence of a relatively stable long-term employment pattern and a high prevalence of precariousness. The health and economic risks of precarious work fall disproportionately on older workers. Future researchers should continue to analyze whether the COVID-19 pandemic will lead to long-term changes in the workforce to improve our understanding of and response to working in later life and its health effects.

Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy.

Small-interfering RNA (siRNA) therapy is considered a powerful therapeutic strategy for treating cardiac hypertrophy, an important risk factor for subsequent cardiac morbidity and mortality. However, the lack of safe and efficient in vivo delivery of siRNAs is a major challenge for broadening its clinical applications. Small extracellular vesicles (sEVs) are a promising delivery system for siRNAs but have limited cell/tissue-specific targeting ability. In this study, a new generation of heart-targeting sEVs (CEVs) has been developed by conjugating cardiac-targeting peptide (CTP) to human peripheral blood-derived sEVs (PB-EVs), using a simple, rapid and scalable method based on bio-orthogonal copper-free click chemistry. The experimental results show that CEVs have typical sEVs properties and excellent heart-targeting ability. Furthermore, to treat cardiac hypertrophy, CEVs are loaded with NADPH Oxidase 4 (NOX4) siRNA (siNOX4). Consequently, CEVs@siNOX4 treatment enhances the in vitro anti-hypertrophic effects by CEVs with siRNA protection and heart-targeting ability. In addition, the intravenous injection of CEVs@siNOX4 into angiotensin II (Ang II)-treated mice significantly improves cardiac function and reduces fibrosis and cardiomyocyte cross-sectional area, with limited side effects. In conclusion, the utilization of CEVs represents an efficient strategy for heart-targeted delivery of therapeutic siRNAs and holds great promise for the treatment of cardiac hypertrophy.

Complete genome sequence of pectin-degrading Flavobacteriaceae bacterium GSB9.

Pectic oligosaccharides, which are considered to be potential prebiotics, may be generated by pectin-degrading enzymes. Here, we report the complete genome sequence of the pectin-degrading marine bacterium, Flavobacteriaceae bacterium GSB9, which was isolated from seawater of South Korea. The complete genome sequence revealed that the chromosome was 3,630,376 bp in size, had a G + C content of 36.6 mol%, and was predicted to encode 3100 protein-coding sequences (CDSs), 40 tRNAs, and six 16S-23S-5S rRNAs. Genome sequence analysis revealed that this strain possesses multiple genes predicted to encode pectin-degrading enzymes. Our analysis may facilitate the future application of this strain against pectin in various industries.

Diabetes severity is strongly associated with the risk of active tuberculosis in people with type 2 diabetes: a nationwide cohort study with a 6-year follow-up.

BACKGROUND: Many have the rising coincidence of diabetes mellitus (DM) and endemic tuberculosis (TB). We evaluated whether the severity of diabetes is associated with an increased risk of active TB infection. METHODS:  Using a nationally representative database from the Korean National Health Insurance System, 2, 489, 718 people with type 2 DM who underwent a regular health checkup during 2009-2012 were followed up until the end of 2018. The diabetes severity score parameters included the number of oral hypoglycemic agents (≥ 3), insulin use, diabetes duration (≥ 5 years), and the presence of chronic kidney disease (CKD) or cardiovascular disease. Each of these characteristics was scored as one point, and their sum (0-5) was used as the diabetes severity score. RESULTS: We identified 21, 231 cases of active TB during a median follow-up of 6.8 years. Each parameter of the diabetes severity score was associated with an increased risk of active TB (all P < 0.001). Insulin use was the most significant factor related to the risk of TB, followed by CKD. The risk of TB increased progressively with increasing diabetes severity score. After adjusting for possible confounding factors, the hazard ratio (95% confidence interval) for TB were 1.23 (1.19-1.27) in participants with one parameter, 1.39 (1.33-1.44) in those with two parameters, 1.65 (1.56-1.73) in those with three parameters, 2.05 (1.88-2.23) in those with four parameters, and 2.62 (2.10-3.27) in those with five parameters compared with participants with no parameters. CONCLUSION: Diabetes severity was strongly associated in a dose-dependent manner with the occurrence of active TB. People with a higher diabetes severity score may be a targeted group for active TB screening.

