Advancement in the development of single chain antibodies using phage display technology.

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.

Preparation of PLCL/ECM nerve conduits by electrostatic spinning technique and evaluationin vitroandin vivo.

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. Thein vivoresults 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.

Competition between lanes and transient jammed clusters in driven binary mixtures.

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.

Exosomes: The emerging mechanisms and potential clinical applications in dermatology.

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.

Neutrophil extracellular trap-related mechanisms in acne vulgaris inspire a novel treatment strategy with adipose-derived stem cells.

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.

Analysis of Effective Pyrolysis Zone and Heat Loss in Oil Shale Reservoir with Random Fractures.

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.

Exosomes Derived from E2F1-/- Adipose-Derived Stem Cells Promote Skin Wound Healing via miR-130b-5p/TGFBR3 Axis.

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 ADSCWT-Exos and ADSCE2F1-/--Exos. The wound healing effects of ADSCWT-Exos and ADSCE2F1-/--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 ADSCWT-Exos and ADSCE2F1-/--Exos to promote cell activities, collagen formation, and angiogenesis were evaluated. The potential mechanism by which ADSCE2F1-/--Exos promote wound healing was determined by miRNA sequencing, ChIP‒qPCR, and dual-luciferase assays. Results: ADSCE2F1-/--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 ADSCWT-Exo group, ADSCE2F1-/--Exos significantly increased the wound healing rate to 72.5%. In vitro, ADSCE2F1-/--Exos activated the function of fibroblasts and vascular endothelial cells. The loss of E2F1 promoted miR-130b-5p expression in ADSCE2F1-/--Exos through transcriptional regulation. MiRNA high-throughput sequencing identified 12 differently expressed miRNAs between ADSCE2F1-/- and ADSCWT. ADSCE2F1-/--Exos enhanced fibroblast activities via the miR-130b-5p/TGFBR3 axis and TGF-ß activation. Conclusion: Our results indicated that ADSCE2F1-/--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.

Macroscopic fracture mechanism of coal body and evolution characteristics analysis of impact force in deep coal and gas outburst.

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% CO2 and 55% N2. 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.

HTRA3 transcriptionally inhibited by FOXP1 suppresses tumorigenesis of osteosarcoma via the PTEN/PI3K/AKT pathway.

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.

Synthetic biodegradable polymer materials in the repair of tumor-associated bone defects.

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.

Gas pressure evolution characteristics of deep true triaxial coal and gas outburst based on acoustic emission monitoring.

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.

Current landscape of personalized clinical treatments for triple-negative breast cancer.

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.

Clinical significance for diagnosis and prognosis of POP1 and its potential role in breast cancer: a comprehensive analysis based on multiple databases.

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.

Perpendicular and parallel phase separation in two-species driven diffusive lattice gases.

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.

Landscape of the epigenetic regulation in wound healing.

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.

Complement Receptor 3 Pathway and NMDA Receptor 2B Subunit Involve Neuropathic Pain Associated with Spinal Cord Injury.

Background: Neuropathic pain (NP) after spinal cord injury (SCI-evoked NP) is clinically challenging; the underlying mechanisms are not fully understood, leading to a lack of promising treatment options. NP occurs in only a subset of patients with SCI. The injured spinal cord exhibits a series of histopathological changes, and the complement system has been shown to play an important role in these processes. In addition, NMDA receptor subunit 2B (NR2B) is involved in the development and maintenance of NP. This preliminary study was performed to investigate the correlations of the complement receptor 3/complement component 3 (CR3/C3) pathway and NR2B with SCI-evoked NP. Methods: A trauma-induced SCI animal model was established and SCI-evoked NP was evaluated by behavioural analysis. Transcriptome analysis was performed to identify genes in the CR3/C3 pathway related to synaptic modification, while the expression and distribution of NR2B in the injured spinal cord, and the relation to NP, were examined by immunohistochemical analysis. Results: Nine of seventeen SCI rats (52.9%) developed NP. C3 mRNA expression was significantly decreased in SCI-evoked NP rats and significantly increased in the non-NP SCI rats. C1q mRNA and CR3 mRNA expression were significantly increased in all SCI rats, but higher levels of expression were observed in the non-NP SCI rats. NR2B mRNA expression was significantly increased in the SCI-evoked NP rats and significantly decreased in the non-NP SCI rats. In addition, significantly elevated expression of NR2B-positive cells was seen in lamina II of the superficial dorsal horn in SCI-evoked NP rats in comparison with non-NP SCI rats. Conclusion: NP occurred in only a subset of SCI rats, and the CR3/C3 pathway and NR2B were involved in SCI-evoked NP. Further studies are required to determine the mechanisms underlying the SCI-evoked NP associated with the CR3/C3 pathway and NR2B.

Immunotherapy landscape analyses of necroptosis characteristics for breast cancer patients.

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.

Production of Triple-Gene (GGTA1, B2M and CIITA)-Modified Donor Pigs for Xenotransplantation.

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.

Analysis of Tumor Glycosylation Characteristics and Implications for Immune Checkpoint Inhibitor's Efficacy for Breast Cancer.

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.