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As a germ-cell-specific maternal mRNA and RNA binding protein, dead end (dnd1) plays an important role in migration and maintenance of primordial germ cells (PGCs), and therefore, disruption of dnd1 results in no germ cells. However, the effect and duration of maternal dnd1 on PGCs in zygote mutants have not been reported yet. In the present study, CRISPR/Cas9 was used to mutate dnd1 in tilapia. Supported by the maternal dnd1 mRNA, germ cells of the dnd1 homozygous mutants proliferated and migrated normally before 7 dpf (days post fertilization). After the degradation of maternal dnd1 mRNA, significant downregulation of piwil1, piwil2, nanos2, nanos3 and vasa mRNA, and blocked migration of germ cells were observed at 14 dpf. In dnd1 zygote mutants, the number of germ cells was decreased significantly compared with wild type (WT) at 25 dpf, and no germ cell was observed at 30 dpf. Consistently, no expression of vasa and nanos3 was detected in the adult dnd1 mutants by qPCR. Interestingly, Cyp19a1a/cyp19a1a and foxl2 were expressed in dnd1-deficient XX gonads, while Cyp11c1/cyp11c1 and dmrt1 were expressed in dnd1-deficient XY gonads as in WT gonads. Correspondingly, both serum E2 level in XX and 11-KT level in XY fish did not differ significantly between dnd1 mutant and WT fish. Taken together, the present study demonstrates that disruption of dnd1 lead to progressive loss of germ cells, while the somatic cells are not affected in tilapia. The sexual fate of gonads is determined by the genetic sex instead of germ cell number, which makes tilapia a potential surrogate model for germ cell transplantation.

Abstract Background The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability. Results Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA. Conclusions CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.

Identifying objectively the processes of regional climate events and evaluating quantitatively their intensity are the basis for accurately monitoring and assessing the impacts of events. Using the data of daily mean precipitation and temperature from 80 national meteorological stations in Anhui Province during 1961-2020, the processes of regional extreme climate events are identified. According to the averaged intensity, duration and average influence range, the intensity evaluation model of regional process is constructed and the intensity grade is divided by the percentile method. The 775 regional rainstorm events are captured, and the annual average frequency is 12.9 times during 1961-2020, including 34 extremely strong regional events, about once in two years. The average duration of single regional rainstorm event is about 1-2 days, and the daily average spatial coverage of a single event covers 37 stations. The frequency and intensity of regional rainstorm event has increased significantly in nearly 60 years. In the past 60 years, there have been 152 regional drought events in Anhui Province, including 9 extremely strong regional events with a return period of about 6~7 years. The average duration of single regional drought event is about 15-60 days. The linear change trend of annual frequency of regional drought event is not obvious, but the inter-annual variation is large. Compared with the summary of climate monitoring services and historical cases, the assessment model is reasonable and consistent with historical extreme cases, having good correspondence with historical disaster data in time, range, and intensity. This index will provide a reference for the quantitative assessment of regional extreme climate events in other areas, and it is also an important technical support for timely monitoring and early warning for the extreme climate events.

Although previous studies reported that gut fungal microbiota was associated with Crohn’s disease (CD), only a few studies have focused on the correlation between gut fungi and clinical phenotypes of CD. Here, we aimed to analyze the association between intestinal fungi and the occurrence of CD, disease activity, biological behaviors, and perianal lesions. Stool samples from subjects meeting the inclusion and exclusion criteria were collected for running internal transcribed spacer 2 (ITS2) high-throughput sequencing. Then, correlation analysis was conducted between intestinal fungi and different clinical groups. There were 45 patients with CD and 17 healthy controls (HCs) enrolled. Results showed that two phyla, Rozellomycota and Mortierellomycota, were not present in patients with CD compared to HCs. At the same time, there was a higher abundance of fungal genera and species belonging to the phylum Ascomycota in patients with CD. SparCC network analysis showed fewer interactions among the fungal communities in patients with CD compared to HCs. Exophiala dermatitidis was positively associated with the clinical active stage and platelet count. The genus Candida was with significantly higher abundance in the non-B1 CD group based on the Montreal classification. Clonostachys, Humicola, and Lophiostoma were significantly enriched in patients with CD with perianal lesions. Our results demonstrated that the composition of the intestinal fungal microbiota in patients with CD and HCs was markedly different, some of which might play a pathogenic role in the occurrence of CD and perianal lesions. Exophiala dermatitidis and genus Candida might be associated with active disease stage and type non-B1 CD (CD with intestinal stenosis or penetrating lesions, or both), respectively.

