Mitofusin 1 and 2 overexpression reduces AßO-mediated ER stress and apoptosis in N2a APPswe cells.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, and amyloid beta oligomers (AßO), which are pathological markers of AD, are known to be highly toxic. AßO increase mitochondrial dysfunction, which is accompanied by a decrease in mitochondrial fusion. Although mitofusin (Mfn) 1 and Mfn2 are mitochondrial fusion proteins, Mfn2 is known to regulate endoplasmic reticulum (ER) function, as it is located in the ER. Several studies have shown that AßO exacerbates ER stress, however, the exact mechanism requires further elucidation. In this study, we used mouse neuroblastoma cells stably overexpressing the amyloid precursor protein (APP) with the Swedish mutation (N2a APPswe cells) to investigate the role of Mfn in ER stress. Our results revealed that  amyloid beta (Aß) caused cellular toxicity in N2a APPswe cells, upregulated ER stress-related proteins, and promoted ER expansion. The AßO-mediated ER stress was reduced when Mfn1 and Mfn2 were overexpressed. Moreover, Mfn1 and Mfn2 overexpressed resulted in reduced apoptosis of N2a APPswe cells. In conclusion, our results indicate that both Mfn1 and Mfn2 reduce ER stress and apoptosis. Our data provide a foundation for future studies on the roles of Mfn1 and Mfn2 in the molecular mechanisms underlying AßO-mediated ER stress and the pathogenesis of AD.

TIPRL Regulates Stemness and Survival in Lung Cancer Stem Cells through CaMKK2-CaMK4-CREB Feedback Loop Activation.

Frequent recurrence and metastasis caused by cancer stem cells (CSCs) are major challenges in lung cancer treatment. Therefore, identifying and characterizing specific CSC targets are crucial for the success of prospective targeted therapies. In this study, it is found that upregulated TOR Signaling Pathway Regulator-Like (TIPRL) in lung CSCs causes sustained activation of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) signaling pathway by binding to CaMKK2, thereby maintaining stemness and survival. CaMKK2-mediated activation of CaM kinase 4 (CaMK4) leads to phosphorylation of cAMP response element-binding protein (CREB) at Ser129 and Ser133, which is necessary for its maximum activation and the downstream constitutive expression of its target genes (Bcl2 and HMG20A). TIPRL depletion sensitizes lung CSCs to afatinib-induced cell death and reduces distal metastasis of lung cancer in vivo. It is determined that CREB activates the transcription of TIPRL in lung CSCs. The positive feedback loop consisting of CREB and TIPRL induces the sustained activation of the CaMKK2-CaMK4-CREB axis as a driving force and upregulates the expression of stemness- and survival-related genes, promoting tumorigenesis in patients with lung cancer. Thus, TIPRL and the CaMKK2 signaling axis may be promising targets for overcoming drug resistance and reducing metastasis in lung cancer.

Effect of lipid emulsion on vasoconstriction induced by epinephrine or norepinephrine in isolated rat aorta.

Background: Epinephrine (EPI) or norepinephrine (NOR) is widely used to treat cardiovascular collapse during lipid emulsion (LE) resuscitation for drug toxicity. However, the effect of LE on the vasoconstriction caused by EPI or NOR remains unknown. The purpose of this study was to examine the effect of an LE (Intralipid) on the vasoconstriction caused by EPI and NOR in isolated rat aorta. Methods: The effect of LE on the vasoconstriction caused by EPI or NOR in isolated rat aorta was examined. Additionally, the effect of LE on the calcium increase caused by EPI or NOR was investigated. The distribution constant (KD: lipid to aqueous phase) of EPI or NOR between a LE (1%) and an aqueous phase was determined. Results: LE (1 and 2%) did not significantly alter vasoconstriction caused by EPI or NOR in isolated endothelium-intact aorta. Moreover, the LE did not significantly alter the increased calcium level caused by EPI or NOR. The log KD of EPI in the LE (1%) was -0.71, -0.99, and -1.00 at 20, 50, and 100 mM ionic strength, respectively. The log KD of NOR in the LE (1%) was -1.22, -1.25, and -0.96 at 20, 50, and 100 mM ionic strength, respectively. Conclusions: Taken together, the Intralipid emulsion did not alter vasoconstriction induced by EPI or NOR that seems to be due to the hydrophilicity of EPI or NOR, leading to sustained hemodynamic support produced by EPI or NOR used during LE resuscitation.

Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.

Robust and Efficient Authentication and Group-Proof Scheme Using Physical Unclonable Functions for Wearable Computing.

Wearable computing has garnered a lot of attention due to its various advantages, including automatic recognition and categorization of human actions from sensor data. However, wearable computing environments can be fragile to cyber security attacks since adversaries attempt to block, delete, or intercept the exchanged information via insecure communication channels. In addition to cyber security attacks, wearable sensor devices cannot resist physical threats since they are batched in unattended circumstances. Furthermore, existing schemes are not suited for resource-constrained wearable sensor devices with regard to communication and computational costs and are inefficient regarding the verification of multiple sensor devices simultaneously. Thus, we designed an efficient and robust authentication and group-proof scheme using physical unclonable functions (PUFs) for wearable computing, denoted as AGPS-PUFs, to provide high-security and cost-effective efficiency compared to the previous schemes. We evaluated the security of the AGPS-PUF using a formal security analysis, including the ROR Oracle model and AVISPA. We carried out the testbed experiments using MIRACL on Raspberry PI4 and then presented a comparative analysis of the performance between the AGPS-PUF scheme and the previous schemes. Consequently, the AGPS-PUF offers superior security and efficiency than existing schemes and can be applied to practical wearable computing environments.

Luteolin supplementation during porcine oocyte maturation improves the developmental competence of parthenogenetic activation and cloned embryos.

Luteolin (Lut), a polyphenolic compound that belongs to the flavone subclass of flavonoids, possesses anti-inflammatory, cytoprotective, and antioxidant activities. However, little is known regarding its role in mammalian oocyte maturation. This study examined the effect of Lut supplementation during in vitro maturation (IVM) on oocyte maturation and subsequent developmental competence after somatic cell nuclear transfer (SCNT) in pigs. Lut supplementation significantly increased the proportions of complete cumulus cell expansion and metaphase II (MII) oocytes, compared with control oocytes. After parthenogenetic activation or SCNT, the developmental competence of Lut-supplemented MII oocytes was significantly enhanced, as indicated by higher rates of cleavage, blastocyst formation, expanded or hatching blastocysts, and cell survival, as well as increased cell numbers. Lut-supplemented MII oocytes exhibited significantly lower levels of reactive oxygen species and higher levels of glutathione than control MII oocytes. Lut supplementation also activated lipid metabolism, assessed according to the levels of lipid droplets, fatty acids, and ATP. The active mitochondria content and mitochondrial membrane potential were significantly increased, whereas cytochrome c and cleaved caspase-3 levels were significantly decreased, by Lut supplementation. These results suggest that Lut supplementation during IVM improves porcine oocyte maturation through the reduction of oxidative stress and mitochondria-mediated apoptosis.

Blockchain-Based Data Access Control and Key Agreement System in IoT Environment.

Recently, with the increasing application of the Internet of Things (IoT), various IoT environments such as smart factories, smart homes, and smart grids are being generated. In the IoT environment, a lot of data are generated in real time, and the generated IoT data can be used as source data for various services such as artificial intelligence, remote medical care, and finance, and can also be used for purposes such as electricity bill generation. Therefore, data access control is required to grant access rights to various data users in the IoT environment who need such IoT data. In addition, IoT data contain sensitive information such as personal information, so privacy protection is also essential. Ciphertext-policy attribute-based encryption (CP-ABE) technology has been utilized to address these requirements. Furthermore, system structures applying blockchains with CP-ABE are being studied to prevent bottlenecks and single failures of cloud servers, as well as to support data auditing. However, these systems do not stipulate authentication and key agreement to ensure the security of the data transmission process and data outsourcing. Accordingly, we propose a data access control and key agreement scheme using CP-ABE to ensure data security in a blockchain-based system. In addition, we propose a system that can provide data nonrepudiation, data accountability, and data verification functions by utilizing blockchains. Both formal and informal security verifications are performed to demonstrate the security of the proposed system. We also compare the security, functional aspects, and computational and communication costs of previous systems. Furthermore, we perform cryptographic calculations to analyze the system in practical terms. As a result, our proposed protocol is safer against attacks such as guessing attacks and tracing attacks than other protocols, and can provide mutual authentication and key agreement functions. In addition, the proposed protocol is more efficient than other protocols, so it can be applied to practical IoT environments.