Agarolytic Pathway in the Newly Isolated Aquimarina sp. Bacterial Strain ERC-38 and Characterization of a Putative ß-agarase.

Marine microbes, particularly Bacteroidetes, are a rich source of enzymes that can degrade diverse marine polysaccharides. Aquimarina sp. ERC-38, which belongs to the Bacteroidetes phylum, was isolated from seawater in South Korea. It showed agar-degrading activity and required an additional carbon source for growth on marine broth 2216. Here, the genome of the strain was sequenced to understand its agar degradation mechanism, and 3615 protein-coding sequences were predicted, which were assigned putative functions according to their annotated functional feature categories. In silico genome analysis revealed that the ERC-38 strain has several carrageenan-degrading enzymes but could not degrade carrageenan because it lacked genes encoding κ-carrageenanase and S1_19A type sulfatase. Moreover, the strain possesses multiple genes predicted to encode enzymes involved in agarose degradation, which are located in a polysaccharide utilization locus. Among the enzymes, Aq1840, which is closest to ZgAgaC within the glycoside hydrolase 16 family, was characterized using a recombinant enzyme expressed in Escherichia coli BL21 (DE3) cells. An enzyme assay revealed that recombinant Aq1840 mainly converts agarose to NA4. Moreover, recombinant Aq1840 could weakly hydrolyze A5 into A3 and NA2. These results showed that Aq1840 is involved in at least the initial agar degradation step prior to the metabolic pathway that uses agarose as a carbon source for growth of the strain. Thus, this enzyme can be applied to development and manufacturing industry for prebiotic and antioxidant food additive. Furthermore, our genome sequence analysis revealed that the strain is a potential resource for research on marine polysaccharide degradation mechanisms and carbon cycling.

Systematic Multiomic Analysis of PKHD1L1 Gene Expression and Its Role as a Predicting Biomarker for Immune Cell Infiltration in Skin Cutaneous Melanoma and Lung Adenocarcinoma.

The identification of genetic factors that regulate the cancer immune microenvironment is important for understanding the mechanism of tumor progression and establishing an effective treatment strategy. Polycystic kidney and hepatic disease 1-like protein 1 (PKHD1L1) is a large transmembrane protein that is highly expressed in immune cells; however, its association with tumor progression remains unclear. Here, we systematically analyzed the clinical relevance of PKHD1L1 in the tumor microenvironment in multiple cancer types using various bioinformatic tools. We found that the PKHD1L1 mRNA expression levels were significantly lower in skin cutaneous melanoma (SKCM) and lung adenocarcinoma (LUAD) than in normal tissues. The decreased expression of PKHD1L1 was significantly associated with unfavorable overall survival (OS) in SKCM and LUAD. Additionally, PKHD1L1 expression was positively correlated with the levels of infiltrating B cells, cluster of differentiation (CD)-8+ T cells, and natural killer (NK) cells, suggesting that the infiltration of immune cells could be associated with a good prognosis due to increased PKHD1L1 expression. Gene ontology (GO) analysis also revealed the relationship between PKHD1L1-co-altered genes and the activation of lymphocytes, including B and T cells. Collectively, this study shows that PKHD1L1 expression is positively correlated with a good prognosis via the induction of immune infiltration, suggesting that PKHD1L1 has potential prognostic value in SKCM and LUAD.

Association of Underlying Comorbidities and Sites of tuberculosis: an analysis using surveillance data.

