One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.United States. National Institutes of Health (HD 045022)United States. National Institutes of Health (R37CA084198

A mini review of communicative language testing

Compared with traditional language testing, which heavily emphasizes psychometric reliability, communicative language testing (CLT), which uses authentic tasks to measure communicative abilities, has long been dominant in language assessment. Given its widely acknowledged advantages and widespread use, CLT has become less controversial in the language assessment field and thus is receiving decreased scholarly attention. However, real-world communication, in which CLT is grounded, evolves over time, suggesting the need to update our understanding of it. To address this need and facilitate the further development of CLT theories and practices, this paper offers an up-to-date review of CLT, including its various approaches, implementation challenges, and suggestions for future research

LiSum: Open Source Software License Summarization with Multi-Task Learning

Open source software (OSS) licenses regulate the conditions under which users can reuse, modify, and distribute the software legally. However, there exist various OSS licenses in the community, written in a formal language, which are typically long and complicated to understand. In this paper, we conducted a 661-participants online survey to investigate the perspectives and practices of developers towards OSS licenses. The user study revealed an indeed need for an automated tool to facilitate license understanding. Motivated by the user study and the fast growth of licenses in the community, we propose the first study towards automated license summarization. Specifically, we released the first high quality text summarization dataset and designed two tasks, i.e., license text summarization (LTS), aiming at generating a relatively short summary for an arbitrary license, and license term classification (LTC), focusing on the attitude inference towards a predefined set of key license terms (e.g., Distribute). Aiming at the two tasks, we present LiSum, a multi-task learning method to help developers overcome the obstacles of understanding OSS licenses. Comprehensive experiments demonstrated that the proposed jointly training objective boosted the performance on both tasks, surpassing state-of-the-art baselines with gains of at least 5 points w.r.t. F1 scores of four summarization metrics and achieving 95.13% micro average F1 score for classification simultaneously. We released all the datasets, the replication package, and the questionnaires for the community

Does higher demand for medicinal plants lead to more harvest? Evidence from the dual trade of Nardostachy jatamansi and Fritillaria cirrhosa and Tibetan people’s harvesting behavior

IntroductionAs the demand for herbal medicines is surging worldwide, regions of medicinal plants are vulnerable to large-scale and unsustainable exploitation for commercial trade and use. Yet, we still lack the understanding about the relationship between indigenous people harvesting and trade practices of medicinal plants and their influencing factors for possible intervention measures.MethodsHere, we combined qualitative and quantitative methods to survey traders (N = 20) and local harvesters (N = 923) from nine Tibetan townships in Hongyuan county, Sichuan Province, on the eastern Qinghai-Tibetan plateau in China. Specifically, we elucidated the local value chain of medicinal plants trade and harvest of Nardostachy jatamansi and Fritillaria cirrhosa, and explored the factors influencing harvester’s willingness to harvest these plants. Furthermore, we empirically tested the constructs of the COM-B model (Capability, Opportunity, Motivation -Behavior) in predicting the sustainable harvesting behavior of medicinal plants.Results and DiscussionOur results revealed that the trade characteristics of N. jatamansi and F. cirrhosa were contrasting, and the sustainability of the former species was largely dependent on the latter one. Importantly, the traders’ practices were affected by the supply, while the harvesters’ willingness to harvest were mainly influenced by harvest incomes, past harvesting experience, and grassland tenure. Finally, though motivation was not directly affecting harvesting behavior, the harvesters’ ecological worldview indirectly affected their harvesting behavior, particularly through the mediation of the level of compliance of village rules and customs. Overall, our results provided crucial insights for the conservation and sustainable management of the valuable wild medicinal plants

Generation of Genetically Modified Mice by Oocyte Injection of Androgenetic Haploid Embryonic Stem Cells

SummaryHaploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.PaperCli

Honokiol Crosses BBB and BCSFB, and Inhibits Brain Tumor Growth in Rat 9L Intracerebral Gliosarcoma Model and Human U251 Xenograft Glioma Model