ATF6 is a critical regulator of cadmium-mediated apoptosis in spermatocytes.

In this study, we examined the mechanisms of cadmium exposure-induced endoplasmic reticulum (ER) stress response and apoptosis in spermatocytes. Responses to cadmium toxicity were investigated using spermatocytes overexpressing p50ATF6, ATF4, and spliced XBP1s, belonging to the 3 unfolded protein response pathways. The ER stress and apoptosis response to cadmium were most strongly stimulated through the activating transcription factor 6 (ATF6) pathway; in contrast, siRNA-induced inhibition of protein expression could reduce apoptosis under stressful conditions. An in vivo experiment using mice confirmed that upregulation of p50ATF6 in the testis increased apoptosis in response to cadmium exposure. Further, when confirming the correlation between ER stress and MAPK in cadmium toxicity, p38 MAPK phosphorylation was strongly regulated by p50ATF6; p-p38 also mediated the activity of p50ATF6. Overall, these findings suggest that modulating the activity of p38 MAPK and p50ATF6 in cadmium exposure-induced toxicity can be considered a potential strategy to treat infertility.

Mitochondrial peroxiredoxin 5 overexpression suppresses insulin-induced adipogenesis by downregulating the phosphorylation of p38.

Obesity is caused by the accumulation of excess lipids due to an energy imbalance. Differentiation of pre-adipocytes induces abnormal lipid accumulation, and reactive oxygen species (ROS) generated in this process promote the differentiation of pre-adipocytes through mitogen-activated protein kinase (MAPK) signaling. Peroxiredoxin (Prx) is a potent antioxidant enzyme, and peroxiredoxin 5 (Prx5), which is mainly expressed in cytosol and mitochondria, inhibits adipogenesis by regulating ROS levels. Based on previous findings, the present study was performed to investigate whether cytosolic Prx5 (CytPrx5) or mitochondrial Prx5 (MtPrx5) has a greater effect on the inhibition of adipogenesis. In this study, MtPrx5 decreased insulin-mediated ROS levels to reduce adipogenic gene expression and lipid accumulation more effectively than CytPrx5. In addition, we found that p38 MAPK mainly participates in adipogenesis. Furthermore, we verified that MtPrx5 overexpression suppressed the phosphorylation of p38 during adipogenesis. Thus, we suggest that MtPrx5 inhibits insulin-induced adipogenesis more effectively than CytPrx5.

Sample size calculation in clinical trial using R.

Since the era of evidence-based medicine, it has become a matter of course to use statistics to create objective evidence in clinical research. As an extension of this, it has become essential in clinical research to calculate the correct sample size to demonstrate a clinically significant difference before starting the study. Also, because sample size calculation methods vary from study design to study design, there is no formula for sample size calculation that applies to all designs. It is very important for us to understand this. In this review, each sample size calculation method suitable for various study designs was introduced using the R program (R Foundation for Statistical Computing). In order for clinical researchers to directly utilize it according to future research, we presented practice codes, output results, and interpretation of results for each situation.

Provably Secure Mutual Authentication and Key Agreement Scheme Using PUF in Internet of Drones Deployments.