BACKGROUND: Tuberculosis (TB) is a highly heterogeneous disease that can affect any organ. Extrapulmonary TB (EPTB) is more difficult to diagnose due to various clinical presentations. Depending on the characteristics of the patient, the involved site of TB may vary. However, data on clinical characteristics of EPTB are inconsistent and insufficient. This study aimed to identify the characteristics of patients with pulmonary TB (PTB) and EPTB and describe characteristic differences for each involved site. METHODS: We systemically collected data of TB patients included in the national surveillance system in South Korea from July 2018 to June 2019 and compared the characteristics of patients with EPTB with that of PTB. RESULTS: A total of 7674 patients with a mean age of 60.9 years were included. Among them, 6038 (78.7%) patients were diagnosed with PTB and 1636 (21.3%) with EPTB. In PTB group, the mean age (61.7 ± 18.7 vs. 57.8 ± 19.9) and proportion of male sex (63.3% vs. 50.1%) were higher, but the body mass index was lower (21.2 ± 3.4 vs. 22.7 ± 3.5) than that of the EPTB group. Prevalence of diabetes (20.5% vs. 16.9%) and chronic lung disease (5.1% vs. 2.9%) were higher in PTB group, meanwhile, those of chronic kidney disease (CKD) (2.7% vs. 5.4%) and long-term steroid use (0.4% vs. 1.0%) were higher in EPTB group. Abdominal TB was more prevalent in patients with chronic liver disease (odds ratio [OR]: 2.69, 95% CI: 1.52-4.74), and urogenital TB was more prevalent in patients with CKD (OR: 2.75, 95% CI: 1.08-6.99). CONCLUSIONS: We found that underlying comorbidities were closely associated with the location of TB development, and therefore, the possibility of EPTB should be carefully evaluated while monitoring for underlying disease in TB-endemic areas.

Severity of underweight affects the development of nontuberculous mycobacterial pulmonary disease; a nationwide longitudinal study.

Regarding to known association between underweight and non-tuberculous mycobacterial pulmonary disease (NTM-PD), the underweight was simply categorized as body mass index (BMI) less than 18.5 kg/m2, mainly because of its low prevalence. We aimed to better define the impact of BMI severity on NTM-PD development. We analysed health data from 4,332,529 individuals examined in 2009 and followed up until December 2017 to determine the incidence of NTM-PD. Based on the BMI in kg/m2, the population was categorized into mild (17.00-18.49), moderate (16.00-16.99), and severe underweight (< 16.00) groups. Using Cox proportional-hazards analyses, hazard ratios for NTM-PD were calculated according to the severity of underweight in reference to normal BMI (18.50-22.99). Over a median follow-up of 5.6 ± 1.2 years, 6223 participants developed NTM-PD. Risk of NTM-PD increased significantly with the severity of underweight: multivariate adjusted hazard ratios (95% confidence intervals) for mild, moderate, and severe underweight were 2.34 (2.17-2.52), 3.50 (3.07-3.99), and 4.35 (3.63-5.21), respectively. In subgroup analysis, being younger (< 65 years old) or male exacerbated the effect of severe underweight on the risk of NTM-PD. This study proved that as underweight categories became more severe, the risk of NTM-PD increased proportionally.

Generation of two PITX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

PITX2 is a homeobox gene located in the human 4q25 locus and is commonly associated with atrial fibrillation (AF). Here, we generated two PITX2 knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 genome editing. The edited iPSCs maintained fullpluripotency, normal karyotype and spontaneousdifferentiation capability. This cell line provides a suitable model for investigating the physiopathologyof PITX2 mutation in atrial fibrillation.

The cascade of care for latent tuberculosis infection in congregate settings: A national cohort analysis, Korea, 2017-2018.

Background: In 2017, Korea implemented a nationwide project to screen and treat latent tuberculosis infection (LTBI) in high-risk for transmission public congregate settings. We aimed to assess programme success using a cascade of care framework. Materials and methods: We undertook a cohort study of people from three congregate settings screened between March 2017 and December 2018: (1) first-grade high school students, (2) employees of educational institutions, (3) employees of social welfare facilities. We report percentages of participants with LTBI completing each step in the cascade of care model. Poisson regression models were used to determine factors associated with not visiting clinics, not initiating treatment, and not completing treatment. Results: Among the 96,439 participants who had a positive interferon-gamma release assay result, the percentage visiting clinics for further assessment, to initiate treatment, and who then completed treatment were 50.7, 34.7, and 28.9%, respectively. Compared to those aged 20-34 years, individuals aged < 20 years and aged ≥ 65 years were less likely to visit clinics, though more likely to complete treatment once initiated. Using public health centres rather than private hospitals was associated with people "not initiating treatment" (adjusted risk ratio [aRR], 3.72; 95% confidence interval [CI], 3.95-3.86). Nine-month isoniazid monotherapy therapy was associated with "not completing treatment," compared to 3-month isoniazid and rifampin therapy (aRR, 1.28; 95% CI, 1.16-1.41). Conclusion: Among participants with LTBI from three congregate settings, less than one third completed treatment. Age, treatment centre, and initial regimen were important determinants of losses to care through the cascade.

