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Translation of the CRISPR–Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci ( HBB , IL2RG , CCR5 , HEXB , and TRAC ) in human CD34 + hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications. A bacterial screen yields a Cas9 variant that retains high on-target activity when delivered in the RNP format. As proof of principle, this Cas9 variant enables high-level correction of the sickle cell disease mutation in patient-derived HSPCs.

River water surface extent can be extracted from optical and radar satellite images; this is useful for estimating streamflow from space. The radiation characteristics of open water from the visible and microwave bands are different and provide independent information. In this study, for the purpose of improving streamflow estimation from space for data-sparse regions, a method that combines satellite optical and radar images data for streamflow estimation using a machine learning technique was proposed. The method was demonstratedthrough a case study in the river segment upstream of the Ganzi gauging station on the Yalong River, China. Utilizing the support vector regression (SVR) model, the feasibility of different combinations of water surface area derived from Sentinel-1 synthetic aperture radar images (AREA_SAR), modified normalized difference water index derived from Landsat 8 images (MNDWI), and reflectance ratios between NIR and SWIR channels derived from MODIS images (RNIR/RSWIR) for streamflow estimation were evaluated through three experiments. In Experiment I, three models using AREA_SAR (Model 1), MNDWI (Model 2), and a combination of AREA_SAR and MNDWI (Model 3) were built; the mean relative error (MRE) and mean absolute error (MAE) of streamflow estimates corresponding to the SVR model using both AREA_SAR and MNDWI (Model 3) were 0.19 and 31.6 m3/s for the testing dataset, respectively, and were lower than two models using AREA_SAR (Model 1) or MNDWI (Model 2) solely as inputs. In Experiment II, three models with AREA_SAR (Model 4), RNIR/RSWIR (Model 5), and a combination of AREA_SAR and RNIR/RSWIR (Model 6) as inputs were developed; the MRE and MAE for the model using AREA_SAR and RNIR/RSWIR (Model 6) were 0.25 and 56.5 m3/s, respectively, which outperformed the two models treating AREA_SAR (Model 4) or MNDWI (Model 5) as single types of inputs. In Experiment III, three models using AREA_SAR (Model 7), MNDWI, and RNIR/RSWIR (Model 8) and the combination of AREA_SAR, MNDWI and RNIR/RSWIR (Model 9) were built; combining all three types of satellite observations (Model 9) exhibited the highest accuracy, for which the MRE and MAE were 0.18 and 18.4 m3/s, respectively. The results of all three experiments demonstrated that integrating optical and microwave observations could improve the accuracy of streamflow estimates using a data-driven model; the proposed method has great potential for near-real-time estimations of flood magnitude or to reconstruct past variations in streamflow using historical satellite images in data-sparse regions.

Satellite and reanalysis-based precipitation products have played a crucial role in addressing the challenges associated with limited ground-based observational data. These products are widely utilized in hydrometeorological research, particularly in data-scarce regions like the Qinghai–Tibetan Plateau (QTP). This study proposed an ensemble streamflow simulation method using remote sensing precipitation data as input. By employing a 1D Convolutional Neural Networks (1D CNN), streamflow simulations from multiple models are integrated and a Shapley Additive exPlanations (SHAP) interpretability analysis was conducted to examine the contributions of individual models on ensemble streamflow simulation. The method is demonstrated using GPM IMERG (Global Precipitation Measurement Integrated Multi-satellite Retrievals) remote sensing precipitation data for streamflow estimation in the upstream region of the Ganzi gauging station in the Yalong River basin of QTP for the period from 2010 to 2019. Streamflow simulations were carried out using models with diverse structures, including the physically based BTOPMC (Block-wise use of TOPMODEL) and two machine learning models, i.e., Random Forest (RF) and Long Short-Term Memory Neural Networks (LSTM). Furthermore, ensemble simulations were compared: the Simple Average Method (SAM), Weighted Average Method (WAM), and the proposed 1D CNN method. The results revealed that, for the hydrological simulation of each individual models, the Kling–Gupta Efficiency (KGE) values during the validation period were 0.66 for BTOPMC, 0.71 for RF, and 0.74 for LSTM. Among the ensemble approaches, the validation period KGE values for SAM, WAM, and the 1D CNN-based nonlinear method were 0.74, 0.73, and 0.82, respectively, indicating that the nonlinear 1D CNN approach achieved the highest accuracy. The SHAP-based interpretability analysis further demonstrated that RF made the most significant contribution to the ensemble simulation, while LSTM contributed the least. These findings highlight that the proposed 1D CNN ensemble simulation framework has great potential to improve streamflow estimations using remote sensing precipitation data as input and may provide new insight into how deep learning methods advance the application of remote sensing in hydrological research.

