Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by ...TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.
Nuclear factor kappa B (NF-κB), a key nuclear transcription factor, is associated with prognosis in a variety of human cancers. However, the clinical value of NF-κB in non-small cell lung cancer ...(NSCLC) is still controversial. Therefore, the aim of this meta-analysis was to obtain an accurate evaluation of the relationship between NF-κB expression and survival prognosis of NSCLC patients based on published articles. PubMed, EMBASE and Web of Science databases were systematically searched for potential articles. A total of 1159 patients from 7 eligible studies comparing prognostic significance of NF-κB expression levels in NSCLC were included in our meta-analysis. I2 statistic and P value were performed to evaluate heterogeneity. The results of analysis were presented as hazard ratio (HR) or odds ratios (OR) with 95% confidence interval (95% CI). Subgroup analysis based on ethnicity of NSCLC patients and NF-kB cellular localization within cancer cells were conducted to illustrate the potential discrepancy. Significant heterogeneity was considered at I2>50% and P<0.05, and random-effects model was used. The combined results indicated that higher NF-κB expression was associated with shorter overall survival (OS) of NSCLC patients (HR = 2.78, 95% CI = 1.51-5.12, P = 0.001). Moreover, NF-κB expression was closely associated with tumor stage (HR = 0.32, 95% CI = 0.18-0.57, P<0.0001), lymph node metastasis (HR = 0.56, 95% CI = 0.38-0.83, P = 0.004) and 5-year OS for NSCLC patients (OR = 1.83, 95% CI = 1.02-3.31, P = 0.04). We conclude that NF-κB expression may be a potential unfavorable prognostic marker for NSCLC patients.
Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca ²⁺) dysregulation followed by rapid ...loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the F O of the F ₁F O ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca ²⁺ enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F ₁, providing a mechanism for mPTP opening. In contrast, recombinant F ₁ beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca ²⁺-induced IMM depolarization and inhibits Ca ²⁺ and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.
Rhoptry proteins (ROPs), secreted by specific rhoptry organelles of apicomplexan parasites, are determinants of parasite pathogenesis and sources of vaccine candidates. Twenty-eight ROPs of
Eimeria ...tenella
have been predicted by genomic approaches, and in the present study,
E. tenella
rhoptry protein 30 (EtROP30) was characterized. Subcellular localizations of EtROP30 in sporozoites and merozoites were in the apical complex and rhoptry-like bulb, suggesting that EtROP30 is a member of ROPs in
E. tenella
. Sequence analysis showed that EtROP30 contained an N-terminal secretory signal, a protein kinase domain with eight
E. tenella
-specific rhoptry kinase 1 subfamily (ROPK-Eten1) motifs, and a C-terminal nuclear localization sequence (NLS), making EtROP30 the only ROP that contains both a secretory signal and an NLS in
E. tenella
. Subsequent experiments showed that EtROP30 was a secreted protein in the sporozoite stage, relying on NLS for migration to the host nucleus. In addition, EtROP30 showed significantly higher expression levels in the parasite merozoite stage, indicating that EtROP30 plays a critical role during parasite reinvasion and development and may be a viable option as a vaccine candidate for anti-parasitic infection. The immunization protection efficacies of EtROP30 were evaluated. Significant improvements in mean body weight gain, reduction of cecum lesion score, and number of oocysts excreted were observed, indicating that EtROP30 has good immunogenicity against
E. tenella
. In the present study, a ROP of
E. tenella
with secretory and nuclear localization characteristics has been identified, and proved to be an effective vaccine candidate against this parasite.
