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Precise genetic modifications in model organisms are essential for biomedical research. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here we first optimize SpG and SpRY systems in zebrafish by purifying protein combined with synthetically modified gRNA. SpG shows high editing efficiency at NGN PAM sites, whereas SpRY efficiently edit PAM-less sites in the zebrafish genome. Then, we generate the SpRY-mediated cytosine base editor SpRY-CBE4max and SpRY-mediated adenine base editor zSpRY-ABE8e. Both target relaxed PAM with up to 96% editing efficiency and high product purity. With these tools, some previously inaccessible disease-relevant genetic variants are generated in zebrafish, supporting the utility of high-resolution targeting across genome-editing applications. Our study significantly improves CRISPR-Cas targeting in the genomic landscape of zebrafish, promoting the application of this model organism in revealing gene function, physiological mechanisms, and disease pathogenesis. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here the authors show that SpG and SpRY could edit NGN and NNN PAMs in zebrafish using the purified protein with synthetically modified gRNA, respectively.

To examine whether the use of natural language processing (NLP) technology is effective in assisting rapid title and abstract screening when updating a systematic review. Using the searched literature from a published systematic review, we trained and tested an NLP model that enables rapid title and abstract screening when updating a systematic review. The model was a light gradient boosting machine (LightGBM), an ensemble learning classifier which integrates four pretrained Bidirectional Encoder Representations from Transformers (BERT) models. We divided the searched citations into two sets (ie, training and test sets). The model was trained using the training set and assessed for screening performance using the test set. The searched citations, whose eligibility was determined by two independent reviewers, were treated as the reference standard. The test set included 947 citations; our model included 340 citations, excluded 607 citations, and achieved 96% sensitivity, and 78% specificity. If the classifier assessment in the case study was accepted, reviewers would lose 8 of 180 eligible citations (4%), none of which were ultimately included in the systematic review after full-text consideration, while decreasing the workload by 64.1%. NLP technology using the ensemble learning method may effectively assist in rapid literature screening when updating systematic reviews.

Unified visual perception requires integration of bottom-up and top-down inputs in the primary visual cortex (V1), yet the organization of top-down inputs in V1 remains unclear. Here, we used optogenetics-assisted circuit mapping to identify how multiple top-down inputs from higher-order cortical and thalamic areas engage V1 excitatory and inhibitory neurons. Top-down inputs overlap in superficial layers yet segregate in deep layers. Inputs from the medial secondary visual cortex (V2M) and anterior cingulate cortex (ACA) converge on L6 Pyrs, whereas ventrolateral orbitofrontal cortex (ORBvl) and lateral posterior thalamic nucleus (LP) inputs are processed in parallel in Pyr-type-specific subnetworks (Pyr ←ORBvl and Pyr ←LP ) and drive mutual inhibition between them via local interneurons. Our study deepens understanding of the top-down modulation mechanisms of visual processing and establishes that V2M and ACA inputs in L6 employ integrated processing distinct from the parallel processing of LP and ORBvl inputs in L5. The organization of top-down inputs in primary visual cortex (V1) remains unclear. Here the authors characterized corticocortical and thalamocortical top-down inputs recruiting V1 neurons with cell-type and layer-specificities, and revealed distinct forms of top-down input processing.

The emergence of super-resolution (SR) fluorescence microscopy has rejuvenated the search for new cellular sub-structures. However, SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment. Thus, we developed SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional optical diffraction tomography (ODT) with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving the mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum, and lysosomes. Using dual-mode correlated live-cell imaging for a prolonged period of time, we observed novel subcellular structures named dark-vacuole bodies, the majority of which originate from densely populated perinuclear regions, and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane. This work demonstrates the unique capabilities of SR-FACT, which suggests its wide applicability in cell biology in general.Microscopy: New tool furthers our understanding of living cellsScientists from China have developed a new microscopy tool that could help to further expand our understanding of cell biological processes in live cells. Although the emergence of superresolution (SR) fluorescence microscopy is revolutionising the life sciences, the technique can only be used to observe a handful of biomolecules simultaneously and cannot provide a holistic map of the cellular landscape. Furthermore, the imaging of live cells in three dimensions (3D) over time also remains very challenging. By combining two different techniques, a team of Chinese researchers led by Liangyi Chen and Kebin Shi from Peking University has created a novel dual-mode high-speed SR microscopy technique. The tool is capable of revealing previously unseen sub-cellular structures and dynamic processes, allowing both the cellular landscape and molecular identity in live cells to be observed in 3D over prolonged periods.

