Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient ...genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.
The modern day drug delivery technology is only 60years old. During this period numerous drug delivery systems have been developed. The first generation (1950–1980) has been very productive in ...developing many oral and transdermal controlled release formulations for clinical applications. On the other hand, the second generation (1980–2010) has not been as successful in generating clinical products. This is in large part due to the nature of the problems to overcome. The first generation of drug delivery technologies dealt with physicochemical problems, while the second struggled with biological barriers. Controlled drug delivery systems can be made with controllable physicochemical properties, but they cannot overcome the biological barriers. The third generation (from 2010) drug delivery systems need to overcome both physicochemical and biological barriers. The physicochemical problems stem from poor water solubility of drugs, large molecular weight of peptide and protein drugs, and difficulty of controlling drug release kinetics. The biological barriers to overcome include distribution of drug delivery systems by the body rather than by formulation properties, limiting delivery to a specific target in the body. In addition, the body's reaction to formulations limits their functions in vivo. The prosperous future of drug delivery systems depends on whether new delivery systems can overcome limits set by human physiology, and the development process can be accelerated with new ways of thinking.
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Remaining useful life (RUL) prediction of lithium-ion batteries can reduce the risk of battery failure by predicting the end of life. In this paper, we propose novel RUL prediction techniques based ...on long short-term memory (LSTM). To estimate RUL even in the presence of capacity regeneration phenomenon, we consider multiple measurable data from battery management system such as voltage, current and temperature charging profiles whose patterns vary as aging. Unlike the traditional LSTM prediction that matches input layer with output layer as one-to-one structure, we leverage many-to-one structure to be flexible for various input types and to substantially reduce the number of parameters for better generalization. Using the NASA lithium-ion battery datasets, we verify the accuracy of the proposed LSTM-based RUL prediction. The experimental results show that the proposed single-channel LSTM model improves the mean absolute percentage error (MAPE) by 39.2% compared to the baseline LSTM model. Furthermore, the proposed multi-channel LSTM model significantly improves the MAPE, e.g., by 63.7% compared to the baseline; the proposed model achieves 0.47-1.88% of MAPE while the state-of-the-art baseline LSTM shows 0.6-6.45% of MAPE.
Medium cut-off (MCO) dialyzers help remove larger middle molecules associated with symptoms related to the accumulation of uremic retention solutes. We investigated the effect of an MCO dialyzer on ...the improvement of quality of life (QOL) in maintenance hemodialysis (HD) patients. Forty-nine HD patients with high-flux dialysis were randomly assigned to either an MCO (Theranova 400, Baxter) or a high-flux (FX CorDiax 80 or 60, Fresenius Medical Care) dialyzer and completed the study. QOL was assessed at baseline and after 12 weeks of treatment using the Kidney Disease Quality of Life Short Form-36, and pruritus was assessed using a questionnaire and visual analog scale. The reduction ratios of middle molecules were also evaluated. Laboratory markers, including serum albumin, did not differ between the two groups after 12 weeks. Removals of kappa and lambda free light chains were greater for MCO dialyzer than high-flux dialyzer. The MCO group had higher scores than the high-flux group in the domains of physical functioning and physical role (75.2 ± 20.8 vs. 59.8 ± 30.1, P = 0.042; 61.5 ± 37.6 vs. 39.0 ± 39.6, P = 0.047, respectively), and the MCO group had lower mean scores for morning pruritus distribution and the frequency of scratching during sleep (1.29 ± 0.46 vs. 1.64 ± 0.64, P = 0.034; 0.25 ± 0.53 vs. 1.00 ± 1.47, P = 0.023, respectively). MCO dialyzers may improve patient-reported outcomes, particularly the physical components of QOL and uremic pruritus, in patients with high-flux dialyzers.