The study describes the use of integrative approaches to search for candidate therapeutic targets for DIPG, and the identification of an HDAC inhibitor as a potential treatment strategy Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi–histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Icariin (IC) promotes osteogenic differentiation, and it may be a potential small molecule drug for local application in bone regeneration. Icariin-loaded hydroxyapatite/alginate (IC/HAA) porous composite scaffolds were designed in this study for the potential application of the sustainable release of icariin and subsequent bone regeneration. An icariin-loaded hydroxyapatite/alginate porous composite scaffold was prepared and characterized by SEM and HPLC for morphology and release behavior, respectively. The mechanical properties, degradation in PBS and cytotoxicity on BMSCs were also evaluated by MTT assay, compression strength and calculation of weight remaining ratio, respectively. Rabbit BMSCs were cocultured with IC/HAA scaffolds, and ALP activity and Alizarin Red staining were performed to evaluate osteogenic differentiation induction. The mRNA and protein expression level of an osteogenic gene was detected by RT-PCR and Western blotting, respectively. In vivo animal models of critical bone defects in the radius of rabbit were used. Four and 12 weeks after the implantation of IC/HAA scaffolds in the bone defect, radiographic images of the radius were obtained and scored by using the Lane and Sandhu X-ray scoring system. Tissue samples were also evaluated using H&E and Masson staining, and an osteogenic gene and Wnt signaling pathway genes were detected. A hydroxyapatite/alginate (HAA) porous composite scaffold-loaded icariin was fabricated using a freeze-drying method. Our data indicated that the icariin was loaded in alginate scaffold without compromising the macro/microstructure or mechanical properties of the scaffold. Notably, the IC/HAA promoted the proliferation of rBMSCs without exerting cytotoxicity on rBMSCs. In vivo, rabbit radius bone defect experiments demonstrated that the IC/HAA scaffold exhibited better capacity for bone regeneration than HAA, and IC/HAA upregulated the relative expression levels of an osteogenic gene and the Wnt signaling pathway genes. Most notably, the IC/HAA scaffold also inhibited osteoclast activity in vivo. Our data suggests a promising application for the use of HAA scaffolds to load icariin and promote bone regeneration in situ through mediation of the coupling processes of osteogenesis induction and osteoclast activity inhibition.