Accumulating evidence indicates that mitotic checkpoint serine/threonine kinase B (BUB1B) plays a critical role in multiple types of cancer. However, the biological function and molecular regulatory mechanism of BUB1B in glioblastoma (GBM) remain unclear. In the present study, we identified that BUB1B expression was enriched in GBM tumors and was functionally required for tumor proliferation both in vitro and in vivo. Clinically, BUB1B expression was associated with poor prognosis in GBM patients and BUB1B-dependent radioresistance in GBM was decreased by targeting BUB1B via shRNAs. Mechanistically, forkhead box protein M1 (FOXM1) transcriptionally regulated BUB1B expression by binding to and then activating the BUB1B promoter. Therapeutically, we found that FOXM1 inhibitor attenuated tumorigenesis and radioresistance of GBM both in vitro and in vivo. Altogether, BUB1B promotes tumor proliferation and induces radioresistance in GBM, indicating that BUB1B could be a potential therapeutic target for GBM.

Interhemispheric communication through the corpus callosum is required for both sensory and cognitive processes. Impaired transcallosal inhibition causing interhemispheric imbalance is believed to underlie visuospatial bias after frontoparietal cortical damage, but the synaptic circuits involved remain largely unknown. Here, we show that lesions in the mouse anterior cingulate area (ACA) cause severe visuospatial bias mediated by a transcallosal inhibition loop. In a visual-change-detection task, ACA callosal-projection neurons (CPNs) were more active with contralateral visual field changes than with ipsilateral changes. Unilateral CPN inactivation impaired contralateral change detection but improved ipsilateral detection by altering interhemispheric interaction through callosal projections. CPNs strongly activated contralateral parvalbumin-positive (PV+) neurons, and callosal-input-driven PV+ neurons preferentially inhibited ipsilateral CPNs, thus mediating transcallosal inhibition. Unilateral PV+ neuron activation caused a similar behavioral bias to contralateral CPN activation and ipsilateral CPN inactivation, and bilateral PV+ neuron activation eliminated this bias. Notably, restoring interhemispheric balance by activating contralesional PV+ neurons significantly improved contralesional detection in ACA-lesioned animals. Thus, a frontal transcallosal inhibition loop comprising CPNs and callosal-input-driven PV+ neurons mediates interhemispheric balance in visuospatial processing, and enhancing contralesional transcallosal inhibition restores interhemispheric balance while also reversing lesion-induced bias. Impaired transcallosal inhibition is believed to underlie visuospatial bias after frontoparietal damage, but the synaptic circuits involved remain largely unknown. Here, authors show a transcallosal inhibition loop in the anterior cingulate area that functions in visuospatial processing by maintaining balanced interhemispheric interactions.

Dihydroartemisinin (DHA), the primary of artemisinin extracted from the traditional Chinese medicine Artemisia annua, has been used in malaria treatment for a long time. Recently, many studies have indicated that, in addition to antimalarial effects, DHA also exhibits anticancer activity in certain types of neoplasms, including ovarian cancer. However, the precise anti‐ovarian cancer mechanism of DHA is still unclear. Abnormal activation of the hedgehog (Hh) pathway is closely related to tumorigenesis and progression of ovarian cancer. We performed this study to elucidate the effects of DHA on the biological behavior of ovarian cancer cells and to determine its effects on the Hh signaling pathway. CCK8 assays and flow cytometry were used to evaluate the effects of DHA on cell viability and apoptosis in both ovarian cancer cells and HOSEPICs (human ovarian surface epithelial cells) in response to DHA treatment. Transwell membrane chambers were used to analyze the effects of DHA on the migration and invasion of epithelial ovarian cancer cells following treatment with DHA. The impact of DHA on Hh signaling was analyzed by RT‐qPCR and Western blot. DHA significantly inhibited proliferation, migration, and invasion of ovarian cancer cells, and induced apoptosis in vitro. In contrast, DHA had few effects on cell proliferation and apoptosis in HOSEPICs. DHA inhibited the hedgehog signaling pathway. Furthermore, DHA inhibited purmorphamine (Hh signaling pathway agonist)‐induced cell proliferation, cell migration, and cell invasion and the inhibition of apoptosis. Importantly, DHA enhanced GANT61 (hedgehog signaling pathway inhibitor)‐induced apoptosis and the inhibition of cell viability, migratory capacity, and invasive ability. This study demonstrates that DHA inhibits cell viability, migration, and invasion, as well as induces apoptosis in epithelial ovarian cancer through suppression of the Hh signaling pathway. Dihydroartemisinin (DHA), the main active products of artemisinin extracted from traditional Chinese medicine Artemisia annua, has been used as an anti‐malarial drug since ancient China. In recent years, DHA has also been shown to exert anticancer effects in certain types of cancer, including ovarian cancer. However, the precise molecular mechanisms of DHA exerts anti‐cancer effects in ovarian cancer are still unclear. The present study demonstrates that DHA induces apoptosis and inhibits proliferation, migration, and invasion in epithelial ovarian cancer via inhibiting the hedgehog signaling pathway.