Internet of Drones (IoD), designed to coordinate the access of unmanned aerial vehicles (UAVs), is a specific application of the Internet of Things (IoT). Drones are used to control airspace and offer services such as rescue, traffic surveillance, environmental monitoring, delivery and so on. However, IoD continues to suffer from privacy and security issues. Firstly, messages are transmitted over public channels in IoD environments, which compromises data security. Further, sensitive data can also be extracted from stolen mobile devices of remote users. Moreover, drones are susceptible to physical capture and manipulation by adversaries, which are called drone capture attacks. Thus, the development of a secure and lightweight authentication scheme is essential to overcoming these security vulnerabilities, even on resource-constrained drones. In 2021, Akram et al. proposed a secure and lightweight user-drone authentication scheme for drone networks. However, we discovered that Akram et al.'s scheme is susceptible to user and drone impersonation, verification table leakage, and denial of service (DoS) attacks. Furthermore, their scheme cannot provide perfect forward secrecy. To overcome the aforementioned security vulnerabilities, we propose a secure mutual authentication and key agreement scheme between user and drone pairs. The proposed scheme utilizes physical unclonable function (PUF) to give drones uniqueness and resistance against drone stolen attacks. Moreover, the proposed scheme uses a fuzzy extractor to utilize the biometrics of users as secret parameters. We analyze the security of the proposed scheme using informal security analysis, Burrows-Abadi-Needham (BAN) logic, a Real-or-Random (RoR) model, and Automated Verification of Internet Security Protocols and Applications (AVISPA) simulation. We also compared the security features and performance of the proposed scheme and the existing related schemes. Therefore, we demonstrate that the proposed scheme is suitable for IoD environments that can provide users with secure and convenient wireless communications.

Clinical and genetic factors associated with radiographic damage in patients with ankylosing spondylitis.

OBJECTIVES: To identify clinical and genetic factors associated with severe radiographic damage in patients with ankylosing spondylitis (AS). METHODS: We newly generated genome-wide single nucleotide polymorphism data (833K) for 444 patients with AS. The severity of radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). To identify clinical and genetic factors associated with severe radiographic damage, multiple linear regression analyses were performed. Human AS-osteoprogenitor and control-osteoprogenitor cells were used for functional validation. RESULTS: The significant clinical factors of final mSASSS were baseline mSASSS (ß=0.796, p=3.22×10-75), peripheral joint arthritis (ß=-0.246, p=6.85×10-6), uveitis (ß=0.157, p=1.95×10-3), and smoking (ß=0.130, p=2.72×10-2) after adjusting for sex, age and disease duration. After adjusting significant clinical factors, the Ryanodine receptor 3 (RYR3) gene was associated with severe radiographic damage (p=1.00×10-6). For pathway analysis, the PI3K-Akt signalling pathway was associated with severe radiographic damage in AS (p=2.21×10-4, false discovery rate=0.040). Treatment with rhodamine B, a ligand of RYR3, dose-dependently induced matrix mineralisation of AS osteoprogenitors. However, the rhodamine B-induced accelerated matrix mineralisation was not definitive in control osteoprogenitors. Knockdown of RYR3 inhibited matrix mineralisation in SaOS2 cell lines. CONCLUSIONS: This study identified clinical and genetic factors that contributed to better understanding of the pathogenesis and biology associated with radiographic damage in AS.

MUC5B promoter variant rs35705950, rare but significant susceptibility locus in rheumatoid arthritis-interstitial lung disease with usual interstitial pneumonia in Asian populations.

BACKGROUND: MUC5B variant rs35705950 is the common and most significant risk variant for rheumatoid arthritis-interstitial lung disease (RA-ILD) in Western populations. However, little is known about its significant association with RA-ILD in Asian populations. We here investigate the association of rs35705950 with Korean patients with RA-ILD. METHODS: In this cross-sectional study, we genotyped rs35705950 in 2444 patients with RA. Among them, 683 patients with RA who have chest CT were divided into RA-ILD and RA-noILD. RA-ILD was classified as usual interstitial pneumonia (UIP) and other than UIP. The associations of rs35705950 with RA-ILD and its subtype were analysed using multivariable regression adjusted for age at RA diagnosis. Meta-analysis of a previously reported Japanese dataset and Korean dataset obtained for this study was conducted. RESULTS: The minor allele (T) frequency of rs35705950 was 0.37%, 1.43% and 2.38% in 2444 patients with RA, 105 patients with RA-ILD and 63 patients with UIP, respectively. Genotypic association of rs35705950 with RA-ILD was insignificant (OR 2.49, 95% CI 0.64 to 9.69, p=0.187), but showed significant association with UIP (OR 4.90, 95% CI 1.23 to 19.59, p=0.024) compared with RA-noILD. In meta-analysis (123 UIP and 878 RA-noILD) combining our data with previously reported Japanese data, this variant was found to be significantly associated with UIP (OR 3.51, 95% CI 1.19 to 10.37, p=0.023). CONCLUSION: MUC5B variant rs35705950 is a rare but significant risk factor for Asian patients with RA-ILD with UIP, suggesting a sharing of the genetic background between Asian and Western populations.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

