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Background: Bacteria can develop resistance to various antibiotics under selective pressure, leading to multifaceted changes in resistance mechanisms. Transcriptomic sequencing allows for the observation of transcriptional level alterations in cells under antibiotic stress. Understanding the bacterial response to such stress is essential for deciphering their strategy against drug-resistant antibiotics and identifying potential targets for antibiotic development., Methods: This study using wild-type (WT) Escherichia coli ( E. coli ) discovered that continuous in vitro induction screening for imipenem-resistant strains resulted in bacteria with enhanced biofilm-forming ability and mutations in antibiotic target sites. Transcriptomic sequencing of the resistant bacteria revealed significant changes in carbon and amino acid metabolism, nutrient assimilation, substance transport, nucleotide metabolism, protein biosynthesis, and cell wall biosynthesis. The up-regulated drug efflux genes were disrupted using gene knockout technology. Drug sensitivity tests indicated that drug efflux has a minimal effect on imipenem resistance., Results: This suggests a strategy for E. coli drug resistance involving the reduction of unnecessary substance synthesis and metabolism, coupled with an increase in activities that aid in resisting foreign threats., Competing Interests: The authors declare there are no competing interests., (©2024 Tong et al.)

Osteosarcoma (OS) is one of the deadliest malignancies in adolescents and its treatment status and prognosis remain unsatisfactory. N-acetylgalactosamine transferase 6 (GALNT6), one of the key enzymes regulating O-glycosylation, functions vary in different types of cancer. Currently, the function of GALNT6 in OS is unclear. Our results showed that GALNT6 was highly expressed in OS tissues, and the patients with higher GALNT6 expression exhibited a lower overall survival rate than patients with lower GALNT6 expression. We constructed the GALNT6-knockdown and GALNT6-overexpression vectors based on Tet-on system and packaged lentiviral particles to modulate GALNT6 expression. GALNT6 silencing impaired OC cell growth and metastasis both in vivo and vitro. Kruppel-like factor 9 (KLF9), a transcription factor known to suppress OS progression, was found to block GALNT6 transcription by binding to its promoter. Meanwhile, GALNT6 overexpression restored the effects caused by KLF9 upregulation. GALNT6 was known to affect protein stability by O-glycosylation regulation, thus the label-free proteomics combined with co-immunoprecipitation/mass-spectrum (MS) analysis were conducted to identify the potential mechanism of GALNT6 in promoting OS progression. EGF-containing fibulin extracellular matrix protein 1 (EFEMP1), contained several O-glycosylation sites and was upregulated in GALNT6 overexpressing cells (Log 2 FC = 1.3195, p = 0.0160), attracted our attention. We demonstrated that GALNT6 interacted with EFEMP1 at protein level. The O-glycosylation of EFEMP1 was increased by GALNT6 overexpression, which slowed the degradation rate of EFEMP1. EFEMP1 knockdown reversed the effects of GALNT6 overexpression. Collectively, our observations demonstrate that KLF9/GALNT6/EFEMP1 may be a promising direction for OS treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46 bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46 bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext +/- ). Off-target effect analysis did not reveal off-target mutations in Ext +/- mice caused by the two sgRNAs used. Compared to wild-type mice, Ext +/- mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext +/- mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext +/- mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext +/- mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext +/- mice serve as a valuable model for further biomedical investigations related to the Ext1 gene., Competing Interests: Declarations. Conflict of Interest: The authors declare no conflict of interests. Ethical Approval The animal procedures conducted in the present study were approved by the Animal Care and Use Committee of the Guilin Medical University (Approval No. GLMC201803033)., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The wearability of the flexible electronic skin (e-skin) allows it to attach to the skin for human motion monitoring, which is essential for studying human motion and especially for assessing how well patients are recovering from rehabilitation therapy. However, the use of non-degradable synthetic materials in e-skin may raise skin safety concerns. Natural biodegradable polymers with advantages such as biodegradability, biocompatibility, sustainability, natural abundance, and low cost have the potential to be alternative materials for constructing flexible e-skin and applying them to human motion monitoring. This review summarizes the applications of natural biodegradable polymers in e-skin for human motion monitoring over the past three years, focusing on the discussion of cellulose, chitosan, silk fibroin, gelatin, and sodium alginate. Finally, we summarize the opportunities and challenges of e-skin based on natural biodegradable polymers. It is hoped that this review will provide insights for the future development of flexible e-skin in the field of human motion monitoring., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries., Competing Interests: The authors declare that they have no competing interests., (© 2024 Zheng et al.)

Objective . Artificial nerve scaffolds composed of polymers have attracted great attention as an alternative for autologous nerve grafts recently. Due to their poor bioactivity, satisfactory nerve repair could not be achieved. To solve this problem, we introduced extracellular matrix (ECM) to optimize the materials. Approach. In this study, the ECM extracted from porcine nerves was mixed with Poly(L-Lactide-co- ϵ -caprolactone) (PLCL), and the innovative PLCL/ECM nerve repair conduits were prepared by electrostatic spinning technology. The novel conduits were characterized by scanning electron microscopy (SEM), tensile properties, and suture retention strength test for micromorphology and mechanical strength. The biosafety and biocompatibility of PLCL/ECM nerve conduits were evaluated by cytotoxicity assay with Mouse fibroblast cells and cell adhesion assay with RSC 96 cells, and the effects of PLCL/ECM nerve conduits on the gene expression in Schwann cells was analyzed by real-time polymerase chain reaction (RT-PCR). Moreover, a 10 mm rat (Male Wistar rat) sciatic defect was bridged with a PLCL/ECM nerve conduit, and nerve regeneration was evaluated by walking track, mid-shank circumference, electrophysiology, and histomorphology analyses. Main results. The results showed that PLCL/ECM conduits have similar microstructure and mechanical strength compared with PLCL conduits. The cytotoxicity assay demonstrates better biosafety and biocompatibility of PLCL/ECM nerve conduits. And the cell adhesion assay further verifies that the addition of ECM is more beneficial to cell adhesion and proliferation. RT-PCR showed that the PLCL/ECM nerve conduit was more favorable to the gene expression of functional proteins of Schwann cells. The in vivo results indicated that PLCL/ECM nerve conduits possess excellent biocompatibility and exhibit a superior capacity to promote peripheral nerve repair. Significance. The addition of ECM significantly improved the biocompatibility and bioactivity of PLCL, while the PLCL/ECM nerve conduit gained the appropriate mechanical strength from PLCL, which has great potential for clinical repair of peripheral nerve injuries., (© 2024 IOP Publishing Ltd.)

