Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Prognostic Value of the Geriatric Nutritional Risk Index in Patients with Non-Small Cell Lung Cancer: A Meta-Analysis.

This study aimed to quantitatively identify the prognostic and clinicopathological value of the geriatric nutritional risk index (GNRI) in non-small cell lung cancer (NSCLC) through a meta-analysis. The electronic databases PubMed, Web of Science, Embase, and Cochrane Library were thoroughly searched from inception to December 14, 2021. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the prognostic value of GNRI. Odds ratios (ORs) and 95%CIs were combined to estimate the clinicopathological significance of the GNRI in NSCLC. Seven studies with 2,023 patients were included in the meta-analysis. A low GNRI score was significantly associated with poor overall survival (OS) (HR = 2.01, 95%CI = 1.65-2.44, p < 0.001) and worse progression-free survival (PFS), recurrence-free survival (RFS), and cancer-specific survival (CSS) (HR = 1.81, 95%CI = 1.48-2.22, p < 0.001) in NSCLC. Furthermore, a low GNRI score was significantly associated with the histological type of non-adenocarcinoma (OR= 1.55, 95%CI = 1.19-2.03, p = 0.001) and Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2 (OR= 2.81, 95%CI= 1.49-5.32, p = 0.001). A low GNRI score is a significant and effective prognostic marker for poor survival outcomes in patients with NSCLC. In addition, low GNRI score was correlated with higher ECOG PS scores.

Modulation of Immune Reaction in Hydrodynamic Gene Therapy for Hemophilia A.

Hemophilia A (HA) is a monogenic disease characterized by plasma clotting factor 8 (F8) deficiency due to F8 mutation. We have been attempting to cure HA permanently using a CRISPR-Cas9 gene-editing strategy. In this study, we induced targeted integration of BDDF8 (B-domain-deleted F8) gene into the albumin locus of HA mice by hydrodynamic tail vein injection of editing plasmid vectors. One week after treatment, a high F8 activity ranging from 70% to 280% of normal serum levels was observed in all treated HA mice but dropped to background levels 3-5 weeks later. We found that the humoral immune reaction targeting F8 is the predominant cause of the decreased F8 activity. We hypothesized that hydrodynamic injection-induced liver damage triggered the release of large quantities of inflammatory cytokines. However, coinjection of plasmids expressing a dozen immunomodulatory factors failed to curtail the immune reaction and stabilize F8 activity effectively. The spCas9 plasmid carrying a miR-142-3p target sequence alleviated the cellular immune response but could not deliver therapeutic efficacy. Strikingly, immunosuppressant cyclophosphamide virtually abolished the immune response, leading to a year-long stable F8 level. Our findings should have important implications in developing therapies in mouse models using the hydrodynamic gene delivery approach, highlighting the necessity of modulating the innate immune response triggered by liver damage.

"Lighting-up" methylene blue-embedded zirconium based organic framework triggered by Al3+ for advancing the sensitivity of E. coli O157:H7 analysis in dual-signal lateral flow immunochromatographic assay.

The sensitive detection of foodborne pathogens is of great significance for ensuring food safety and quality. Herein, on the basis of methylene blue-embedded zirconium based organic framework (UIO@MB) as the remarkable capture carrier and signal indicator, with the Al3+-assisted the fluorescent signal response, we developed a label-free and dual-signal lateral flow immunochromatographic assay (LDLFIA) for sensitive detection of Escherichia coli (E. coli) O157:H7. The UIO@MB sensing carrier without monoclonal antibodies (mAbs) was manufactured, which adhered to bacteria to form the UIO@MB-E. coli O157:H7 conjugate, resulting in visible blue band. Then the fluorescent response of the OH-rich UIO@MB was excited by introducing Al3+, arising from capturing of Al3+ by -OH through coordination and electrostatic affinity, thus generating a green fluorescent band. Impressively, a smartphone-based portable reading system was developed that can reflect the test results of UIO@MB-LDLFIA immediately. Under optimum conditions, UIO@MB-LDLFIA can complete colorimetric and fluorescent mode detection within 90 min, with a detection sensitivity of 103 CFU/mL, which were 100 times lower than traditional gold nanoparticles-based LFIA and polymerase chain reaction (PCR). Moreover, the feasibility of the method was further evaluated by the determination of E. coli O157: H7 in drinking water and cabbage with average recoveries of 85.1-123.0%.

Two New Oleanane-Triterpenoid Saponins from Clinopodium gracile.