Peroxiredoxin 2 Ameliorates AßO-Mediated Autophagy by Inhibiting ROS via the ROS-NRF2-p62 Pathway in N2a-APP Swedish Cells.

In Alzheimer's disease, reactive oxygen species (ROS) are generated by the deposition of amyloid-beta oligomers (AßOs), which represent one of the important causes of neuronal cell death. Additionally, AßOs are known to induce autophagy via ROS induction. Previous studies have shown that autophagy upregulation aggravates neuronal cell death. In this study, the effects of peroxiredoxin 2 (Prx2), a member of the peroxidase family of antioxidant enzymes, on regulating AßO-mediated autophagy were investigated. Prx2 decreased AßO-mediated oxidative stress and autophagy in N2a-APPswe cells. Further, we examined the relationship between the neuronal protective effect of Prx2 and a decrease in autophagy. Similar to the effects of N-acetyl cysteine, Prx2 decreased AßO-induced ROS and inhibited p62 protein expression levels by downregulating the activation of NRF2 and its translocation to the nucleus. In addition, treatment with 3-methyladenine, an autophagy inhibitor, ameliorates neuronal cell death. Overall, these results demonstrate that the Prx2-induced decrease in autophagy was associated with the inhibition of ROS via the ROS-NRF2-p62 pathway in N2a-APPswe cells. Therefore, our results revealed that Prx2 is a potential therapeutic target in anti-Alzheimer therapy.

PUFTAP-IoT: PUF-Based Three-Factor Authentication Protocol in IoT Environment Focused on Sensing Devices.

In IoT-based environments, smart services can be provided to users under various environments, such as smart homes, smart factories, smart cities, smart transportation, and healthcare, by utilizing sensing devices. Nevertheless, a series of security problems may arise because of the nature of the wireless channel in the Wireless Sensor Network (WSN) for utilizing IoT services. Authentication and key agreements are essential elements for providing secure services in WSNs. Accordingly, two-factor and three-factor-based authentication protocol research is being actively conducted. However, IoT service users can be vulnerable to ID/password pair guessing attacks by setting easy-to-remember identities and passwords. In addition, sensors and sensing devices deployed in IoT environments are vulnerable to capture attacks. To address this issue, in this paper, we analyze the protocols of Chunka et al., Amintoosi et al., and Hajian et al. and describe their security vulnerabilities. Moreover, this paper introduces PUF and honey list techniques with three-factor authentication to design protocols resistant to ID/password pair guessing, brute-force, and capture attacks. Accordingly, we introduce PUFTAP-IoT, which can provide secure services in the IoT environment. To prove the security of PUFTAP-IoT, we perform formal analyses through Burrows Abadi Needham (BAN) logic, Real-Or-Random (ROR) model, and scyther simulation tools. In addition, we demonstrate the efficiency of the protocol compared with other authentication protocols in terms of security, computational cost, and communication cost, showing that it can provide secure services in IoT environments.

iAKA-CIoT: An Improved Authentication and Key Agreement Scheme for Cloud Enabled Internet of Things Using Physical Unclonable Function.