Skin tissue, composed of epidermis, dermis, and subcutaneous tissue, is the largest organ of the human body. It serves as a protective barrier against pathogens and physical trauma and plays a crucial role in maintaining homeostasis. Skin diseases, such as psoriasis, dermatitis, and vitiligo, are prevalent and can seriously impact the quality of patient life. Exosomes are lipid bilayer vesicles derived from multiple cells with conserved biomarkers and are important mediators of intercellular communication. Exosomes from skin cells, blood, and stem cells, are the main types of exosomes that are involved in modulating the skin microenvironment. The dysregulation of exosome occurrence and transmission, as well as alterations in their cargoes, are crucial in the complex pathogenesis of inflammatory and autoimmune skin diseases. Therefore, exosomes are promising diagnostic and therapeutic targets for skin diseases. Importantly, exogenous exosomes, derived from skin cells or stem cells, play a role in improving the skin environment and repairing damaged tissues by carrying various specific active substances and involving a variety of pathways. In the domain of clinical practice, exosomes have garnered attention as diagnostic biomarkers and prospective therapeutic agents for skin diseases, including psoriasis and vitiligo. Furthermore, clinical investigations have substantiated the regenerative efficacy of stem cell-derived exosomes in skin repair. In this review, we mainly summarize the latest studies about the mechanisms and applications of exosomes in dermatology, including psoriasis, atopic dermatitis, vitiligo, systemic lupus erythematosus, systemic sclerosis, diabetic wound healing, hypertrophic scar and keloid, and skin aging. This will provide a novel perspective of exosomes in the diagnosis and treatment of dermatosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

We consider mixtures of oppositely driven particles, showing that their nonequilibrium steady states form lanes parallel to the drive, which coexist with transient jammed clusters where particles are temporarily immobilized. We analyze the interplay between these two types of nonequilibrium pattern formation, including their implications for macroscopic demixing perpendicular to the drive. Finite-size scaling analysis indicates that there is no critical driving force associated with demixing, which appears as a crossover in finite systems. We attribute this effect to the disruption of long-ranged order by the transient jammed clusters.

Acne vulgaris is a type of chronic skin disorder caused by Propionibacterium acnes (P. acnes). Neutrophil extrinsic traps (NETs) play key role in many types of inflammatory skin diseases. Adipose-derived stem cells (ADSCs) was reported modulate immune responses and neutrophil activity. Here, we explored the potential role of ADSCs and the potential mechanism associated with neutrophil extracellular traps (NETs) in relieving acne vulgaris. In the P. acnes-infected ear skin model, histological staining was used to evaluate the inflammatory infiltration and NET formation in control, P. acnes, and P. acnes + ADSCs groups. Besides, western blot was used to detect the expression levels of cit-H3, MPO, and Nrf2 in ear tissue. In vitro, the immunofluorescence staining of MPO and cit-H3, and SYTOX green staining were performed to measure the NET formation. CCK-8 assay, EdU staining, and wound healing assay were used to detect the proliferation and migration abilities of keratinocytes. ELISA assay was utilized to detect the secretion of inflammatory cytokines. In P. acnes-infected ear skin, ADSC treatment significantly attenuated inflammation and NET formation via activating Nrf2 signaling pathway. In vitro, the conditioned medium of ADSCs reduced the formation of P. acne-induced NETs. Besides, ADSCs could inhibit that the NETs efficiently promoted the proliferation, migration, and inflammatory cytokine secretion of keratinocytes. Our study suggested that ADSCs could attenuate P. acne-related inflammation by inhibiting NET formation. This study provides a novel therapeutic perspective of ADSCs in combating acne vulgaris., (© 2024. The Author(s).)

Given the unclear variation law of the effective pyrolysis zone in the process of in situ heat injection mining of oil shale, the actual pyrolysis effect cannot be accurately judged. In this paper, considering the influence of two different random fractures, the thermal-fluid-solid coupling mechanical model of oil shale in situ heat injection mining is established. The effective pyrolysis zone, steam injection pressure, and temperature-affected zone of the roof and floor rocks in the process of in situ heat injection mining of oil shale are analyzed. The results showed that interconnected and high-density fractures are important channels for superheated steam seepage, which is conducive to the efficient penetration of superheated steam in oil shale reservoirs. There are multiple pyrolysis paths in oil shale reservoirs in bedding fractures, while oil shale reservoirs in hydraulic fractures are uniformly pyrolyzed by a high-temperature network formed by superheated steam. When the bedding fracture model and the hydraulic fracture model are injected with heat for 233 and 90 days, the oil shale reservoir reaches the effective pyrolysis temperature, and the pyrolysis efficiency of the hydraulic fracture network is 2.59 times that of the bedding fracture network. The average temperature of the affected area of the overlying and overlying strata is 471.98 and 467.02 °C, respectively. When in situ heat injection mining of oil shale is carried out, it is necessary to adjust the heat injection time reasonably and keep the hydraulic fracture away from the upper and lower boundaries of the oil shale reservoir to avoid the heat dissipation of superheated steam., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Background: Skin wound is a widespread health problem and brings extraordinary burdens to patients. Exosomes derived from adipose-derived stem cells (ADSC-Exos) are considered promising strategies for repairing skin wounds. E2F1 is a member of the E2F family of transcription factors involved in cell growth and apoptosis. E2F1 deficiency in mice enhances wound healing by improving collagen deposition and angiogenesis. Additionally, E2F1 can regulate the transcription and paracrine activity of multiple miRNAs, which will inevitably reshape the paracrine expression profile of ADSC-Exos. This study aimed to investigate the impact of transcription factor E2F1 deficiency on the functions of ADSC-Exos in promoting wound healing., Methods: First, we obtained ADSCs from subcutaneous adipose tissues of WT and E2F1 -/- C57BL/6 mice and separated their exosomes, denoted as ADSC WT -Exos and ADSC E2F1-/- -Exos. The wound healing effects of ADSC WT -Exos and ADSC E2F1-/- -Exos in full-thickness skin wound models were investigated by wound images, H&E staining, and immunohistochemical staining. For the in vitro study, the abilities of ADSC WT -Exos and ADSC E2F1-/- -Exos to promote cell activities, collagen formation, and angiogenesis were evaluated. The potential mechanism by which ADSC E2F1-/- -Exos promote wound healing was determined by miRNA sequencing, ChIP‒qPCR, and dual-luciferase assays., Results: ADSC E2F1-/- -Exos accelerated wound healing by promoting collagen formation and angiogenesis. As a result, compared with the lower wound healing rate of 30.5% within 7 days in the control group and 42.3% in the ADSC WT -Exo group, ADSC E2F1-/- -Exos significantly increased the wound healing rate to 72.5%. In vitro, ADSC E2F1-/- -Exos activated the function of fibroblasts and vascular endothelial cells. The loss of E2F1 promoted miR-130b-5p expression in ADSC E2F1-/- -Exos through transcriptional regulation. MiRNA high-throughput sequencing identified 12 differently expressed miRNAs between ADSC E2F1-/- and ADSC WT . ADSC E2F1-/- -Exos enhanced fibroblast activities via the miR-130b-5p/TGFBR3 axis and TGF-β activation., Conclusion: Our results indicated that ADSC E2F1-/- -Exos effectively promoted wound healing by regulating the miR-130b-5p/TGFBR3 axis, thus providing a novel strategy of gene-engineered stem cell exosomes for accelerating wound healing., Competing Interests: The authors declare that they have no conflicts of interest in this work., (© 2023 Yu et al.)