Abstract Background World Health Organization recommends countries introducing new drug and short treatment regimen for drug resistant tuberculosis (DR-TB) should develop and implement a system for active pharmacovigilance that allows for detection, reporting and management of adverse events. The aim of the study is to evaluate the frequency and severity of adverse events (AEs) of bedaquiline-containing regimen in a cohort of Chinese patients with multidrug-resistant (MDR)/extensively drug-resistant (XDR)-TB based on active drug safety monitoring (aDSM) system of New Drug Introduction and Protection Program (NDIP). Methods AEs were prospectively collected with demographic, bacteriological, radiological and clinical data from 54 sites throughout China at patient enrollment and during treatment between February, 2018 and December, 2019. This is an interim analysis including patients who are still on treatment and those that have completed treatment. A descriptive analysis was performed on the patients evaluated in the cohort. Results By December 31, 2019, a total of 1162 patients received bedaquiline-containing anti-TB treatment. Overall, 1563 AEs were reported, 66.9% were classified as minor (Grade 1–2) and 33.1% as serious (Grade 3–5). The median duration of bedaquiline treatment was 167.0 [interquartile range (IQR): 75–169] days. 86 (7.4%) patients received 36-week prolonged treatment with bedaquiline. The incidence of AEs and serious AEs was 47.1% and 7.8%, respectively. The most frequently reported AEs were QT prolongation (24.7%) and hepatotoxicity (16.4%). There were 14 (1.2%) AEs leading to death. Out of patients with available corrected QT interval by Fridericia's formula (QTcF) data, 3.1% (32/1044) experienced a post-baseline QTcF ≥ 500 ms, and 15.7% (132/839) had at least one change of QTcF ≥ 60 ms from baseline. 49 (4.2%) patients had QT prolonged AEs leading to bedaquiline withdrawal. One hundred and ninety patients reported 361 AEs with hepatotoxicity ranking the second with high occurrence. Thirty-four patients reported 43 AEs of hepatic injury referred to bedaquiline, much lower than that referred to protionamide, pyrazinamide and para-aminosalicylic acid individually. Conclusions Bedaquiline was generally well-tolerated with few safety concerns in this clinical patient population without any new safety signal identified. The mortality rate was generally low. These data inform significant positive effect to support the WHO recent recommendations for the wide use of bedaquiline.

Based on direct anonymous attestation of trusted computing,a wireless anonymous authentication scheme was proposed,the platform of the mobile node was verified by the foreign network agent and the identity of the mobile node user was authenticated by the home network agent and the foreign network agent together.By using of temporary identi-ties and one time secret keys,identity anonymity and domain separation property are achieved.The analysis shows that the scheme is secure,reliable,and with higher performance.

The current access protocols of WLAN Mesh network and trusted network could not satisfy the requirements of performance and security when the trusted access is needed in WLAN Mesh networks.Then,a provably secure trusted access protocol MN-TAP for the WLAN Mesh networks was proposed.Such protocol will achieve authentication and key confirmation among the access requestor,policy enforcement point and policy decision point.At the same time,the protocol could realize the platform authentication and platform integrity verification in the first round of protocol interaction,which would improve the efficiency of the protocol and decrease the overhead of the servers.A provably secure model was used,named universally composable secure model,to analyze the security of the protocol.And the performance of the protocol was analyed.The results show that: the protocol is a UC-secure protocol,and the performance has great advantages over the current protocols.

With computer and network as setting in collaborative learning, due to the changes on learning environment and mode, the roles of teachers online are faced with challenges. This study is to examine functions of online teachers’ roles in computer based college English collaborative learning. In the test carried in college English writing course, teachers perform roles of guide, assistant, facilitator, coordinator, etc. in collaborative learning procedure, namely the steps of Imputing, Drafting, Revising, Final drafting and Final evaluation. For functions of teachers’ online and effects of the test, questionnaires on students’ recognition, understanding, satisfaction and expectations of teachers’ roles are employed respectively. From results of questionnaires and interviews, students’ response prove that most learners’ have already confirmed online teachers’ roles, yet to fully realize their responsibilities, adequate measures and strategies must be taken to enrich teachers’ online serving experiences.

This study is to examine the acceptability of peer feedback in computer aided college English writing instruction. For the whole writing procedure, subjects in language labs were to take preparation, writing and evaluation stages, undergoing three editions suggested by peer feedbacks. For the first time reading, peers would comment on such general scale as subject and content of drafts, and the second focuses on specific items, for example the morphological, lexical and syntactical aspects, etc. To compare students’ attitudes towards the trial, before and after the experiment, Pre-Questionnaire and Post-Questionnaire were adopted to analyze differences. From the frequency of students’ views on ten statements, it is drawn that majority of subjects accept and enjoy the innovative writing process, since peer feedback is an essential and encouraging part for writing.