Objective To study the outcome of human papillomavirus (HPV) infection in women with cervical pathology results of non-cervical intraepithelial neoplasia (CIN) or cervical cancer and positive high-risk HPV test, as well as analyze the associated risk factors affecting the outcome of infection. Methods To investigate the outcome of high-risk (HR)-HPV infection in the female genital tract and analyze the associated risk factors affecting their outcome, a total of 196 women with positive HR-HPV test results and non-CIN or cervical cancer cervical pathology results were selected for follow-up at the Cervical Disease Clinic of the Obstetrics and Gynecology Hospital, Zhejiang University School of Medicine from January 2017 to March 2020. The follow-up interval was every 6 months, and both cervical cytology (TCT) and HR-HPV testing were performed at each follow-up visit. If the cervical cytology results were normal upon recheck and the HR-HPV test was negative, the woman was considered to be cleared of the HPV infection and was entered into the routine cervical screening population. When the repeat HR-HPV test remained positive after 6 months, the woman was defined as having a persistent HR-HPV infection. If HR-HPV persisted but the TCT results were normal, follow-up was continued. If HR-HPV persisted and the TCT results were abnormal, a colposcopy-guided biopsy was performed immediately. In this situation, if the histological results were still non-CIN or cervical cancer, the follow-up was continued. If the histological results confirmed the development of CIN or invasive cancer, then enter another study follow-up to further track its development and outcome, and the woman commenced the treatment process. The HPV infection clearance time was analyzed by the Kaplan-Meier method, and the comparison of the HPV clearance rate and infection clearance time between each of the different groups was performed using aχ 2 test or Fisher’s exact test, as appropriate. After the univariate analysis, several significant factors were included in the Cox model and independent risk factors were analyzed. Results A total of 163 women were enrolled in this study. The median age was 40.0 years (22–67 years) and the median follow-up time was 11.5 months (6–31 months). The spontaneous clearance rate of HR-HPV infection was 51.5%, and the median time to viral clearance was 14.5 months. Age and the initial viral load were high risk factors affecting the spontaneous clearance of HR-HPV infection. The factors significantly associated with HPV clearance rate and time to HPV clearance consisted of menopause and full-term delivery ( P  < 0.05). Conclusions In women with normal or low-grade lesions on the cell smear, the spontaneous clearance rate of HR-HPV infection was 51.5% and the time to clearance was 14.5 months. Age and the initial viral load were independent associated factors affecting the spontaneous clearance of HR-HPV infection in the female genital tract. These findings suggest that non-young women or those with high viral loads have a higher rate of persistent HR-HPV infection. Thus, intensive screening should be recommended.

Previous studies have reported that adenosine mono-phosphate-activated protein kinase (AMPK) activation can enhance osteoblast differentiation and mineralization; however, the underlying mechanism is not fully understood. Autophagy also serves an important role in osteoblast mineralization and bone homeostasis. The present study aimed to explore whether activation of AMPK could enhance osteoblast differentiation and mineralization via the induction of autophagy. The fracture healing and nonunion animal models were established and verified by X-ray imaging. Bone maturation was measured by Masson staining and the expression of AMPK, p-AMPK, microtubule-associated proteins 1A/1B light chain 3B II, and p62 in the fracture ends were detected by immunohistochemical staining. The mRNA expression levels of alkaline phosphatase (ALP), osteocalcin, runt-related transcription factor 2 and BCN1 were determined by reverse transcription-quantitative polymerase chain reaction. 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to determine ALP activity and alizarin red staining was adopted to examine mineralization. Western blot analysis was performed to detect protein expression. Autophagosome was observed by Transmission electron microscopy. Small interfering (si)RNA was used to knock down the expression of target gene. In vivo experiments demonstrated that new bone mineralization and maturation was markedly restrained in the nonunion group, alongside decreased AMPK activation and autophagic activity, compared with in the fracture healing group. The results of an in vitro study indicated that AMPK activation stimulated the osteogenic differentiation of MC3T3-E1 cells, with increases in ALP activity, mineralization, and the mRNA expression levels of ALP, osteocalcin and runt-related transcription factor 2. Furthermore, AMPK activation induced autophagy, as determined by upregulation of microtubule-associated proteins 1A/1B light chain 3B, increased autophagosome density and downregulation of p62. In addition, inhibition of autophagy reversed the effects of AMPK activation on osteoblast differentiation. These results suggested that AMPK activation may stimulate osteoblast differentiation and mineralization via the induction of autophagy, and provides evidence to suggest that enhancing AMPK activation and autophagic activity may be a potential novel approach to promote fracture healing.