BACKGROUND: Gliosarcoma is one of the most common malignant brain tumors, and anti-angiogenesis is a promising approach for the treatment of gliosarcoma. However, chemotherapy is obstructed by the physical obstacle formed by the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). Honokiol has been known to possess potent activities in the central nervous system diseases, and anti-angiogenic and anti-tumor properties. Here, we hypothesized that honokiol could cross the BBB and BCSFB for the treatment of gliosarcoma. METHODOLOGIES: We first evaluated the abilities of honokiol to cross the BBB and BCSFB by measuring the penetration of honokiol into brain and blood-cerebrospinal fluid, and compared the honokiol amount taken up by brain with that by other tissues. Then we investigated the effect of honokiol on the growth inhibition of rat 9L gliosarcoma cells and human U251 glioma cells in vitro. Finally we established rat 9L intracerebral gliosarcoma model in Fisher 344 rats and human U251 xenograft glioma model in nude mice to investigate the anti-tumor activity. PRINCIPAL FINDINGS: We showed for the first time that honokiol could effectively cross BBB and BCSFB. The ratios of brain/plasma concentration were respectively 1.29, 2.54, 2.56 and 2.72 at 5, 30, 60 and 120 min. And about 10% of honokiol in plasma crossed BCSFB into cerebrospinal fluid (CSF). In vitro, honokiol produced dose-dependent inhibition of the growth of rat 9L gliosarcoma cells and human U251 glioma cells with IC(50) of 15.61 µg/mL and 16.38 µg/mL, respectively. In vivo, treatment with 20 mg/kg body weight of honokiol (honokiol was given twice per week for 3 weeks by intravenous injection) resulted in significant reduction of tumor volume (112.70±10.16 mm(3)) compared with vehicle group (238.63±19.69 mm(3), P = 0.000), with 52.77% inhibiting rate in rat 9L intracerebral gliosarcoma model, and (1450.83±348.36 mm(3)) compared with vehicle group (2914.17±780.52 mm(3), P = 0.002), with 50.21% inhibiting rate in human U251 xenograft glioma model. Honokiol also significantly improved the survival over vehicle group in the two models (P<0.05). CONCLUSIONS/SIGNIFICANCE: This study provided the first evidence that honokiol could effectively cross BBB and BCSFB and inhibit brain tumor growth in rat 9L intracerebral gliosarcoma model and human U251 xenograft glioma model. It suggested a significant strategy for offering a potential new therapy for the treatment of gliosarcoma

Disruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice

We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn , SMN2 ). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases. -/- tg/

Methylprednisolone as Adjunct to Endovascular Thrombectomy for Large-Vessel Occlusion Stroke

Importance It is uncertain whether intravenous methylprednisolone improves outcomes for patients with acute ischemic stroke due to large-vessel occlusion (LVO) undergoing endovascular thrombectomy. Objective To assess the efficacy and adverse events of adjunctive intravenous low-dose methylprednisolone to endovascular thrombectomy for acute ischemic stroke secondary to LVO. Design, Setting, and Participants This investigator-initiated, randomized, double-blind, placebo-controlled trial was implemented at 82 hospitals in China, enrolling 1680 patients with stroke and proximal intracranial LVO presenting within 24 hours of time last known to be well. Recruitment took place between February 9, 2022, and June 30, 2023, with a final follow-up on September 30, 2023.InterventionsEligible patients were randomly assigned to intravenous methylprednisolone (n = 839) at 2 mg/kg/d or placebo (n = 841) for 3 days adjunctive to endovascular thrombectomy. Main Outcomes and Measures The primary efficacy outcome was disability level at 90 days as measured by the overall distribution of the modified Rankin Scale scores (range, 0 [no symptoms] to 6 [death]). The primary safety outcomes included mortality at 90 days and the incidence of symptomatic intracranial hemorrhage within 48 hours. Results Among 1680 patients randomized (median age, 69 years; 727 female [43.3%]), 1673 (99.6%) completed the trial. The median 90-day modified Rankin Scale score was 3 (IQR, 1-5) in the methylprednisolone group vs 3 (IQR, 1-6) in the placebo group (adjusted generalized odds ratio for a lower level of disability, 1.10 [95% CI, 0.96-1.25]; P = .17). In the methylprednisolone group, there was a lower mortality rate (23.2% vs 28.5%; adjusted risk ratio, 0.84 [95% CI, 0.71-0.98]; P = .03) and a lower rate of symptomatic intracranial hemorrhage (8.6% vs 11.7%; adjusted risk ratio, 0.74 [95% CI, 0.55-0.99]; P = .04) compared with placebo. Conclusions and Relevance Among patients with acute ischemic stroke due to LVO undergoing endovascular thrombectomy, adjunctive methylprednisolone added to endovascular thrombectomy did not significantly improve the degree of overall disability.Trial RegistrationChiCTR.org.cn Identifier: ChiCTR210005172