PurposeTo evaluate the efficacy of metformin plus first-line chemotherapy versus chemotherapy alone in the treatment of epithelial ovarian cancer.MethodsEpithelial ovarian cancer patients without diabetes mellitus were allocated to non-metformin group (paclitaxel plus carboplatin) or metformin group (paclitaxel plus carboplatin plus metformin). The primary endpoint was progression-free survival (PFS) and disease-free survival (DFS).ResultsA total of 20 patients were assigned to metformin group and 24 patients to non-metformin group. The baseline information in two groups had no significant difference. The PFS and DFS of patients with metformin intake versus without metformin intake was 23 versus 21 months (p = 0.68) and 29 versus 26 months (p = 0.61), respectively. The PFS and DFS of patients with normal weight versus obese/overweight were 23 versus 17 months (p = 0.14) and 27 versus 23 months (p = 0.50), respectively. Metformin effectively inhibited the increase of IGF-1 and maintained the IGFBP-1.ConclusionsWithin the limitations of the small sample size, there was no evidence of meaningful effect on PFS by metformin even though evidence of modulation of IGF-1 signaling axis was apparent.

Switchgrass is a promising energy crop has the potential to mitigate global warming and energy security, improve local ecology and generate profit. Its quantitative traits, such as biomass productivity and environmental adaptability, are determined by genotype‐by‐environment interaction (GEI) or response of genotypes grown across different target environments. To simulate the yield of switchgrass outside its original habitat, a genotype‐specific growth model, SwitchFor that captures GEI was developed by parameterising the MiscanFor model. Input parameters were used to describe genotype‐specific characteristics under different soil and climate conditions, which enables the model to predict the yield in a wide range of environmental and climate conditions. The model was validated using global field trail data and applied to estimate the switchgrass yield potentials on the marginal land of the Loess Plateau in China. The results suggest that upland and lowland switchgrass have significant differences in the spatial distribution of the adaptation zone and site‐specific biomass yield. The area of the adaption zone of upland switchgrass was 4.5 times of the lowland ecotype's. The yield difference between upland and lowland ecotypes ranges from 0 to 34 Mg ha−1. The weighted average yield of the lowland ecotype (20 Mg ha−1) is significantly higher than the upland type (5 Mg ha−1). The optimal yield map, generated by comparing the yield of upland and lowland ecotypes based on 1 km2 grid locations, illustrates that the total yield potential of the optimal switchgrass is 61.6–106.4 Tg on the marginal land of the Loess Plateau, which is approximately twice that of the individual ecotypes. Compared with the existing models, the accuracy of the yield prediction of switchgrass is significantly improved by using the SwitchFor model. This spatially explicit and cultivar‐specific model provides valuable information on land management and crop breeding and a robust and extendable framework for yield mapping of other cultivars. A new cultivar‐specific switchgrass plant growth model, SwitchFor model, was developed and validated to give an accurate estimation of the switchgrass yield in a wide environment. The SwitchFor model was applied to the Loess Plateau region, which has about 12.8–20.8 Mha marginal land to estimate the yield potential. It was estimated that the Loess Plateau has a potential to produce up to 62–106 Tg switchgrass by considering the upland and lowland ecotype.

Cytosine base editing is a powerful tool for making precise single nucleotide changes in cells and model organisms like zebrafish, which are valuable for studying human diseases. However, current base editors struggle to edit cytosines in certain DNA contexts, particularly those with GC and CC pairs, limiting their use in modelling disease-related mutations. Here we show the development of zevoCDA1, an optimized cytosine base editor for zebrafish that improves editing efficiency across various DNA contexts and reduces restrictions imposed by the protospacer adjacent motif. We also create zevoCDA1-198, a more precise editor with a narrower editing window of five nucleotides, minimizing off-target effects. Using these advanced tools, we successfully generate zebrafish models of diseases that were previously challenging to create due to sequence limitations. This work enhances the ability to introduce human pathogenic mutations in zebrafish, broadening the scope for genomic research with improved precision and efficiency. Cytosine base editing is crucial for modeling human diseases in zebrafish. Here, the authors present zevoCDA1 and zevoCDA1-198, optimized editors that improve editing efficiency and precision, allowing zebrafish modeling for disease-related mutations which were previously limited by DNA sequence contexts.