Clinical characteristics and survival of patients concurrently diagnosed with lung cancer and active pulmonary tuberculosis.

Background: The diagnosis of pulmonary tuberculosis (TB) in patients suspected of lung cancer is difficult because of the similarities in signs, symptoms, and radiologic results. The clinical and radiologic characteristics of the co-occurrence of pulmonary TB and lung cancer have not been fully evaluated. Methods: Patients diagnosed with lung cancer and active pulmonary TB from January 2009 to December 2017 in four hospitals of the Catholic University of Korea were retrospectively reviewed. The clinical characteristics, including the TB diagnosis methods, lung cancer pathology, staging, initial radiographic features, and survival were analyzed and compared to 575 lung cancer patients without active pulmonary TB from the same hospitals. Results: Forty-eight (0.48%) of the 9,936 lung cancer patients had active pulmonary TB confirmed for M. tuberculosis at the time of the initial cancer diagnosis. The majority of the patients (95.9%) had non-small cell lung cancer and the most frequent findings were a mass-like lesion (79.2%) and separate nodules (75%). When compared to lung cancer patients without TB, the body mass index (BMI) was lower (21.4 vs. 23.1, P=0.001) and clinical stages were advanced in the lung cancer patients with TB; T3-4 (70.9% vs. 50.6%, P=0.002), N2-3 (85.2% vs. 55.6%, P<0.001); M1 (65.9% vs. 44.5%, P=0.007). Interestingly, lung cancer with TB was associated with lower mortality [hazard ratio (HR) =0.35, 95% CI: 0.21-0.60]. Conclusions: Rarely diagnosed concurrent active TB in lung cancer patients was associated with lower BMI and advanced clinical stages. Active investigation of and treatment for active pulmonary TB in lung cancer patients might possibly improve patient outcomes.

Generation of three TTN knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

TTN mutations are the common genetic cause for various types of cardiomyopathies (e.g., dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy) and skeletal myopathies. Here, we generated three TTN knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 system. These cell lines, which exhibit normal karyotype, typical morphology and pluripotency, could provide useful platform for investigating the role of TTN in associated disorders.

Comparison of different regimens with or without fluoroquinolone in isoniazid-resistant tuberculosis: A multicenter cohort study.

In 2018, the World Health Organization recommended a 6-month four-drug regimen (rifampicin, ethambutol, pyrazinamide, and levofloxacin) for the treatment of isoniazid-monoresistant tuberculosis. However, the regimen had very low certainty. This cohort study assessed the impact of fluoroquinolone use and initial baseline regimen on treatment effectiveness in isoniazid-monoresistant tuberculosis. This multicenter retrospective cohort study included 318 patients with isoniazid-monoresistant tuberculosis notified between 2011 and 2018 in Korea. Baseline regimens were classified into two groups, namely 6-9-month rifampicin, ethambutol, and pyrazinamide (6-9REZ) and a combination regimen of 2-month rifampicin, ethambutol, pyrazinamide and 7-10-month rifampicin and ethambutol (2REZ/7-10RE). Multivariable logistic regression was performed to assess factors associated with positive treatment outcomes. Of 318 enrolled patients, 234 (73.6%) were treated with the 6-9REZ and 103 (32.4%) with additional fluoroquinolone. In a multivariable logistic regression model comparing the 6-9REZ and 2REZ/7-10RE groups, there was no difference in the odds of positive outcomes (adjusted odds ratio = 1.08, 95% confidence interval = 0.65-1.82). Addition use of fluoroquinolone was not associated with positive treatment outcomes in the whole cohort (adjusted odds ratio = 1.41, 95% confidence interval = 0.87-2.27); however, its additional use was beneficial in the 2REZ/7-10RE subgroup (adjusted odds ratio = 3.58, 95% confidence interval = 1.32-9.75). Both initial baseline regimens, 6-9REZ and 2REZ/7-10RE, were similarly effective. Shortening of the pyrazinamide administration duration with additional fluoroquinolone use could be a safe alternative for patients with potential hepatotoxicity related to pyrazinamide.