Despite recent advances in understanding the biological behavior of osteosarcoma (OS), OS is still the most common primary bone sarcoma that endangers the health of children and adolescents. High-temperature requirement A (HTRA) protease family plays an important regulatory role in numerous malignancies and acts as a prognostic biomarker. However, the function and underlying mechanisms of the HTRA family in OS development remain unknown. Through analyzing the GSE126209 dataset obtained from different Gene Expression Omnibus (GEO) databases, we found that HTRA3 as a member of the HTRA family was downregulated in OS tissues compared with that in normal tissues. Functional experiments indicated that HTRA3 overexpression suppressed malignant behaviors of OS cells in vitro and tumor growth in vivo. Mechanistically, we found that HTRA3 co-localized with the X-linked inhibitor of apoptosis protein (XIAP) and decreased XIAP stability. Further investigation showed that XIAP knockdown inhibited the degradation of phosphatase and tensin homolog (PTEN) and that HTRA3 caused the blockage of PTEN/phosphoinositide 3-kinase (PI3K)/AKT pathway, characterized as the reverse of cell function caused by HTRA3 overexpression after PTEN inhibitor BpV (HOpic) treatment. Detailed investigations showed that forkhead box protein 1 (FOXP1), an oncogene in OS progression, downregulated HTRA3 expression and inhibited the transcriptional activity of HTRA3, suggesting that HTRA3 was regulated negatively by FOXP1. In conclusion, our study demonstrates that HTRA3 is a repressor involved in OS development via the PTEN/PI3K/AKT pathway under the modulation of transcription factor FOXP1, and it may provide a therapeutic direction for OS patients., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts., (Copyright © 2023 Elsevier B.V. All rights reserved.)

With the increase of mining depth and intensity, coal and gas outburst dynamic disasters occur frequently. In order to deeply study the macroscopic fracture mechanism of coal body and evolution characteristics analysis of impact force, taking the outburst coal seam of Pingmei No. 11 Coal Mine and Sunjiawan coal seam of Hengda Coal Mine as the research objects, the simulation roadway test system of self-developed true triaxial coal and gas outburst is applied to carry out the simulation test of deep coal and gas outburst with buried depths of 1000 m, 1200 m, 1400 m and 1600 m. During the test, the overlying strata stress is simulated by axial compression, the surrounding rock stress is simulated by confining pressure, the gas pressure is simulated by pore pressure, the impact force and acoustic emission monitoring technology are introduced, and the coal seam gas pressure is simulated by mixture pressure of 45% CO 2 and 55% N 2 . From the viewpoint of fracture mechanics, the crack propagation mechanism of coal in the outburst launching area is discussed, the evolution characteristics of impact force and gas pressure are analyzed, and the influence law between acoustic emission signal and impact force is revealed. From the viewpoint of energy conversion, the transformation character of gas internal energy to impact kinetic energy (gas pressure to impact force) are analyzed. The results show that the generation of I-type crack is a prerequisite for outburst catastrophe. With the crack propagation, I-type and II-type cracks intersect and penetrate, resulting in internal structural damage and skeleton instability of coal. Gas wrapped fragmentized coal body thrown, outburst occurs. There is obvious negative pressure in the roadway after outburst. The occurrence of negative pressure is greatly affected by the physical and mechanical properties of coal, ground stress and gas pressure. Impact kinetic energy is mainly provided by gas internal energy. Part of the gas pressure is converted into impact force. The strength and duration of the impact force are determined by the gas pressure. Under the condition of deep working conditions (high ground stress and low gas pressure), the propagation of impact force in the roadway is more hindered. Both impact force and acoustic emission signals can monitor the occurrence of outburst. The peak point of acoustic emission ringing count is earlier than the impact force. The acoustic emission signal can monitor the outburst hazard earlier. The impact force can more specifically reflect the coal fracture., (© 2023. Springer Nature Limited.)

The repair and reconstruction of bone defects and the inhibition of local tumor recurrence are two common problems in bone surgery. The rapid development of biomedicine, clinical medicine, and material science has promoted the research and development of synthetic degradable polymer anti-tumor bone repair materials. Compared with natural polymer materials, synthetic polymer materials have machinable mechanical properties, highly controllable degradation properties, and uniform structure, which has attracted more attention from researchers. In addition, adopting new technologies is an effective strategy for developing new bone repair materials. The application of nanotechnology, 3D printing technology, and genetic engineering technology is beneficial to modify the performance of materials. Photothermal therapy, magnetothermal therapy, and anti-tumor drug delivery may provide new directions for the research and development of anti-tumor bone repair materials. This review focuses on recent advances in synthetic biodegradable polymer bone repair materials and their antitumor properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yu, Liu, Shen and Ao.)