Obesity can cause chronic metabolic disorders such as type 2 diabetes, hyperlipidemia, and nonalcoholic fatty liver diseases. The aim of this study was to investigate the antiobesity and antidiabetic ...effects of the dairy bacterium P. freudenreichii MJ2 isolated from raw milk using 3T3-L1 cells and high-fat diet (HFD)-induced obese mice. Lipid accumulation and the expression levels of genes related to lipid metabolism, such as preadipocytic gene (Pref-1), adipogenic genes (PPARγ and C/EBPα), and lipogenic genes (FAS, SCD-1, and ACC), significantly decreased in heat-killed P. freudenreichii MJ2 (hkMJ2)-treated adipocytes. Live P. freudenreichii MJ2 (MJ2), hkMJ2, and Lactobacillus plantarum (LP) decreased body weight gain in HFD-induced obese mice compared with the model group. The liver and epididymal white adipose tissue weights in the MJ2-, hkMJ2- and LP-treated groups were significantly lower than those in the model group. The expression levels of genes and proteins related to adipogenesis and lipogenesis significantly decreased and lipolysis (HSL and ATGL) increased in the MJ2-, hkMJ2-, and LP-treated groups. The expression levels of genes related to fatty acid β-oxidation (CPT-1α and ACOX1) increased in the MJ2-, hkMJ2-, and LP-treated groups. In addition, blood glucose and fasting insulin levels in the MJ2- and hkMJ2-treated groups decreased compared with those in the model group. P. freudenreichii MJ2 ameliorate insulin resistance by obesity. In conclusion, both MJ2 and hkMJ2 alleviate obesity and metabolic syndrome.
Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic ...agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.
Recent evidence suggests that gut microbiota dysbiosis adversely affects the efficacy of immune checkpoint inhibitors (ICIs). Our objective was to investigate the association between concomitant use ...of proton pump inhibitors (PPIs) and ICIs, and poor prognosis in patients with nonsmall cell lung cancer (NSCLC). We conducted a cohort study using a completely enumerated lung cancer cohort from a nationwide healthcare database in South Korea. We identified 2963 patients treated with ICIs as second‐line or later therapy for stage ≥IIIB NSCLC. PPI use was ascertained within 30‐days before and on the date of ICI initiation, and nonuse was defined as no prescription of PPIs during this period. Using national vital statistics in South Korea, we assessed the risk of all‐cause mortality associated with concomitant PPI use through a propensity score‐matched Cox proportional hazard model. Among 1646 patients included after 1:1 propensity score‐matching, concomitant PPI use was associated with a 28% increased risk of all‐cause mortality, compared to nonuse (adjusted hazard ratio HR 1.28; 95% confidence intervals CIs, 1.13‐1.46). We observed an increased risk when we restricted the analysis to new users of PPI (adjusted HR = 1.64; 95% CI = 1.25‐2.17). Subgroup analysis showed that PPI use was associated with high mortality risk among patients with viral hepatitis (adjusted HR = 2.72; 95% CI = 1.54‐4.78; Pinteraction = .048). Our study indicates that PPI use is associated with poor prognosis in NSCLC patients treated with ICIs. Further prospective studies are required to determine the risk‐benefit balance of concomitant use of PPIs and ICIs.
What's new?
The efficacy of immune checkpoint inhibitors (ICIs) in the treatment of non‐small cell lung cancer (NSCLC) is potentially affected by imbalances in the human gut microbiota. Such imbalances may be induced by proton pump inhibitors (PPIs), though whether concomitant PPI and ICI use affects ICI efficacy remains unknown. Here, the authors investigated associations between concomitant PPI and ICI therapy and poor prognosis among NSCLC patients in South Korea. Propensity score‐matched cohort study shows that PPI use is associated with elevated risk of early mortality in ICI‐treated NSCLC patients. Increased risk was most evident among patients with viral hepatitis.
Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).
Tobacco and alcohol may interact to increase the risk of liver cancer, which might be modified by other risk factors. Their combined effects in the context of metabolic syndrome (MetS) remain ...unclear. Given the increasing prevalence of MetS, this nested case–control study was conducted to evaluate the combined effects of smoking and alcohol consumption on liver cancer risk with stratification by MetS. We included 15,352 liver cancer patients and 92,112 matched controls who attended the nationwide general health examination during 2009–2019, using a customized database (N = 5,545,835) from the Korean National Health Insurance Service. Liver cancer risk according to smoking and alcohol consumption was estimated using conditional multivariable logistic regression. Additive and multiplicative interactions between these two factors were assessed. Results showed that in men, dual current users were at a significantly higher risk of liver cancer compared with dual nonusers, adjusted odds ratio (aOR) = 1.61, 95% confidence interval: (1.50, 1.72). Interactions were detected between light‐to‐moderate alcohol consumption (0.1–28 g/day) and heavy smoking (>20 pack‐years) on additive scale, relative excess risk due to interaction = 0.34 (0.16, 0.51), attributable proportion = 0.22 (0.11, 0.33), synergy index = 2.75 (1.85, 3.66), and multiplicative scale, aOR for the product term = 1.28 (1.11, 1.49). An additive interaction was also revealed between light‐to‐moderate drinking and light‐to‐moderate smoking in the MetS subgroup. In women, light‐to‐moderate drinking/nonsmoking was negatively associated with the risk in the non‐MetS subgroup. In conclusion, a holistic health promotion program should target male dual users of tobacco cigarettes and alcohol, including light‐to‐moderate users, especially those with MetS.