The aim of the present study was to identify the effect of solute Ni element on the plastic deformation and its thermal activation process of 18Cr ferritic stainless steels. Single-crystal ...micropillars with different diameters of approximately 2 and 5 μm were fabricated on the surface of Fe–18%Cr–Ni alloy sheets containing different contents of Ni (0–2 mass%). Compression tests were performed on the micropillars at different initial strain rates to investigate the strain rate sensitivity (m) of stress for the slip initiation and the specimen-size dependence of the m value. In micropillars with a small diameter of 2 μm, the Fe–18%Cr binary alloy specimen exhibited a relatively higher strain-rate sensitivity of initial slip stress (m = 0.12), whereas m was reduced to 0.01 by adding 2% Ni. The same trend was observed in micropillars with a larger size of approximately 5 μm. The experimental results were utilized to evaluate the activation volume of plastic deformation. The activation volumes of the Fe–18%Cr alloy and alloy containing 1% Ni followed the specimen-size dependence trend of activation volume in bcc metals; however, the alloy containing 2% Ni exhibited larger activation volumes, suggesting a different thermal activation process presumably due to the significant interaction that occurred between dislocations and solute Ni atoms in bcc solid-solutions.
A new Athelia rolfsii exopolysaccharides (AEPS) were purified by Sephacryl S-300 and S-200. The physicochemical characteristics of AEPS fractions were assayed by HPGPC and GC methods. The structures ...of AEPS and AEPS‑zinc complex were characterized by SEM, FTIR and NMR. Moreover, the bioactivities of complex were also evaluated by experiments in vitro and in vivo. AEPSI consisted of glucose, galacturonic acid, talose, galactose, mannose and xylose, the relative contents of them were 24.74, 19.60, 33.65, 8.77, 7.97 and 5.28%, respectively. AEPSII consisted of glucose, inositol, galacturonic acid, ribitol, gluconic acid, talose and xylose, whose relative contents were 36.06, 21.21, 12.78, 11.07, 6.58, 5.45 and 6.82%, respectively. The Mw and Mn of AEPSI were 6.1324×104 and 1.4218×104Da, those of AEPSII were 517 and 248Da. SEM observations showed that microstructures of AEPS and AEPS‑zinc complex were obviously different both in size and shape. FTIR and NMR analysis indicated that AEPS might chelate with zinc ion through hydroxy and carboxy group. In vitro experiments showed that AEPS‑zinc complex had a good bioavailability, in vivo experiments showed that it had good effect on improving zinc deficiency and antioxidant activities, which suggested that it could be used as zinc supplementation with high antioxidant activities.
•Attention mechanism with LSTM (ALSTM) has been applied to improve wheat yield estimation.•ALSTM model produced more accurate crop yield estimates compared with LSTM model.•Attention mechanism ...facilitatesd interpretability of the LSTM network’s inner working.•ALSTM model provided stable performance at different sampling sites and years.
The rapid and effective acquisition of crop yield information is critical to the stability of food markets and development and implementation of related policies. It is an important baseline observation that is used for ensuring regional and global food security. In this study, a novel deep learning framework was developed for winter wheat yield estimation using meteorological data and two remotely sensed indices, Vegetation Temperature Condition Index (VTCI) and Leaf Area Index (LAI) at the main growth stages of winter wheat in the Guanzhong Plain. The proposed deep learning model was based on Long Short-Term Memory (LSTM) neural network with an attention mechanism (ALSTM), which the main idea is to assign attention to the key parts of the input sequence that affect the target vectors so that the specific features can be accurately extracted. The ALSTM model provided an improved estimation accuracy (R2 = 0.63, MAPE = 8.20%, RMSE = 502.71 kg/ha, NRMSE = 11.15%) as compared with the LSTM (R2 = 0.55, MAPE = 13.46%, RMSE = 699.92 kg/ha, NRMSE = 15.52%). A validation based on leave-one-year-out-validation further substantiated the robustness of ALSTM with smaller values of NRMSE and MAPE (13.63% and 11.54%). We demonstrated that the ALSTM model provided good generalization ability for sampling sites under different farming systems, including irrigation and rain-fed sampling sites. In addition, we evaluated the relative importance of each input variable in determining yields based on stepwise sensitivity analysis. It was found that LAI at the heading-filling stage and the milk stage as well as VTCI at the jointing stage contributed more than other input feature variables towards the corresponding yield. In conclusion, our findings highlighted that the attention mechanism helped to improve the interpretability of neural networks and the ALSTM model along with remotely sensed biophysical indices can provide a reliable and robust estimation of crop yield. An accurate estimation of wheat yield is not only helping towards informed crop management decisions but it will improve efficiency and sustainability of farming operations.
Isotope Dilution Laser Ablation ICP-MS (ID-LA-ICP-MS), because of its impressive spatial resolution capacity and precise means for quantification, is one of the most promising tools for in-situ ...quantitative imaging of trace elements in biological samples. In the ID-LA-ICP-MS strategy for tissue section, the tissue must be maintained intact during the whole sample preparation process. Therefore, how to homogeneously distribute enriched isotope spike on tissue section and how to confirm isotope equilibration between sample and spike are two important challenges. In this study, we reported a novel quantitative imaging strategy for biological thin section based on ID-LA-ICP-MS. To distribute the enriched isotope spikes on tissue section homogeneously, a “border” was constructed to make spike droplet stay on the tissue for isotope exchange. Laser ablation and isotope exchange parameters were also investigated to obtain optimal ID-LA-ICP-MS conditions. The prepared homogeneous in-house standard was used to validate the ID-LA-ICP-MS approach and good agreement with the bulk analysis was achieved. On this basis, quantitative imaging of Fe, Cu and Zn in real mouse brain of Alzheimer's Disease (AD) were measured by the improved methodology. Assessment of the method for real sample was undertaken by comparison of the LA-ICP-MS data with that obtained by micro-XRF. Moreover, comparative analysis of elements distribution and immunohistochemical markers in AD mouse brain was also carried out. The similar distributional patterns demonstrated that the proposed methodology is potential to investigate the correlation of biomarker heterogeneity and elements distribution, and may be useful to understand such complex brain mechanisms in the future.
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•A quantitative imaging strategy of intact brain section based on ID-LA-ICP-MS was proposed.•Addition methods of enriched isotope spikes were evaluated.•Isotope exchange condition was investigated to reach satisfactory isotope exchange.•The proposed strategy was validated and assessed by μ-XRF and immunohistochemistry.•Offer potential for absolute quantification in LA-ICP-MS imaging in different bio-analytical analysis.
The Max Planck Institute Grand Ensemble (MPI‐GE) is the largest ensemble of a single comprehensive climate model currently available, with 100 members for the historical simulations (1850–2005) and ...four forcing scenarios. It is currently the only large ensemble available that includes scenario representative concentration pathway (RCP) 2.6 and a 1% CO2 scenario. These advantages make MPI‐GE a powerful tool. We present an overview of MPI‐GE, its components, and detail the experiments completed. We demonstrate how to separate the forced response from internal variability in a large ensemble. This separation allows the quantification of both the forced signal under climate change and the internal variability to unprecedented precision. We then demonstrate multiple ways to evaluate MPI‐GE and put observations in the context of a large ensemble, including a novel approach for comparing model internal variability with estimated observed variability. Finally, we present four novel analyses, which can only be completed using a large ensemble. First, we address whether temperature and precipitation have a pathway dependence using the forcing scenarios. Second, the forced signal of the highly noisy atmospheric circulation is computed, and different drivers are identified to be important for the North Pacific and North Atlantic regions. Third, we use the ensemble dimension to investigate the time dependency of Atlantic Meridional Overturning Circulation variability changes under global warming. Last, sea level pressure is used as an example to demonstrate how MPI‐GE can be utilized to estimate the ensemble size needed for a given scientific problem and provide insights for future ensemble projects.
Key Points
The 100‐member MPI‐GE is currently the largest publicly available ensemble of a comprehensive climate model
MPI‐GE currently has the most forcing scenarios of all large ensemble projects: RCP2.6, RCP4.5, RCP8.5, and 1% CO2
The power of MPI‐GE is to estimate the forced response and internal variability, including changing variability, to unprecedented precision
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