CRISPR RNAs trigger innate immune responses in human cells

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to similar to 80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting St-hydroxyl gRNAs in complex with Cas9 or Cpfl avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4(+) T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

Posttraumatic stress disorder and depression of survivors 12 months after the outbreak of Middle East respiratory syndrome in South Korea

The 2015 outbreak of Middle East Respiratory Syndrome (MERS) in the Republic of Korea is a recent and representative occurrence of nationwide outbreaks of Emerging Infectious Diseases (EIDs). In addition to physical symptoms, posttraumatic stress disorder (PTSD) and depression are common following outbreaks of EID. The present study investigated the long-term mental health outcomes and related risk factors in survivors of MERS. A prospective nationwide cohort study was conducted 12 months after the MERS outbreak at multi-centers throughout Korea. PTSD and depression as the main mental health outcomes were assessed with the Impact of Event Scale-Revised Korean version (IES-R-K) and the Patient Health Questionnaire-9 (PHQ-9) respectively. 42.9% of survivors reported PTSD (IES-R-K ≥ 25) and 27.0% reported depression (PHQ-9 ≥ 10) at 12 months post-MERS. A multivariate analysis revealed that anxiety (adjusted odds ratio [aOR], 5.76; 95%CI, 1.29–25.58; P = 0.021), and a greater recognition of stigma (aOR, 11.09, 95%CI, 2.28–53.90; P = 0.003) during the MERS-affected period were independent predictors of PTSD at 12 months after the MERS outbreak. Having a family member who died from MERS predicted the development of depression (aOR, 12.08, 95%CI, 1.47–99.19; P = 0.020). This finding implies that psychosocial factors, particularly during the outbreak phase, influenced the mental health of patients over a long-term period. Mental health support among the infected subjects and efforts to reduce stigma may improve recovery from psychological distress in an EID outbreak.The study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI15C3227) and a grant from the Korean Mental Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HL19C0007). The funding bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript

A framework for nationwide COVID-19 vaccine safety research in the Republic of Korea: the COVID-19 Vaccine Safety Research Committee

With the introduction of coronavirus disease 2019 (COVID-19) vaccines, the Korea Disease Control and Prevention Agency (KDCA) commissioned the National Academy of Medicine of Korea to gather experts to independently assess post-vaccination adverse events. Accordingly, the COVID-19 Vaccine Safety Research Committee (CoVaSC) was launched in November 2021 to perform safety studies and establish evidence for policy guidance. The CoVaSC established 3 committees for epidemiology, clinical research, and communication. The CoVaSC mainly utilizes pseudonymized data linking KDCA’s COVID-19 vaccination data and the National Health Insurance Service’s claims data. The CoVaSC’s 5-step research process involves defining the target diseases and organizing ad-hoc committees, developing research protocols, performing analyses, assessing causal relationships, and announcing research findings and utilizing them to guide compensation policies. As of 2022, the CoVaSC completed this research process for 15 adverse events. The CoVaSC launched the COVID-19 Vaccine Safety Research Center in September 2022 and has been reorganized into 4 divisions to promote research including international collaborative studies, long-/short-term follow-up studies, and education programs. Through these enhancements, the CoVaSC will continue to swiftly provide scientific evidence for COVID-19 vaccine research and compensation and may serve as a model for preparing for future epidemics of new diseases

Effect of Mechanical Stress on the Proliferation and Expression of Cell Cycle Regulators in Human Periodontal Ligament Cells

Recently, mechanical stress has been found to be associated with the proliferation of human periodontal ligament〔PDL〕cells. Although the detailed mechanism for the proliferation of PDL cells by mechanical stress remains largely unknown, the effect may be due to the increased expression of cell cycle regulatory proteins. To investigate this possibility, we measured the growth pattern and determined the protein levels of cellular p53, p21^WAF1/CIP1, cyclin-dependent kinases〔cdks〕, G_1 cyclins, and proliferating cell nuclear antigen〔PCNA〕expressed by PDL cells exposed to mechanical stress. Human PDL cells of passage number 6 were planted onto 55-mm Petriperm dishes〔1×10^4 cells/dish〕and exposed to mechanical stress〔1kg/dish〕for 12days. Mechanical stress markedly increased the proliferation of the PDL cells exposed to stress for 8-12days, compared to the controls. It also increased the protein level of cellular PCNA in the PDL cells exposed to stress for 6-10days. Mechanical stress also slightly increased the expression of cdks and cyclin D1 in these cells, but the protein levels of p53 and p21^WAF1/CIP1 were unchanged. The increased proliferation of PDL cells by mechanical stress may be due, in part, to the elevated cellular levels of cdks, without corresponding changes in p53 and p21^WAF1/CIP1 levels in human PDL cells

Short Communication: Increase of HIV-1 K103N Transmitted Drug Resistance and Its Association with Efavirenz Use in South Korea.

Previous studies reported a relatively low prevalence of transmitted drug resistance (TDR) in South Korea (<5%). A genotypic resistance test was performed on 131 treatment-naive HIV-1-infected individuals from February 2013 to February 2014. Eleven individuals (8.4%) presented TDR, of whom eight had K103N, revealing a significant increase in K103N TDR compared to previous studies (p<0.001). Using phylogenetic analysis, we identified three distinct clustering pairs with genetic relativeness and a total of five independent strains among the eight K103N cases. Our findings suggest that multiple sources of K103N occurred, most likely as a consequence of increased efavirenz use in South Korea

HIV drug resistance mutations in proviral DNA from a community treatment program.

BACKGROUND:Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations. METHODS:The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu. RESULTS:Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%); the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26%) with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively. CONCLUSIONS:The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence

Molecular epidemiology identifies HIV transmission networks associated with younger age and heterosexual exposure among Korean individuals.

To evaluate if HIV transmission networks could be elucidated from data collected in a short time frame, 131 HIV-1 pol sequences were analyzed which were generated from treatment-naïve Korean individuals who were sequentially identified over 1 year. A transmission linkage was inferred when there was a genetic distance &lt;1.5% and a total of 16 clusters, involving 39/131 (29.8%), were identified. Younger age and heterosexual exposure were independently related with clustering in the inferred network, which demonstrated that molecular epidemiology with currently generated data (i.e., drug resistance genotypes) can be used to identify local transmission networks, even over a short timeframe. J. Med. Virol. 88:1832-1835, 2016. © 2016 Wiley Periodicals, Inc