Coal and gas outburst is one of the geological disasters that seriously threaten the safety of coal mines production. In recent years, with the increase of mining depth, outbursts become frequent. To further explore the occurrence mechanism of deep coal and gas outburst, a self-developed true triaxial coal and gas outburst simulation device was used to simulate the coal and gas outburst at different depths. The results show that with the increase of simulation depth, the critical gas pressure of outburst gradually decreases, and the unit outburst intensity increases sharply. The gas threshold of deep coal and gas outburst is lower. During the incubation and excitation, gas pressure has three special variation rules, namely self-increasing characteristic, stage and instantaneous. In the early incubation, acoustic emission (AE) energy is at a low level, low energy frequency is dominant; in the later incubation, AE energy increases greatly, high energy frequency is dominant. From the perspective of AE energy, a quantitative index that reflects the danger of coal and gas outburst in the incubation is defined, which provides a scientific reference for the prediction and prevention of disasters of coal and gas outburst in deep mining., (© 2022. The Author(s).)

Triple-negative breast cancer (TNBC) is a highly malignant subtype of breast cancer (BC) with vicious behaviors. TNBC is usually associated with relatively poor clinical outcomes, earlier recurrence, and high propensity for visceral metastases than other BC types. TNBC has been increasingly recognized to constitute a very molecular heterogeneous subtype, which may offer additional therapeutic opportunities due to newly discovered cancer-causing drivers and targets. At present, there are multiple novel targeted therapeutic drugs in preclinical researches, clinical trial designs, and clinical practices, such as platinum drugs, poly ADP-ribose polymerase (PARP) inhibitors, immunocheckpoint inhibitors, androgen receptor inhibitors as well as PI3K/AKT/mTOR targeted inhibitors. These personalized, single, or combinational therapies based on molecular heterogeneity are currently showing positive results. The scope of this review is to highlight the latest knowledge about these potential TNBC therapeutic drugs, which will provide comprehensive insights into the personalized therapeutic strategies and options for combating TNBC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Xia, Zhou, Yu, Tan, Du, Zhang and Wu.)

Background: Breast cancer (BC) is one of the most common cancers in women. The discovery of available biomarkers is crucial for early diagnosis and improving prognosis. The effect of POP1 in BC remains unrevealed. Our study aims to explore the expression of POP1 in BC and demonstrate its clinical significance and potential molecular mechanisms., Methods: The Cancer Genome Atlas (TCGA) BC cohort transcriptome data and corresponding clinical information were downloaded. GSE42568 cohort, GSE162228 cohort, GSE7904 cohort, and GSE161533 cohort in the Gene Expression Omnibus (GEO) database were used as verification groups. R software and several web tools were used for statistical analysis. Moreover, the proliferation, transwell, wound healing experiments, and flow cytometry were used for in vitro investigation., Results: Compared with normal breast tissue, POP1 expression was up-regulated in BC tissue with a higher mutation rate. POP1 had good diagnostic value for BC and could be utilized as a new marker. POP1 was significantly correlated with multiple pathways in BC and played an important role in the immune infiltration of BC. High-POP1 expression patients were more prone to be responded to immunotherapy and had a significantly higher percentage of immunotherapy response rate. Moreover, POP1 promoted proliferation and migration and inhibited apoptosis in BC cells., Conclusions: POP1 expression was up-regulated in BC and was associated with a poor prognosis. Patients with high-POP1 expression were more likely to be responded to immunotherapy. Our study can provide a potential marker POP1 for BC, which is beneficial in the diagnosis and treatment of BC.

Wound healing after skin injury is a dynamic and highly coordinated process involving a well-orchestrated series of phases, including hemostasis, inflammation, proliferation, and tissue remodeling. Epigenetic regulation refers to genome-wide molecular events, including DNA methylation, histone modification, and non-coding RNA regulation, represented by microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA). Epigenetic regulation is pervasively occurred in the genome and emerges as a new role in gene expression at the post-transcriptional level. Currently, it is well-recognized that epigenetic factors are determinants in regulating gene expression patterns, and may provide evolutionary mechanisms that influence the wound microenvironments and the entire healing course. Therefore, this review aims to comprehensively summarize the emerging roles and mechanisms of epigenetic remodeling in wound healing. Moreover, we also pose the challenges and future perspectives related to epigenetic modifications in wound healing, which would bring novel insights to accelerated wound healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yu, Wang, Wang, Yi, Liu, Wu, Wu and Zhang.)

We study three different lattice models in which two species of diffusing particles are driven in opposite directions by an electric field. We focus on dynamical phase transitions that involve phase separation into domains that may be parallel or perpendicular to a driving field. In all cases, the perpendicular state appears for weak driving, consistent with previous work. For strong driving, we introduce two models that support the parallel state. In one model, this state occurs because of the inclusion of dynamical rules that enhance lateral diffusion during collisions; in the other, it is a result of a nearest-neighbor attractive or repulsive interaction between particles of the same or opposite species. We discuss the connections between these results and the behavior found in off-lattice systems, including laning and freezing by heating.

Necroptosis plays a major role in breast cancer (BC) progression and metastasis. Besides, necroptosis also regulates inflammatory response and tumor microenvironment. Here, we aim to explore the predictive signature based on necroptosis-related genes (NRGs) for predicting the prognosis and response to therapies. Using Lasso multivariate cox analysis, we firstly established the NRG signature based on TCGA database. A total of 6 NRGs (FASLG, IPMK, FLT3, SLC39A7, HSP90AA1, and LEF1), which were associated with the prognosis of BC patients, were selected to establish our signature. Next, CIBERSORT algorithm was utilized to evaluate immune cell infiltration levels. We compare the response to immunotherapy using IMvigor 210 database, and also compared immune indicators in two risk groups via multiple methods. The biological function of IPMK was explored via in vitro verification. Finally, our results indicated that the signature was an independent prognostic indicator for BC patients with better efficiency than other reported signatures. The immune cell infiltration levels were higher, and the response to immunotherapy and chemotherapy was better in the low-risk groups. Besides, other immunotherapy-related factors, including TMB, TIDE, and expression of immune checkpoints were also increased in the low-risk group. Clinical sample validation showed that CD206 and IPMK in clinical samples were both up-regulated in the high-risk group. In vitro assay showed that IPMK promoted BC cell proliferation and migration, and also enhanced macrophage infiltration and M2 polarization. In summary, we successfully established the NRG signature, which could be used to evaluate BC prognosis and identify patients who will benefit from immunotherapy., (© 2022. The Author(s).)