Generation of a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell line using CRISPR/Cas9 system.

E192K missense mutation of TPM1 has been found in different types of cardiomyopathies (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, and left ventricular non-compaction), leading to systolic dysfunction, diastolic dysfunction, and/or tachyarrhythmias. Here, we generated a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-based genome editing system. The cells exhibit normal karyotype, typical stem cell morphology, expression of pluripotency markers and differentiation ability into three germ layers. Accordingly, this cell line could provide a useful cell resource for exploring the pathogenic role of TPM1-E192K mutation in different types of cardiomyopathies.

Time delays and risk factors in the management of patients with active pulmonary tuberculosis: nationwide cohort study.

Estimating the time delay and identifying associated factors is essential for effective tuberculosis control. We systemically analysed data obtained from the Korea Tuberculosis Cohort in 2019 by classifying delays as presentation and healthcare delays of pulmonary tuberculosis (PTB). Of 6593 patients with active PTB, presentation and healthcare delays were recorded in 4151 and 5571 patients, respectively. The median presentation delay was 16.0 (5.0-40.0) days. Multivariable logistic regression analysis showed that longer presentation delays were associated with neuropsychiatric disease [adjusted odds ratio (OR) 2.098; 95% confidence interval (CI) 1.639-2.687; p < 0.001] and heavy alcohol intake (adjusted OR 1.505; 95% CI 1.187-1.907; p < 0.001). The median healthcare delay was 5.0 (1.0-14.0) days. A longer healthcare delay was associated with malignancy (adjusted OR 1.351; 95% CI 1.069-1.709; p = 0.012), autoimmune disease (adjusted OR 2.445; 95% CI 1.295-4.617; p = 0.006), and low bacterial burden manifested as an acid-fast bacillus smear-negative and tuberculosis polymerase chain reaction-negative status (adjusted OR 1.316; 95% CI 1.104-1.569; p = 0.002). Active case-finding programmes need to focus on patients with heavy alcoholism or neuropsychiatric diseases. To ensure early PTB detection, healthcare providers must carefully monitor patients with malignancy, autoimmune disease, or a high index of suspicion for PTB.

Cluster analysis categorizes five phenotypes of pulmonary tuberculosis.

Tuberculosis (TB) has a heterogeneous phenotype, which makes it challenging to diagnose. Our study aimed to identify TB phenotypes through cluster analysis and compare their initial symptomatic, microbiological and radiographic characteristics. We systemically collected data of notified TB patients notified in Korea and constructed a prospective, observational cohort database. Cluster analysis was performed using K-means clustering, and the variables to be included were determined by correlation network. A total of 4,370 subjects with pulmonary TB were enrolled in the study. Based on the correlation network, age and body mass index (BMI) were selected for the cluster analysis. Five clusters were identified and characterised as follows: (1) middle-aged overweight male dominance, (2) young-aged relatively female dominance without comorbidities, (3) middle-aged underweight male dominance, (4) overweight elderly with comorbidities and (5) underweight elderly with comorbidities. All clusters had distinct demographic and symptomatic characteristics. Initial microbiologic burdens and radiographic features also varied, including the presence of cavities and bilateral infiltration, which reflect TB-related severity. Cluster analysis of age and BMI identified five phenotypes of pulmonary TB with significant differences at initial clinical presentations. Further studies are necessary to validate our results and to assess their clinical implications.