Neuropathic pain (NP) is caused by primary damage and dysfunction of nervous system, in which spinal cord injury (SCI) is a common cause of NP. Evidence shows that neuroinflammation and oxidative stress are related to the pathophysiology of NP, in which the activation of microglia and astrocytes in spinal is significant. Therefore, understanding the molecular mechanism of NP after SCI is of great significance. The rat model of SCI was established and BV2 cell was treated with LPS. The exosomes derived from astrocytes were extracted by centrifugation. The morphology of the exosomes was observed by electron microscope and the surface markers were detected by Western blot. LncRNA in astrocytes and astrocyte-derived exosomes were detected by qRT-PCR. The expression of microglia activation markers CD68 and Iba-1 was detected by immunohistochemistry. The von Frey test was applied to assess mechanical hypersensitivity. The heat plate analgesia instrument was used to evaluate Paw withdrawal latency (PWL). QRT-PCR used to detect expression of LncRNA49rik and miR-10a-5p. Western blot was used to detect MAPK/PI3K/AKT / mTOR signal pathway and COX2, iNOS. The content of MDA and the activity of SOD were detected by oxidative stress kit. The concentrations of IL-6, IL-1β, IL-18 and IFN-αwere detected by ELISA. The targeting relationship between LncRNA49rik and miR-10a-5p was analyzed by bioinformatics and double luciferase activity, Rip and FISH experiments. LncRNA49rik was highly expressed in astrocytes and its derived exosomes. SCI stimulated astrocytes to release exosome containing LncRNA49rik and promote microglia activation to increase inflammatory response. At the same time, overexpression of LncRNA49rik increased the incidence of NP and aggravated the level of inflammation and oxidative stress in rats with SCI. MiR-10a-5p is the target of LncRNA4933431K23Rik. Overexpression of LncRNA49rik significantly inhibited the up-regulation of miR-10a-5p. Overexpression of miR-10a-5p inhibited hyperalgesia and inflammation in SCI rats. In addition, transfection of miR-10a-5p mimics significantly inhibited the expression of MAPK/PI3K/AKT and up-regulated the expression of mTOR. Mechanism studies have shown that overexpression of miR-10a-5p weakens the phenotypic induction of microglia induced by LncRNA4933431K23Rik. LncRNA4933431K23Rik regulates microglial phenotype through inhibiting miR-10a-5p, which is responsible for NP induced by SCI.

Background Dengue virus (DENV) is the most widespread arbovirus. The World Health Organization (WHO) declared dengue one of the top 10 global health threats in 2019. However, it has been underrepresented in bibliometric analyses. This study employs bibliometric analysis to identify research hotspots and trends, offering a comprehensive overview of the current research dynamics in this field. Results We present a report spanning from 1995 to 2023 that provides a unique longitudinal analysis of Dengue virus (DENV) research, revealing significant trends and shifts not extensively covered in previous literature. A total of 10,767 DENV-related documents were considered, with a notable increase in publications, peaking at 747 articles in 2021. Plos Neglected Tropical Diseases has become the leading journal in Dengue virus research, publishing 791 articles in this field—the highest number recorded. Our bibliometric analysis provides a comprehensive mapping of DENV research across multiple dimensions, including vector ecology, virology, and emerging therapies. The study delineates a complex network of immune response genes, including IFNA1, DDX58, IFNB1, STAT1, IRF3, and NFKB1, highlighting significant trends and emerging themes, particularly the impacts of climate change and new outbreaks on disease transmission. Our findings detail the progress and current status of key vaccine candidates, including the licensed Dengvaxia, newer vaccines such as Qdenga and TV003, and updated clinical trials. The study underscores significant advancements in antiviral therapies and vector control strategies for dengue, highlighting innovative drug candidates such as AT-752 and JNJ-1802, and the potential of drug repurposing with agents like Ribavirin, Remdesivir, and Lopinavir. Additionally, it discusses biological control methods, including the introduction of Wolbachia-infected mosquitoes and gene-editing technologies. Conclusion This bibliometric study underscores the critical role of interdisciplinary collaboration in advancing DENV research, identifying key trends and areas needing further exploration, including host-virus dynamics, the development and application of antiviral drugs and vaccines, and the use of artificial intelligence. It advocates for strengthened partnerships across various disciplines to effectively tackle the challenges posed by DENV.