Cutaneous melanoma is an aggressive cancer type derived from melanocytes and its incidence has rapidly increased worldwide. Despite the vast improvement in therapy, melanoma is still confronted with high invasion, metastasis, and recurrence rate. Recent studies have confirmed that the exosomes are naturally occurring membranous extracellular vesicles with nano-sized lipid bilayers, performing as information messagers within cellular reciprocal action. Exosomes are unquestionably endowed with multifaceted roles in various diseases, including melanoma. Notably, tumor-derived exosomes play a pivotal role in conditioning the tumor microenvironment to promote the growth, metastasis, immune escape, and even drug-resistance of melanoma by transferring carcinogenic nucleic acids and proteins. Clinically, the dynamic expressions of exosomal components and loadings in melanoma patients with different tumor stages confer the clinical application of melanoma exosomes as diagnostic biomarkers. Hence, this review highlights the recent complicated roles and mechanisms of melanoma exosomes, as well as their potential as diagnostic and therapeutic targets in melanoma. The in-depth insights into the properties and behaviors of melanoma exosomes are of great potential to yield attractive therapeutic methods for melanoma., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Activation of human immune T-cells by swine leukocyte antigens class I (SLA-I) and class II (SLA-II) leads to xenograft destruction. Here, we generated the GGTA1, B2M, and CIITA (GBC) triple-gene-modified Diannan miniature pigs, analyzed the transcriptome of GBC-modified peripheral blood mononuclear cells (PBMCs) in the pig's spleen, and investigated their effectiveness in anti-immunological rejection. A total of six cloned piglets were successfully generated using somatic cell nuclear transfer, one of them carrying the heterozygous mutations in triple genes and the other five piglets carrying the homozygous mutations in GGTA1 and CIITA genes, but have the heterozygous mutation in the B2M gene. The autopsy of GBC-modified pigs revealed that a lot of spot bleeding in the kidney, severe suppuration and necrosis in the lungs, enlarged peripulmonary lymph nodes, and adhesion between the lungs and chest wall were found. Phenotyping data showed that the mRNA expressions of triple genes and protein expressions of B2M and CIITA genes were still detectable and comparable with wild-type (WT) pigs in multiple tissues, but α1,3-galactosyltransferase was eliminated, SLA-I was significantly decreased, and four subtypes of SLA-II were absent in GBC-modified pigs. In addition, even in swine umbilical vein endothelial cells (SUVEC) induced by recombinant porcine interferon gamma (IFN-γ), the expression of SLA-I in GBC-modified pig was lower than that in WT pigs. Similarly, the expression of SLA-II DR and DQ also cannot be induced by recombinant porcine IFN-γ. Through RNA sequencing (RNA-seq), 150 differentially expressed genes were identified in the PBMCs of the pig's spleen, and most of them were involved in immune- and infection-relevant pathways that include antigen processing and presentation and viral myocarditis, resulting in the pigs with GBC modification being susceptible to pathogenic microorganism. Furthermore, the numbers of human IgM binding to the fibroblast cells of GBC-modified pigs were obviously reduced. The GBC-modified porcine PBMCs triggered the weaker proliferation of human PBMCs than WT PBMCs. These findings indicated that the absence of the expression of α1,3-galactosyltransferase and SLA-II and the downregulation of SLA-I enhanced the ability of immunological tolerance in pig-to-human xenotransplantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Yu, Chen, Zhang, Guo, Yang, Jiao, Nguyen, Zhao, Wang, Wei, Li, Jia, Jamal, Zhao, Huang and Wei.)

The alterations of glycosylation, which is a common post-translational modification of proteins, have been acknowledged as key events in breast cancer (BC) oncogenesis and progression. The aberrant expression of glycosyltransferases leads to aberrant glycosylation patterns, posing the diagnostic potential in BC outcomes. The present study aims to establish a glycosyltransferase-based signature to predict BC prognosis and response to immune checkpoint inhibitors. We firstly screened 9 glycosyltransferase genes from The Cancer Genome Atlas (TCGA) database and accordingly established a glyco-signature for predicting the prognosis in BC patients. Patients with BC were successfully divided into high-risk and low-risk groups based on the median cutoff point for risk scores in this signature. Next, the combinational analyses of univariate and multivariate Cox regression, Kaplan-Meier, and receiver operating characteristic (ROC) curves were used to prove that this glyco-signature possessed excellent predictive performance for prognosis of BC patients, as the high-risk group possessed worse outcomes, in comparison to the low-risk group. Additionally, the Gene Set Enrichment Analysis (GSEA) and immunologic infiltration analysis were adopted and indicated that there was a more immunosuppressive state in the high-risk group than that in the low-risk group. The clinical sample validation verified that glycosyltransferase genes were differentially expressed in patients in the low- and high-risk groups, while the biomarkers of antitumor M1 macrophages were increased and N-glycosyltransferase STT3A decreased in the low-risk group. The final in vitro assay showed that the silencing of STT3A suppressed the proliferation and migration of BC cells. Collectively, our well-constructed glyco-signature is able to distinguish the high- and low-risk groups and accordingly predict BC prognosis, which will synergistically promote the prognosis evaluation and provide new immunotherapeutic targets for combating BC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lv, Yu, Han, Tan, Wu, Zhang, Wu and Zhang.)

Background: Nowadays, wound is associated with a complicated repairing process and still represents a significant biomedical burden worldwide. Bone marrow mesenchymal stem cells (BMSCs) possess multidirectional differentiation potential and secretory function, emerging as potential cellular candidates in treating wounds. Ascorbic acid 2-glucoside (AA2G) is a well-known antioxidant and its function in BMSC-promoting wound healing is worth exploring., Methods: The in vitro cell proliferation, migration, and angiogenesis of BMSCs and AA2G-treated BMSCs were detected by flow cytometry, EDU staining, scratch assay, transwell assay, and immunofluorescence (IF). Besides, the collagen formation effect of AA2G-treated BMSCs conditioned medium (CM) on NIH-3T3 cells was evaluated by hydroxyproline, qRT-PCR and IF staining detection. Next, in the wound healing mouse model, the histological evaluation of wound tissue in PBS, BMSCs, and AA2G-treated BMSCs group were further investigated. Lastly, western blot and ELISA were used to detect the expression levels of 5-hmc, TET2 and VEGF protein, and PI3K/AKT pathway activation in BMSCs treated with or without AA2G., Results: The in vitro results indicated that AA2G-treated BMSCs exhibited stronger proliferation and improved the angiogenesis ability of vascular endothelial cells. In addition, the AA2G-treated BMSCs CM enhanced migration and collagen formation of NIH-3T3 cells. In vivo, the AA2G-treated BMSCs group had a faster wound healing rate and a higher degree of vascularization in the new wound, compared with the PBS and BMSCs group. Moreover, AA2G preconditioning might enhance the demethylation process of BMSCs by regulating TET2 and up-regulating VEGF expression by activating the PI3K/AKT pathway., Conclusions: AA2G-treated BMSCs promoted wound healing by promoting angiogenesis and collagen deposition, thereby providing a feasible strategy to reinforce the biofunctionability of BMSCs in treating wounds., (© 2022. The Author(s).)