Two new oleanane-triterpenoid saponins, clinograsaponins A (1) and B (2), together with twelve known ones (3-14), were isolated from the whole herb of Clinopodium gracile (Bentham) Matsumura. Their structures were determined by spectroscopic analysis and chemical method. All the isolated compounds were evaluated for their activities against ATP-citrate lyase (ACLY) and nuclear factor kappa B (NF-κB).

Mental Health Status of Late-Middle-Aged Adults in China During the Coronavirus Disease 2019 Pandemic.

Background: The novel coronavirus 2019 (COVID-19) pandemic and related compulsory measures have triggered a wide range of psychological issues. However, the effect of COVID-19 on mental health in late-middle-aged adults remains unclear. Methods: This cross-sectional, web-based survey recruited 3,730 participants (≥ 50 years old) between February 28 and March 11 of 2020. The Patient Health Questionnaire-9, Generalized Anxiety Disorder-7, Insomnia Severity Index, and Acute Stress Disorder Scale were used to evaluate depression, anxiety, insomnia, and acute stress symptoms. Multivariate logistic regression analysis was fitted to explore risk factors that were associated with the selected outcomes. Results: The mean age of the participants was 54.44 ± 5.99 years, and 2,026 (54.3%) of the participants were female. The prevalence of depression, anxiety, insomnia, and acute stress symptoms among late-middle-aged adults in China during the COVID-19 pandemic was 20.4, 27.1, 27.5, and 21.2%, respectively. Multivariable logistic regression analyses showed that participants who were quarantined had increased odds ratios for the four mental health symptoms, and those with a good understanding of the COVID-19 pandemic displayed a decreased risk for all mental health symptoms among late-middle-aged adults. In addition, participants with a low income and with a risk of COVID-19 exposure at work had a remarkably high risk of depression, anxiety, and acute stress symptoms. Conclusions: Mental health symptoms in late-middle-aged adults in China during the COVID-19 pandemic are prevalent. Population-specific mental health interventions should be developed to improve mental health outcomes in late-middle-aged adults during this public health emergency.

Neuraxial labor analgesia is associated with a reduced risk of postpartum depression: A multicenter prospective cohort study with propensity score matching.

BACKGROUND: Depression is a common and serious complication in new mothers. We investigated the hypothesis that neuraxial labor analgesia is associated with a decreased risk of postpartum depression. METHODS: In this multicenter prospective cohort study with propensity score matching, 599 nulliparous women with single term cephalic pregnancy who planned vaginal delivery were enrolled and self-selected neuraxial analgesia or not. The primary outcome was 6-week postpartum depression assessed with the Chinese version Edinburgh Postnatal Depression Scale; a score of ≥10 was set as the threshold of postpartum depression. Logistic regression models were established to assess the association between neuraxial labor analgesia and postpartum depression. RESULTS: Of the 577 parturients who completed the study, 417 (72.3%) received neuraxial analgesia and 160 (27.7%) did not. After propensity score matching, 433 parturients were included in the analysis; of whom, 279 (64.4%) received neuraxial analgesia and 154 (35.6%) did not. The incidence of postpartum depression was lower in parturients with neuraxial analgesia than in those without (14.9% [62/417] vs. 23.8% [38/160], P=0.012 before matching; 13.3% [37/279] vs. 23.4% [36/154], P=0.007 after matching). After adjustment for confounding factors, neuraxial analgesia was associated with decreased odds of postpartum depression (odds ratio [OR] 0.50, 95% CI 0.28-0.88, P=0.015 before matching; OR 0.40, 95% CI 0.21-0.77, P=0.006 after matching). LIMITATIONS: As an observational study, unidentified confounders might influence the results. CONCLUSIONS: In nulliparae with single term cephalic pregnancy preparing to give vaginal delivery neuraxial analgesia during labor was associated with a decreased risk of 6-week postpartum depression.

Meroterpenoids with diverse structures and anti-inflammatory activities from Rhododendron anthopogonoides.

Eight pairs of meroterpenoid enantiomers and four achiral meroterpenoids were isolated from Rhododendron anthopogonoides Maxim. Seventeen of them, named (+)-/(-)-anthoponoids A-G, (+)-daurichromene D, and anthoponoids H and I, are undescribed compounds with structural diversity. Their structures were characterized herein by a combined application of spectroscopic techniques, X-ray crystallographic analysis, ECD calculation, and the modified Mosher's method. (+)-/(-)-Anthoponoid A and anthoponoid I are the first Rhododendron meroterpenoids found to possess a hexahydroxanthene motif and a diterpene unit, respectively. Some isolates were identified as NF-κB pathway inhibitors, and (+)-anthoponoid E, (-)-anthoponoid G, and anthoponoid H showed suppressive effects on LPS-induced inflammatory responses in RAW 264.7 macrophages.