What's new?
Tobacco smoking and alcohol consumption may interact to increase liver cancer risk, and their combined effects are potentially modified by factors such as metabolic syndrome (MetS). Here, in a Korean cohort, combined effects of tobacco and alcohol use on liver cancer risk were evaluated according to MetS status. Analyses uncovered excess liver cancer risk for men across all levels of combined alcohol and tobacco use. Synergistic effects were observed between light‐to‐moderate drinking and heavy smoking across strata of MetS and between light‐to‐moderate drinking and light‐to‐moderate smoking in the MetS subgroup among men. The data indicates that dual tobacco and alcohol users, namely men with MetS, even with light‐to‐moderate consumption, are at substantially increased risk of liver malignancy.
Background and Purpose
Paracetamol (acetaminophen)‐induced hepatotoxicity is the leading cause of drug‐induced liver injury worldwide. Autophagy is a degradative process by which various cargoes are ...collected by the autophagic receptors such as p62/SQSTM1/Sequestosome‐1 for lysosomal degradation. Here, we investigated the protective role of p62‐dependent autophagy in paracetamol‐induced liver injury.
Experimental Approach
Paracetamol‐induced hepatotoxicity was induced by a single i.p. injection of paracetamol (500 mg·kg−1) in C57/BL6 male mice. YTK‐2205 (20 mg·kg−1), a p62 agonist targeting ZZ domain, was co‐ or post‐administered with paracetamol. Western blotting and immunocytochemistry were performed to explore the mechanism.
Key Results
N‐terminal arginylation of the molecular chaperone calreticulin retro‐translocated from the endoplasmic reticulum (ER) was induced in the livers undergoing paracetamol‐induced hepatotoxicity, and YTK‐2205 exhibited notable therapeutic efficacy in acute hepatotoxicity as assessed by the levels of serum alanine aminotransferase and hepatic necrosis. This efficacy was significantly attributed to accelerated degradation of ubiquitin (Ub) conjugates as well as damaged mitochondria (mitophagy) and endoplasmic reticulum (ER‐phagy). In primary murine hepatocytes treated with paracetamol, YTK‐2205 induced the co‐localization of p62+LC3+ phagophores to the sites of mitophagy and ER‐phagy. A similar activity of YTK‐2205 was observed with N‐acetyl‐p‐benzoquinone imine, a putative toxic metabolite of paracetamol in Hep3B cells.
Conclusion and Implications
Our results elucidated that p62‐dependent autophagy plays a key role in the removal of cytotoxic materials such as damaged mitochondria in paracetamol‐induced hepatotoxicity. Small molecule ligands to p62 may be developed into drugs to treat this pathological condition.
Background
Null phenotypes are characterized by complete absence of all antigens within a blood group system and caused by null variants (e.g., nonsense, frameshift, initiation codon, and canonical ...splice site variants) in the genes encoding the antigens. Knowing the prevalence and molecular basis of null phenotypes is essential to establish a rare donor program, and the aim of this study was to reveal the prevalence and molecular basis of null phenotypes using the Korean Reference Genome Database (KRGDB) containing whole‐genome sequences of 1722 Korean individuals.
Study Design and Methods
Population allele frequencies of null alleles in 39 blood group systems except ABO, MNS, Rh, Lewis, and FORS were obtained from the KRGDB. The prevalence of null phenotypes was calculated using Hardy–Weinberg equation.
Results
The prevalence of null phenotypes were estimated to be less than 0.001% in all blood group systems except JR and SID. The prevalence of the Jr(a−) and Sd(a−) phenotypes were estimated to be 0.0453% and 0.2323%, respectively. The most frequent null allele of the JR system was ABCG2*01N.01, accounting for approximately 85% of null alleles.
Discussion
Our approach using a public database allowed us to investigate the prevalence and molecular basis of null phenotypes in the Korean population, which will serve as a guide for establishing a rare donor program in Korea. Considering the clinical significance, Jr(a−) is an important null phenotype that should be typed in the Korean population, and molecular assays targeting the most frequent allele ABCG2*01N.01 may be useful in detecting this phenotype.
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