Breast cancer (BC) is the most commonly diagnosed malignancy accompanied by high invasion and metastasis features. Importantly, emerging studies have supported that aging is a key clue that participates in the immune state and development of BC. Nevertheless, there are no studies concerning the aging-related genes (AGs) in constructing the prognosis signature of BC. Here, to address this issue, we initially performed a systematic investigation of the associations between AGs and BC prognosis and accordingly constructed a prognosis risk model with 10 AGs including PLAU, JUND, IL2RG, PCMT1, PTK2, HSPA8, NFKBIA, GCLC, PIK3CA , and DGAT1 by using the least absolute shrinkage and selection operator (LASSO) regression and Cox regression analysis. Meanwhile, our analysis further confirmed that the nomogram possessed a robust performance signature for predicting prognosis compared to clinical characteristics of BC patients, including age, clinical stage, and TNM staging. Moreover, the risk score was confirmed as an independent prognostic index of BC patients and was potentially correlated with immune scores, estimate score, immune cell infiltration level, tumor microenvironment, immunotherapy effect, and drug sensitivity. Furthermore, in the external clinical sample validation, AGs were expressed differentially in patients from different risk groups, and tumor-associated macrophage markers were elevated in high-risk BC tissues with more co-localization of AGs. In addition, the proliferation, transwell, and wound healing assays also confirmed the promoting effect of DGAT1 in BC cell proliferation and migration. Therefore, this well-established risk model could be used for predicting prognosis and immunotherapy in BC, thus providing a powerful instrument for combating BC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lv, Zhao, Tan, Hu, Yu, Zeng, Zhang and Wu.)

Obesity is associated with energy metabolic disturbance and is caused by long-term excessive energy storage in white adipose tissue (WAT). The WAT browning potentially reduces excessive energy accumulation, contributing an attractive target to combat obesity. As a pivotal regulator of cell growth, the transcription factor E2F1 activity dysregulation leads to metabolic complications. The regulatory effect and underlying mechanism of E2F1 knockout on WAT browning, have not been fully elucidated. To address this issue, in this study, the in vivo adipose morphology, mitochondria quantities, uncoupling protein 1 (UCP-1), autophagy-related genes in WAT of wild-type (WT) and E2F1 -/- mice were detected. Furthermore, we evaluated the UCP-1, and autophagy-related gene expression in WT and E2F1 -/- adipocyte in vitro . The results demonstrated that E2F1 knockout could increase mitochondria and UCP-1 expression in WAT through autophagy suppression in mice, thus promoting WAT browning. Besides, adipocytes lacking E2F1 showed upregulated UCP-1 and downregulated autophagy-related genes expression in vitro . These results verified that E2F1 knockout exerted effects on inducing mice WAT browning through autophagy inhibition in vivo and in vitro . These findings regarding the molecular mechanism of E2F1-modulated autophagy in controlling WAT plasticity, provide a novel insight into the functional network with the potential therapeutic application against obesity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xiong, Hu, Tan, Yu, Zhang, Zhao, Yi, Wang, Wu and Wu.)

Pickering emulsifiers are significant for organic reactions in an aqueous medium because they have the ability of emulsifying water-oil biphasic systems. For this reason, 2,5-bis[( E )-2-(triethoxysilyl)vinyl]pyridine [BTOSVP] containing a pyridine bridging group was selected as a precursor to prepare a novel salt of organosilicon framework (SOF), an amphiphilic mesoporous pyridine hydrobromide nanosphere. We first synthesized a mesoporous organosilicon framework made up of organic groups containing vinyl groups, pyridine groups, and so forth. Then, hydrobromic acid was added to protonate the pyridine groups in the mesoporous organosilicon framework. Eventually, pyridine hydrobromide salt was formed on the surfaces of channels, and the SOF was successfully prepared for the first time. Pyridine hydrobromide salt can be ionized in water into protonated pyridine cations located on the SOF surfaces and free Br-anions swimming around the protonated pyridine cations because of the electrostatic interaction. In the water-oil biphasic systems, hydrophilicity of SOF originates from the protonated pyridine cations and the lipophilicity of SOF comes from organic groups in the framework; thus, this new kind of SOF can be used as a new generation of solid Pickering emulsifiers. Most importantly, the mesoporous SOF nanosphere can also be used as a catalyst for significantly improved dibromination of olefins in an aqueous medium.

Carbon-metal composites are promising multifunctional electrocatalysts, but it is still challenging to prepare carbon-metal composites with tunable structure and strong metal-carbon interactions. Here, we present a unique gas-foaming assembly strategy to prepare cytosine-Co chelate derived Co and N codoped carbon nanotube (Co-NCNT). The structure for Co-NCNTs could be easily controlled by regulating cytosine-Co coordination or the carbonization temperature. The optimal Co-NCNT possesses homogeneous distributed NCNTs (10 nm), CoO x (5 nm) and CoN x moieties to synergistically boost electrochemical processes, and offer mesoporous nanosheet architecture to guarantee fast mass migrate and electron transfer. As a result, Co-NCNT shows remarkable ORR performance (onset potential of 0.93 V in 0.1 M KOH electrolyte) along with significant OER and HER activity. More important, it was found that CoN x moieties are responsible for the remarkable electrocatalytic activity in Co-NCNTs, because CoN x could alter active center, enhance metal-carbon synergy, decrease interfacial resistance and reinforce the strength of composites. Therefore, this paper not just demonstrates an advanced multi-functional electrocatalyst, but could also give deep understanding on the designing of multifunctional electrocatalysts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecB B ) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecB B mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.