The Internet of Things (IoT) with cloud services are important functionalities in the latest IoT systems for providing various convenient services. These cloud-enabled IoT environments collect, analyze, and monitor surrounding data, resulting in the most effective handling of large amounts of heterogeneous data. In these environments, secure authentication with a key agreement mechanism is essential to ensure user and data privacy when transmitting data between the cloud server and IoT nodes. In this study, we prove that the previous scheme contains various security threats, and hence cannot guarantee essential security requirements. To overcome these security threats, we propose an improved authentication and key agreement scheme for cloud-enabled IoT using PUF. Furthermore, we evaluate its security by performing informal, formal (mathematical), and simulation analyses using the AVISPA tool and ROR model. The performance and security properties of our scheme are subsequently compared with those of other related schemes. The comparison confirms that our scheme is suitable for a practical cloud-enabled IoT environment because it provides a superior security level and is more efficient than contemporary schemes.

Biological insights into systemic lupus erythematosus through an immune cell-specific transcriptome-wide association study.

OBJECTIVE: Genome-wide association studies (GWAS) have identified >100 risk loci for systemic lupus erythematosus (SLE), but the disease genes at most loci remain unclear, hampering translation of these genetic discoveries. We aimed to prioritise genes underlying the 110 SLE loci that were identified in the latest East Asian GWAS meta-analysis. METHODS: We built gene expression predictive models in blood B cells, CD4+ and CD8+ T cells, monocytes, natural killer cells and peripheral blood cells of 105 Japanese individuals. We performed a transcriptome-wide association study (TWAS) using data from the latest genome-wide association meta-analysis of 208 370 East Asians and searched for candidate genes using TWAS and three data-driven computational approaches. RESULTS: TWAS identified 171 genes for SLE (p<1.0×10-5); 114 (66.7%) showed significance only in a single cell type; 127 (74.3%) were in SLE GWAS loci. TWAS identified a strong association between CD83 and SLE (p<7.7×10-8). Meta-analysis of genetic associations in the existing 208 370 East Asian and additional 1498 cases and 3330 controls found a novel single-variant association at rs72836542 (OR=1.11, p=4.5×10-9) around CD83. For the 110 SLE loci, we identified 276 gene candidates, including 104 genes at recently-identified SLE novel loci. We demonstrated in vitro that putative causal variant rs61759532 exhibited an allele-specific regulatory effect on ACAP1, and that presence of the SLE risk allele decreased ACAP1 expression. CONCLUSIONS: Cell-level TWAS in six types of immune cells complemented SLE gene discovery and guided the identification of novel genetic associations. The gene findings shed biological insights into SLE genetic associations.

Expansion of the prime editing modality with Cas9 from Francisella novicida.

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.

Amyloid beta oligomers-induced parkin aggravates ER stress-mediated cell death through a positive feedback loop.

Recently, Parkin has been reported to induce endoplasmic reticulum (ER) stress. In addition, amyloid beta oligomers (AßO), hallmarks of Alzheimer's disease (AD), also increase ER stress in neurons. Because a mutation in the Parkin gene is a well-known predominant cause of familial Parkinson's disease (PD), Parkin has been well studied in PD but has not been well researched in AD. In this study, we investigated the role of AßO-mediated Parkin associated with ER stress in AD. For AD-based research, we used AßO treatments in mouse hippocampus-derived HT-22 cells. We stably expressed Parkin in HT-22 cells to confirm the hypothesis and used siParkin for downregulation of Parkin expression. Moreover, using hippocampi from amyloid precursor protein/presenilin 1/Tau triple transgenic mice (3xTg-AD mice), which are used for AD models, we confirmed the relationship between ER stress and Parkin in vivo. We observed that ATF4 upregulated AßO-increases in Parkin. Parkin overexpression aggravated ER stress in AßO-treated HT-22 cells and the hippocampi of 3xTg-AD mice. Parkin downregulation led to no significant change when compared to AßO-treated cells. Moreover, Parkin-mediated ER stress was not related to oxidative stress. Our study indicates that AßO-induced ATF4 upregulated Parkin levels and that Parkin increases ER stress as a positive feedback loop. Through this study, our findings provide a foundation for future studies on the specific mechanisms related to the role of Parkin in AD.