Simple <i>in Vivo</i> Gene Editing <i>via</i> Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment

Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for <i>in vivo</i> applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing <i>in vivo</i>. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) <i>via in vitro</i> and <i>in vivo</i> editing of the <i>KRAS</i> gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly­(l-lactide-<i>co</i>-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5–7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell–cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types

Data_Sheet_1_Risk of newly diagnosed interstitial lung disease after COVID-19 and impact of vaccination: a nationwide population-based cohort study.docx

ObjectivesPrevious studies suggested that coronavirus disease 2019 (COVID-19) could lead to pulmonary fibrosis, but the incidence of newly diagnosed interstitial lung disease (ILD) after COVID-19 is unclear. We aimed to determine whether COVID-19 increases the risk of newly diagnosed ILD and whether vaccination against COVID-19 can reduce this risk.MethodsThis retrospective cohort study used data from the Korean National Health Insurance claim-based database. Two study groups and propensity score (PS)-matched control groups were constructed: Study 1: participants diagnosed with COVID-19 (COVID-19 cohort) and their PS-matched controls; Study 2: COVID-19 vaccinated participants (vaccination cohort) and their PS-matched controls.ResultsIn Study 1, during a median 6 months of follow-up, 0.50% of the COVID-19 cohort (300/60,518) and 0.04% of controls (27/60,518) developed newly diagnosed ILD, with an incidence of 9.76 and 0.88 per 1,000 person-years, respectively. The COVID-19 cohort had a higher risk of ILD [adjusted hazard ratio (aHR), 11.01; 95% confidence interval (CI), 7.42–16.32] than controls. In Study 2, the vaccination cohort had a lower risk of newly diagnosed ILD than controls (aHR, 0.44; 95% CI, 0.34–0.57).ConclusionUsing nationwide data, we demonstrated that COVID-19 was associated with a higher incidence rate of newly diagnosed ILD, but that this risk could be mitigated by COVID-19 vaccination.</p

Table_2_Impact of air pollution on healthcare utilization in patients with bronchiectasis.DOCX

IntroductionAir pollutants are increasingly recognized to affect long-term outcomes in patients with bronchiectasis. We aimed to figure out the association between air pollutants and the risk of healthcare utilization in patients with bronchiectasis.MethodsData for 1,029 subjects with bronchiectasis in Seoul were extracted. The air pollutants included particulate matter of 10 μm or less in diameter (PM10), particulate matter of 2.5 μm or less in diameter (PM2.5), sulfur dioxide (SO2), carbon monoxide (CO), ozone (O3), and nitrogen dioxide (NO2). The outcome was all-cause healthcare uses, defined as outpatient visit, emergency department visit, or hospitalization. The concentration–response curves between each air pollutant and relative risks for healthcare utilization were obtained.ResultsThere were significant correlations between air pollutant concentrations and the risk of healthcare utilization, particularly for PM10, NO2, SO2, and CO. This risk was observed even at concentrations below the recommended safe thresholds for the general population. The slopes for the association between PM10 and NO2 and the risk of healthcare use showed a logarithmic growth pattern, with the steepest increase up to 30 μg/m3 and 0.030 parts per million (ppm), respectively. The curves for SO2 and CO showed an inverted U-shaped pattern, with a peak at 0.0045 ppm and a slow upward curve, respectively. No specific trends were observed for PM2.5 and O3 and the risk of healthcare use.DiscussionIncreased concentrations of PM10, NO2, SO2, and CO were associated with increased healthcare utilization in patients with bronchiectasis. For patients with bronchiectasis, there were no safety thresholds for those air pollutants, and even low levels of air pollutant exposure can negatively impact bronchiectasis outcomes.</p

Table_1_Impact of air pollution on healthcare utilization in patients with bronchiectasis.DOCX

IntroductionAir pollutants are increasingly recognized to affect long-term outcomes in patients with bronchiectasis. We aimed to figure out the association between air pollutants and the risk of healthcare utilization in patients with bronchiectasis.MethodsData for 1,029 subjects with bronchiectasis in Seoul were extracted. The air pollutants included particulate matter of 10 μm or less in diameter (PM10), particulate matter of 2.5 μm or less in diameter (PM2.5), sulfur dioxide (SO2), carbon monoxide (CO), ozone (O3), and nitrogen dioxide (NO2). The outcome was all-cause healthcare uses, defined as outpatient visit, emergency department visit, or hospitalization. The concentration–response curves between each air pollutant and relative risks for healthcare utilization were obtained.ResultsThere were significant correlations between air pollutant concentrations and the risk of healthcare utilization, particularly for PM10, NO2, SO2, and CO. This risk was observed even at concentrations below the recommended safe thresholds for the general population. The slopes for the association between PM10 and NO2 and the risk of healthcare use showed a logarithmic growth pattern, with the steepest increase up to 30 μg/m3 and 0.030 parts per million (ppm), respectively. The curves for SO2 and CO showed an inverted U-shaped pattern, with a peak at 0.0045 ppm and a slow upward curve, respectively. No specific trends were observed for PM2.5 and O3 and the risk of healthcare use.DiscussionIncreased concentrations of PM10, NO2, SO2, and CO were associated with increased healthcare utilization in patients with bronchiectasis. For patients with bronchiectasis, there were no safety thresholds for those air pollutants, and even low levels of air pollutant exposure can negatively impact bronchiectasis outcomes.</p

Additional file 1 of In vivo safety and biodistribution profile of Klotho-enhanced human urine-derived stem cells for clinical application

Additional file 1. Table S1: Antibodies for flow cytometry, western blotting, and immunofluorescence analysis