Here, we demonstrate an all-silicon photonic switch, working at an infrared communication wavelength and pumped by spatial light, where a ring resonator and a metasurface absorber are both designed in photonic crystals and monolithically integrated on a silicon-on-insulator wafer. Through selective doping, the absorber gets a pump absorption completely different from near zero of the resonator. Based on the thermo-optical effect, the device is capable of tuning the wavelength of the guided mode by $\sim{341}\;{\rm pm/mW}$∼341pm/mW and switching in time $ {\lt} {1.0}\;\unicode{x00B5} {\rm s}$<1.0µs to the pump response. The high responsivity and switching speed as well as all-silicon processing techniques make the design potentially for free-space optical communication and detection.

Background: Osteosarcoma is a malignant bone tumor. Increasing evidences have revealed that a disintegrin and metalloproteinase 10 (ADAM10) is implicated in tumor development. The main purpose of this study is to explore the effects of ADAM10 on osteosarcoma cell functions and the underlying molecular mechanisms., Methods: Western blot and quantitative real-time PCR were performed to detect the expression of ADAM10 in one osteoblast (hFOB 1.19) and six osteosarcoma cells (Saos-2, SW1353, HOS, U-2OS, MG63, and 143B). The biological functions of ADAM10 in osteosarcoma cells were measured by cell counting kit-8 assay, flow cytometry, wound healing assay, and transwell assay. The interaction between miR-122-5p and ADAM10 was validated using dual-luciferase reporter assay. The effect of ADAM10 on the tumorigenicity of osteosarcoma cells was evaluated in a nude mice model in vivo., Results: We found that the expression of ADAM10 was relatively high in osteosarcoma cells compared with that in osteoblast. ADAM10 promoted osteosarcoma cell growth, migration, and invasion. Mechanism studies showed that knockdown of ADAM10 inactivated E-cadherin/β-catenin signaling pathway, as evidenced by increased the level of E-cadherin, reduced nuclear translocation of β-catenin, and decreased the levels of MMP-9, Cyclin D1, c-Myc, and Survivin. Downregulation of ADAM10 suppressed the tumorigenicity of osteosarcoma cells in vivo. Furthermore, ADAM10 was validated to be a downstream target of microRNA-122-5p (miR-122-5p). MiR-122-5p-induced inhibition of cell proliferation, migration, and invasion was reversed by overexpression of ADAM10 in osteosarcoma cells., Conclusions: Collectively, the key findings of this study are that ADAM10 promotes osteosarcoma cell proliferation, migration, and invasion by regulating E-cadherin/β-catenin signaling pathway, and miR-122-5p can target ADAM10, indicating that miR-122-5p/ADAM10 axis might serve as a therapeutic target of osteosarcoma., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Background: MicroRNAs (miRNAs) can act as negative regulators of gene expression, and play a crucial role in cancer progression. The aim of this study was to investigate the role of miR-1294/pyruvate kinase M2 (PKM2) axis in osteosarcoma cells in vitro and in vivo., Methods: The function of miR-1294 and its association with PKM2 in osteosarcoma cells were studied by real-time PCR, CCK-8, Western blot, scratch test, transwell assay, flow cytometry, and dual-luciferase reporter assays. The effect of miR-1294 on tumor growth in vivo was evaluated in a subcutaneous xenograft model of osteosarcoma., Results: miR-1294 was downregulated in osteosarcoma cells. Forced overexpression of miR-1294 inhibited cell proliferation, migration, and invasion, and induced G0/G1 arrest and apoptosis. Consistently, protein expression levels of proliferating cell nuclear antigen, c-Myc, cyclin D1, active matrix metalloproteinase 2, and active matrix metalloproteinase 9 were decreased, and cleaved caspase 3 and cleaved PARP were increased following miR-1294 overexpression. Moreover, we demonstrated that PKM2 was a target of miR-1294 in osteosarcoma cells, and the effects caused by miR-1294 mimic were reversed by the overexpression of PKM2. Furthermore, we found that upregulation of miR-1294 inhibited tumorigenesis of osteosarcoma cells in vivo, which was accompanied by downregulation of PKM2., Conclusion: Our results revealed that miR-1294/PKM2 signaling cascade exerts important roles in the regulation of tumor progression, implying that this pathway may serve as a potential therapeutic target in osteosarcoma., Competing Interests: The authors declare that they have no competing interests in this work., (© 2020 Yuan et al.)

The ability to alter single bases without homology directed repair (HDR) of double-strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5, which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor (BE3) plasmid and four different sgRNAs into caprine fibroblasts were ineffective in altering FGF5. In contrast, all five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF5 expression. PCR amplicon and whole-genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single-cell embryos. Our results support further optimization of BEs for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation., (© 2019 Federation of European Biochemical Societies.)

MicroRNAs (miRNAs) are known to be associated with certain cancers, including osteosarcoma. We examined osteosarcoma tissues and cell lines, and found that most expressed lower levels of miR-548d-3p than adjacent tissues and normal cell lines. KRAS was identified as a potential target gene of miR-548d-3p. In osteosarcoma cells, miR-548d-3p exerted tumor-suppressive effects by downregulating KRAS . Functional assays revealed that miR-548d-3p mimics dramatically reduced cell growth and migration in vitro . These results suggest that miR-548d-3p mimics could be applied for osteosarcoma treatment.

Since its emergence, CRISPR/Cas9-mediated base editors (BEs) with cytosine deaminase activity have been used to precisely and efficiently introduce single-base mutations in genomes, including those of human cells, mice, and crop species. Most production traits in livestock are induced by point mutations, and genome editing using BEs without homology-directed repair of double-strand breaks can directly alter single nucleotides. The p.96R > C variant of Suppressor cytokine signaling 2 (SOCS2) has profound effects on body weight, body size, and milk production in sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.96R > C substitution in SOCS2 by the co-injection of BE3 mRNA and a single guide RNA (sgRNA) into sheep zygotes. The observed efficiency of the single nucleotide exchange in newborn animals was as high as 25%. Observations of body size and body weight in the edited group showed that gene modification contributes to enhanced growth traits in sheep. Moreover, targeted deep sequencing and unbiased family trio-based whole genome sequencing revealed undetectable off-target mutations in the edited animals. This study demonstrates the potential for the application of BE-mediated point mutations in large animals for the improvement of production traits in livestock species.

