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Background Millions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens. Results To improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons of BRCA1 and BRCA2 genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants for BRCA1/2 , including 151 variants in the noncoding part of the genes such as the 5′ untranslated regions. Many of them are identified in cancer patients and are reported as “benign/likely benign” or “variants of uncertain significance” by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer. Conclusions Our results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.

Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR -deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.

Botrytis cinerea is a fungal pathogen that can infect healthy fruits and vegetables, causing significant economic losses through the development of gray mold disease. Over the past few decades, antagonistic yeast has been successfully employed to control this disease. In this study, an exhaustive screening process was conducted on seven yeast strains, ultimately identifying Hanseniaspora uvarum as the strain with the highest antimicrobial activity. Subsequent investigations focused on understanding its inhibitory mechanisms against cucumber gray mold. The results demonstrated that H. uvarum exhibited remarkable suppression of spore germination and germ tube elongation in B. cinerea . Most notably, H. uvarum displayed rapid colonization on the surface of cucumber fruits and effectively curbed the invasion of cucumber gray mold by inducing ROS metabolism, enhancing the activity of disease-resistant enzymes (PPO and POD) and upregulating the expression levels of relevant genes ( CsPLA , CsPR1 , CsCHI , and CsGLU ), while promoting the accumulation of total phenolics and flavonoids. These collective actions served to maintain the storage quality of the fruits. In conclusion, H. uvarum holds significant potential in mitigating the harm caused by cucumber gray mold.

Imaging-based sensors convert physicochemical parameters of analytes into visible patterns, yet a high sensitivity remains constrained. Here, we introduce the concept of differential guided-mode resonance with thickness modulation at a tens-nanometer scale to greatly enhance the sensitivity, alleviating the sensitivity-dynamic range tradeoff. Experimental results reveal a sensitivity of up to a million-level pixels per refractive index unit (RIU), surpassing existing technologies by nearly three orders of magnitude, with a large dynamic range reconfigured by the incident angle. With the present method, a moderate value (about 100) of the Q factor suffices to make a record high sensitivity and the Figure of Merit (FOM) can reach 10 4 RIU −1 level. We also demonstrate a portable device, highlighting its potential for practical applications, including 2D distribution sensing. This method unlocks the potential of imaging-based sensors with both record high sensitivity and tremendous dynamic range for accurate medical diagnosis, biochemical analysis, dynamic pollution monitoring, etc. This paper presents an imaging-based sensor enabled by differential guided-mode resonance with nanoscale thickness modulation, achieving high sensitivity and expanded dynamic range for medical diagnostics and biochemical sensing.

ObjectivesTo investigate the knowledge of stroke and the attitudes towards stroke and prehospital delay among patients who had an acute ischaemic stroke (AIS) and their family members.DesignThis cross-sectional study was conducted through a self-designed questionnaire.SettingThe study took place in a Grade-A tertiary hospital in Zhejiang Province, China, between July 2023 and November 2023.ParticipantsA total of 521 valid questionnaires were collected from 367 patients who had an AIS and 154 family members.InterventionsParticipants provided demographic information and answered questions related to stroke knowledge, attitudes towards stroke and prehospital delay.Primary and secondary outcome measuresThe primary outcome measures included scores on stroke knowledge, attitudes towards stroke and attitudes towards prehospital delay. Secondary outcomes focused on identifying correlations and independent factors influencing prehospital delay.ResultsThe average scores for patients were stroke knowledge 8.74±6.16 (range: 0–24), stroke attitude 23.52±2.73 (range: 7–35) and prehospital delay attitude 38.65±7.68 (range: 10–50). Family members scored 12.66±6.85, 23.60±2.57 and 40.02±7.45, respectively. Significant correlations were found between stroke knowledge and attitude (r=0.2262, p<0.001) and between stroke attitude and prehospital delay attitude (r=0.1305, p=0.0028). Multivariate analysis indicated that patients’ prehospital delay attitude (OR=0.91) and choice of the first medical facility (OR=0.30) were associated with prehospital delay, while in family members, prehospital delay attitude (OR=0.91) and wake-up stroke (OR=2.91) were significant.ConclusionsBoth patients who had an AIS and their family members demonstrated insufficient knowledge and moderate attitudes towards stroke and prehospital delay, which were associated with extended prehospital delay. Educational interventions are necessary to enhance stroke knowledge. Targeted stroke awareness programmes and rapid response training could help improve early recognition and timely medical intervention, reducing prehospital delay and improving patient outcomes.

ObjectiveHepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further.DesignHere, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice.ResultsThese HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials.ConclusionHBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient’s life.

Background Harnessing hepatocytes for basic research and regenerative medicine demands a complete understanding of the genetic determinants underlying hepatocyte differentiation and maturation. Single-cell CRISPR screens in organoids could link genetic perturbations with parallel transcriptomic readout in single cells, providing a powerful method to delineate roles of cell fate regulators. However, a big challenge for identifying key regulators during data analysis is the low expression levels of transcription factors (TFs), which are difficult to accurately estimate due to noise and dropouts in single-cell sequencing. Also, it is often the changes in TF activities in the transcriptional cascade rather than the expression levels of TFs that are relevant to the cell fate transition. Results Here, we develop Organoid-based Single-cell CRISPR screening Analyzed with Regulons (OSCAR), a framework using regulon activities as readouts to dissect gene knockout effects in organoids. In adult-stem-cell-derived liver organoids, we map transcriptomes in 80,576 cells upon 246 perturbations associated with transcriptional regulation of hepatocyte formation. Using OSCAR, we identify known and novel positive and negative regulators, among which Fos and Ubr5 are the top-ranked ones. Further single-gene loss-of-function assays demonstrate that Fos depletion in mouse and human liver organoids promote hepatocyte differentiation by specific upregulation of liver metabolic genes and pathways, and conditional knockout of Ubr5 in mouse liver delays hepatocyte maturation. Conclusions Altogether, we provide a framework to explore lineage specifiers in a rapid and systematic manner, and identify hepatocyte determinators with potential clinical applications.

Background Most cancer risk-associated single nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) are noncoding and it is challenging to assess their functional impacts. To systematically identify the SNPs that affect gene expression by modulating activities of distal regulatory elements, we adapt the self-transcribing active regulatory region sequencing (STARR-seq) strategy, a high-throughput technique to functionally quantify enhancer activities. Results From 10,673 SNPs linked with 996 cancer risk-associated SNPs identified in previous GWAS studies, we identify 575 SNPs in the fragments that positively regulate gene expression, and 758 SNPs in the fragments with negative regulatory activities. Among them, 70 variants are regulatory variants for which the two alleles confer different regulatory activities. We analyze in depth two regulatory variants—breast cancer risk SNP rs11055880 and leukemia risk-associated SNP rs12142375—and demonstrate their endogenous regulatory activities on expression of ATF7IP and PDE4B genes, respectively, using a CRISPR-Cas9 approach. Conclusions By identifying regulatory variants associated with cancer susceptibility and studying their molecular functions, we hope to help the interpretation of GWAS results and provide improved information for cancer risk assessment.

Background The transfibular fracture region (TFFR) approach can be utilized for managing posterior pilon fractures associated with intercalary fragments. However, its long-term outcomes remain unreported. This study aimed to compare the long-term clinical outcomes of the TFFR approach and the posteromedial approach for posterior pilon fractures (Klammer type 2/3, Danis–Weber type B) associated with displaced intercalary fragments over an average 8 year follow-up. Method From 2012 to 2018, a cohort of consecutive patients who underwent open reduction and internal fixation surgery via either the TFFR approach or the posteromedial approach for posterior pilon fracture associated with intercalary fragments were enrolled for this study. Clinical outcomes were evaluated over an average 8 year (range 5–12 years) follow-up. The surgical duration, number of intraoperative fluoroscopies, and postoperative complications were recorded. Functional outcomes were assessed using the Foot and Ankle Outcome Score (FAOS), Foot and Ankle Ability Measure (FAAM), and Short Form-36 (SF-36) score at last follow-up. Results Seventy-nine patients were included in the final analysis, including 43 in the TFFR group and 36 in the posteromedial group. No significant differences between the two groups were observed in the FAOS ( p  = 0.679) or its specific components for symptoms ( p  = 0.264), pain ( p  = 0.963), activities of daily living (ADL, p  = 0.102), sports ( p  = 0.156), or quality of life ( p  = 0.859). There was also no significant difference between the two groups in the FAAM-ADL ( p  = 0.408), FAAM-Sport ( p  = 0.617), and SF-36 scores ( p  = 0.757). Nevertheless, the surgical duration was shorter in the TFFR group ( p  < 0.001). Conclusion The TFFR approach is not inferior to the posteromedial approach. For posterior pilon fractures with lateral malleolar fractures in the same plane, the TFFR approach may be preferred owing to its potential to reduce surgical time and the use of a single incision. Level of Evidence Level III, retrospective cohort study.