Cross-cultural adaptation and validation of the 3D-CAM Chinese version in surgical ICU patients.

BACKGROUND: Accurate diagnosis of delirium is very important for prevention and treatment. Present study was designed to validate the 3-Minute Diagnostic Interview for CAM-defined Delirium Chinese version (3D-CAM-CN) in surgical ICU patients. METHODS: In this prospective diagnostic study, the 3D-CAM was translated into Chinese with culture adaption. Two interviewers (Roles A and B) independently administrated 3D-CAM-CN assessment in adult patients from postoperative days 1 to day 3. At the meantime, a panel of psychiatrists diagnosed delirium according to the Diagnostic and Statistical Manual of Mental Disorders-fifth edition as the reference standard. The sensitivity and specificity were calculated to analyze the diagnostic character of the 3D-CAM-CN. Kappa coefficient was used to evaluate interrater reliability. RESULTS: Two hundred forty-five adult patients were assessed for at least 2 days, resulting a total of 647 paired-assessments. When compared with the reference standard, the sensitivity and specificity of the 3D-CAM-CN assessment were 87.2 and 96.7%, respectively, by Role A and 84.6 and 97.4%, respectively, by Role B, with good interrater reliability (Kappa coefficient = 0.82, P < 0.001). It also performed well in patients with mild cognitive impairment, with the sensitivity from 85.7 to 100% and the specificity from 95.7 to 96.4%. CONCLUSION: Our results showed that the 3D-CAM-CN can be used as a reliable and accurate instrument for delirium assessment in surgical patients. TRIAL REGISTRATION: This trail was approved by the Clinical Research Ethic Committee of Peking University First Hospital (No. 2017-1321) and registered on Chinese clinical trial registry on July 6, 2017 (ChiCTR-OOC-17011887).

Poststroke depression and risk of recurrent stroke at 1 year in a Chinese cohort study.

BACKGROUND: Studies show that poststroke depression (PSD) increases mortality risk at 1 year. However, whether PSD increases the risk of recurrent stroke at 1 year remains unclear. This study was to investigate whether PSD at 2 weeks following a stroke could increase risk of recurrent stroke at 1 year. METHODS AND RESULTS: This was a multi-centered prospective cohort study. A total of 2306 patients with acute stroke were enrolled in our study. PSD was diagnosed according to the criteria set by the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). The outcomes of recurrent stroke were followed up via face-to-face or phone interview. A total of 1713 patients had complete follow-up data, with 481 (28.1%) cases of PSD and 158 (9.2%) cases of cumulative recurrent stroke at 1 year. Multivariate logistic regression analysis showed a 49% increase of OR of recurrent stroke at 1 year in patients with PSD, compared to patients without PSD following a stroke (OR=1.49, 95%CI: 1.03-2.15). There was no significant correlation between anti-depressant drugs and the risk of recurrent stroke at 1 year following a stroke (OR=1.96, 95%: CI 0.95-4.04). CONCLUSIONS: Based on the DSM-IV diagnostic criteria, nearly 3 out of 10 hospitalized stroke patients in China were diagnosed with PSD at 2 weeks following a stroke. PSD is associated with a higher risk of recurrent stroke at 1 year. Our study did not find benefit of anti-depressant drugs in reducing such risk.

Time course of depression and one-year prognosis of patients with stroke in mainland China.

AIMS: To investigate the incidence of depression at different time points within the first year after stroke in mainland China and to identify risk factors related to a poor 1-year prognosis in stroke patients. METHODS: Subjects with acute cerebrovascular diseases were recruited and enrolled from 56 hospitals in mainland China between April 2008 and April 2010. Demographic data, previous disease history, and clinical data were collected. Four follow-up visits were occurred within the first year after stroke. The modified Rankin Scale ≥ 2 represents an unfavorable prognosis. Depression was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, fourth edition and was divided into persistent, recurrent and transient types. RESULTS: The 1-year cumulative incidence of depression in stroke patients was 41.8%. Logistic regression analysis showed that the 1-year prognosis level was associated with age, disability before onset, neurological functional deficit level at admission, and a range of depression types. The odds ratio for persistent depression is the highest (OR = 7.615, P < 0.0001, 95% confidence interval 5.011-11.572). CONCLUSIONS: In our study, depression occurred in >40% of patients within the first year after stroke. Persistent depression is the first independent determinant of prognosis during the first year after stroke.