Here, we present a graphene-based long-wavelength infrared photodetector, for enhancing the infrared absorption of which the design consists of magnetic- and electric-plasmon resonators of metasurface to excite the graphene surface-plasmonic polaritons (SPPs). Through tuning the graphene Fermi energy to achieve the distinct resonances in a matching frequency, peak graphene absorbance exceeding 67.2% is confirmed, even when a lossy dielectric is used, and the field angle of view is up to 90°. If the graphene is of a different carrier mobility, then the absorption frequency is lockable, and the device always can keep the system absorbance close to 100 percent. The significantly enhanced graphene absorbance, up to ~29-fold that of a suspended graphene (general 2.3%), is attributed to the surface-plasmonic coupling between the magnetic and the electric resonances, as well as Fabry-Pérot interference of the coherent SPPs. The plasmonic cavity-mode model and equivalent-circuit method developed in this study will also be useful in guiding other optoelectronic device design.

Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q>R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.

Background: Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome., Methods: To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing., Results: In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR -47/-1 , GHR -47/-46 ) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission., Conclusion: The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome.

Osteosarcoma (OS) is the mostly diagnosed primary bone malignancy. Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. In the present study, the effect of PKM2 knockdown on the proliferation and migration of OS cells were assessed both in vitro and in vivo. Small hairpin RNA (shRNA) technology were employed to suppress the expression of PKM2 in MG-63 and Saos-2 cell lines. In vitro, shRNA-mediated knockdown of PKM2 efficiently inhibited cell proliferation, and induced G1 cell cycle arrest and apoptosis in both cell lines, which was associated with decreased expressions of cyclin D1 and Bcl-2 as well as increased expressions of Bax, cleaved-caspase-3, and cleaved-PARP. The invasion and migration potential of OS cell lines were also inhibited by PKM2 knockdown through the regulating effect of PKM2 on MMP-2 and VEGF signaling. In vivo, knockdown of PKM2 decelerated tumor growth rate and induced structure deterioration in tumor tissues. The current study for the first time showed that the activity of PKM2 was indispensable for the development and metastasis of OS, thereby providing the basic information for the future development of PKM2-based anti-OS therapies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Our previous study found that co-culture with human vascular endothelial cells (HMVECs) is beneficial for dorsal root ganglion cells (DRGCs). The goal of the present study is to investigate whether co-culture with HMVECs could promote the development of DRGCs, and whether this effect is induced by the secretion of BDNF by HMVECs. DRGCs were mono-cultured, co-cultured with HMVECs or co-cultured with HMVECs that pre-transfected with BDNF siRNA, the expression of neurite formation and branching factors were determined. The results showed that transfecting with BDNF siRNA inhibited BDNF expression and reduced BDNF secretion. Co-culture with HMVECs increased the expression of Etv4, Etv5, FN-L, FN-M, and GAP-43 in DRGCs that accompanied by the activation of ERK pathway. However, these changes were all reversed by the inhibition of BDNF in HMVECs. In conclusion, our data demonstrate that HMVECs potentiated DRGCs development at least partly by the secretion of BDNF in the co-culture system.

Herein, we propose a new security enhancing method that employs wavefront aberrations as optical keys to improve the resistance capabilities of conventional double-random phase encoding (DRPE) optical cryptosystems. This study has two main innovations. First, we exploit a special beam-expander afocal-reflecting to produce different types of aberrations, and the wavefront distortion can be altered by changing the shape of the afocal-reflecting system using a deformable mirror. Then, we reconstruct the wavefront aberrations via the surface fitting of Zernike polynomials and use the reconstructed aberrations as novel asymmetric vector keys. The ideal wavefront and the distorted wavefront obtained by wavefront sensing can be regarded as a pair of private and public keys. The wavelength and focal length of the Fourier lens can be used as additional keys to increase the number of degrees of freedom. This novel cryptosystem can enhance the resistance to various attacks aimed at DRPE systems. Finally, we conduct ZEMAX and MATLAB simulations to demonstrate the superiority of this method.

Background: α1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation., Methods: Transcription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting., Results: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet., Conclusions: TALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.

Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes., Competing Interests: The authors have declared that no competing interests exist.

The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27-33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.

The induced pluripotent stem cells (iPSCs), derived by ectopic expression of reprogramming factors in somatic cells, can potentially provide unlimited autologous cells for regenerative medicine. In theory, the autologous cells derived from patient iPSCs should be immune tolerant by the host without any immune rejections. However, our recent studies have found that even syngeneic iPSC-derived cells can be immunogenic in syngeneic hosts by using a teratoma transplantation model (Nature 474:212-215, 2011). Recently two research groups differentiated the iPSCs into different germ layers or cells, transplanted those cells to the syngeneic hosts, and evaluated the immunogenicity of those cells. Both of the two studies support our conclusions that some certain but not all tissues derived from iPSCs can be immunogenic, although they claimed either "negligible" or "lack of" immunogenicity in iPSC derivatives (Nature 494:100-104, 2013; Cell Stem Cell 12:407-412, 2013). To test the immunogenicity of clinically valuable cells differentiated from human iPSCs are emergently required for translation of iPSC technology to clinics.

The plateau pika (Ochotona curzonia) has adapted to high-altitude hypoxia during evolution. Higher microvessel density in specific tissues and a blunted hypoxic pulmonary vasoconstriction response are the critical components of this adaptation. VEGF, vascular endothelial growth factor, has proved to be a key regulator of angiogenesis in response to tissue hypoxia and to play an important role in vascular vasodilation. However, the role of VEGF in adaptation to high-altitude hypoxia in the plateau pika remains unknown. In this study, we cloned cDNAs for VEGF165 and VEGF189 and examined their expression in pikas inhabiting altitudes of 3200 and 4750 m. Phylogenetic analysis reveals that pika VEGF165 and VEGF189 are evolutionarily conserved. Real-time PCR analysis demonstrates that VEGF165 and VEGF189 display tissue and altitude-specific expression patterns. Interestingly, we found that the levels of VEGF189 mRNA are significantly higher than those of VEGF165 in the brain and muscle tissues of the pika, which is different from what was previously observed in sea-level mammals. VEGF189 mRNA levels in brain, muscle, and lung of the pika increased with increased habitat altitude, whereas VEGF165 shows less change. Our study suggests an important role for VEGF189 in adaptation to hypoxia by the plateau pika in the high-altitude environment.