α-Ketoglutarate alleviates osteoarthritis by inhibiting ferroptosis via the ETV4/SLC7A11/GPX4 signaling pathway.

Osteoarthritis (OA) is the most common degenerative joint disorder that causes disability in aged individuals, caused by functional and structural alterations of the knee joint. To investigate whether metabolic drivers might be harnessed to promote cartilage repair, a liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics approach was carried out to screen serum biomarkers in osteoarthritic rats. Based on the correlation analyses, α-ketoglutarate (α-KG) has been demonstrated to have antioxidant and anti-inflammatory properties in various diseases. These properties make α-KG a prime candidate for further investigation of OA. Experimental results indicate that α-KG significantly inhibited H2O2-induced cartilage cell matrix degradation and apoptosis, reduced levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) and glutathione (GSH)/glutathione disulfide (GSSG) levels, and upregulated the expression of ETV4, SLC7A11 and GPX4. Further mechanistic studies observed that α-KG, like Ferrostatin-1 (Fer-1), effectively alleviated Erastin-induced apoptosis and ECM degradation. α-KG and Fer-1 upregulated ETV4, SLC7A11, and GPX4 at the mRNA and protein levels, decreased ferrous ion (Fe2+) accumulation, and preserved mitochondrial membrane potential (MMP) in ATDC5 cells. In vivo, α-KG treatment inhibited ferroptosis in OA rats by activating the ETV4/SLC7A11/GPX4 pathway. Thus, these findings indicate that α-KG inhibits ferroptosis via the ETV4/SLC7A11/GPX4 signaling pathway, thereby alleviating OA. These observations suggest that α-KG exhibits potential therapeutic properties for the treatment and prevention of OA, thereby having potential clinical applications in the future.

Comparative transcriptome analysis provides insight into the molecular targets and signaling pathways of deer TGF-1 regulating chondrocytes proliferation and differentiation.

BACKGROUND: Chondrocytes are the only cell components in the cartilage, which has the poor regeneration ability. Thus, repairing damaged cartilage remains a huge challenge. Sika deer antlers are mainly composed of cartilaginous tissues that have an astonishing capacity for repair and renewal. Our previous study has demonstrated the transforming growth factor ß (TGF-ß1) is considered to be a key molecule involved in rapid growth, with the strongest expression in the cartilage layer. However, it remains to be clarified whether deer TGF-ß1 has significantly different function from other species such as mouse, and what is the molecular mechanism of regulating cartilage growth. METHODS: Primary chondrocytes was collected from new born mouse rib cartilage. The effect of TGF-ß1 on primary chondrocytes viability was elucidated by RNA sequencing (RNA-seq) technology combined with validation methods such as quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence assay (IFA). Differential expression genes were identified using the DEGseq package. RESULTS: Our results demonstrated that the overexpression of deer TGF-ß1 possibly promoted chondrocyte proliferation and extracellular matrix (ECM) synthesis, while simultaneously suppressing chondrocyte differentiation through regulating transcription factors, growth factors, ECM related genes, proliferation and differentiation marker genes, such as Comp, Fgfr3, Atf4, Stat1 etc., and signaling pathways such as the MAPK signaling pathway, inflammatory mediator regulation of TRP channels etc. In addition, by comparing the amino acid sequence and structures between the deer TGF-ß1 and mouse TGF-ß1, we found that deer TGF-ß1 and mouse TGF-ß1 proteins are mainly structurally different in arm domains, which is the main functional domain. Phenotypic identification results showed that deer TGF-ß1 may has stronger function than mouse TGF-ß1. CONCLUSION: ​These results suggested that deer TGF-ß1 has the ability to promote chondrogenesis by regulating chondrocyte proliferation, differentiation and ECM synthesis. This study provides insights into the molecular mechanisms underlying the effects of deer TGF-ß1 on chondrocyte viability.

Novel insights into the effect of deer IGF-1 on chondrocyte viability and IL-1ß-induced inflammation response.

Clinical treatment of Osteoarthritis (OA) remains a challenge due to the poor self-regeneration ability of cartilage. Deer antler is the only cartilage tissue that can completely regenerate each year. Insulin-like growth factor 1 (IGF-1) is one of the major active components in the deer antler that participate in regulating the rapid regeneration of deer antler cartilage. This has led us to speculate that deer IGF-1 might potentially become a candidate drug for reducing damage and inflammation of OA. Thus, we aimed to explore the underlying mechanism of deer IGF-1 in chondrocyte proliferation, differentiation, and inflammation response. Deer, mouse, and human IGF-1 amino acid sequences and protein structures were aligned using CLUSTAL and PSIPRED. The underlying molecular mechanism of deer IGF-1 on primary chondrocytes was investigated by RNA-sequencing (RNA-seq) technology combined with various experiments. Cytokine interleukin-1ß (IL-1ß) was used to induce the inflammation response of primary chondrocytes. We found that deer IGF-1 was more similar to human IGF-1 than mouse IGF-1. qRT-PCR and immunofluorescence assay indicated that deer IGF-1 had stronger effects than mouse IGF-1. We also found that the deer IGF-1 enhanced the expression of cell proliferation, differentiation, and extracellular matrix (ECM)-related genes, but decreased the expression of ECM-degrading genes. Deer IGF-1 also attenuated the IL-1ß-induced inflammatory and ECM degradation in chondrocytes. This study provides insight into the molecular mechanisms of deer IGF-1 on primary chondrocyte viability and presents a candidate for combatting inflammatory responses in OA development.

Comprehensive RNA sequencing in primary murine keratinocytes and fibroblasts identifies novel biomarkers and provides potential therapeutic targets for skin-related diseases.

BACKGROUND: Keratinocytes and fibroblasts represent the major cell types in the epidermis and dermis of the skin and play a significant role in maintenance of skin homeostasis. However, the biological characteristics of keratinocytes and fibroblasts remain to be elucidated. The purpose of this study was to compare the gene expression pattern between keratinocytes and fibroblasts and to explore novel biomarker genes so as to provide potential therapeutic targets for skin-related diseases such as burns, wounds, and aging. METHODS: Skin keratinocytes and fibroblasts were isolated from newborn mice. To fully understand the heterogeneity of gene expression between keratinocytes and fibroblasts, differentially expressed genes (DEGs) between the two cell types were detected by RNA-seq technology. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the known genes of keratinocytes and fibroblasts and verify the RNA-seq results. RESULTS: Transcriptomic data showed a total of 4309 DEGs (fold-change > 1.5 and q-value < 0.05). Among them, 2197 genes were highly expressed in fibroblasts and included 10 genes encoding collagen, 16 genes encoding transcription factors, and 14 genes encoding growth factors. Simultaneously, 2112 genes were highly expressed in keratinocytes and included 7 genes encoding collagen, 14 genes encoding transcription factors, and 8 genes encoding growth factors. Furthermore, we summarized 279 genes specifically expressed in keratinocytes and 33 genes specifically expressed in fibroblasts, which may represent distinct molecular signatures of each cell type. Additionally, we observed some novel specific biomarkers for fibroblasts such as Plac8 (placenta-specific 8), Agtr2 (angiotensin II receptor, type 2), Serping1 (serpin peptidase inhibitor, clade G, member 1), Ly6c1 (lymphocyte antigen 6 complex, locus C1), Dpt (dermatopontin), and some novel specific biomarkers for keratinocytes such as Ly6a (lymphocyte antigen 6 complex, locus A) and Lce3c (late cornified envelope 3C), Ccer2 (coiled-coil glutamate-rich protein 2), Col18a1 (collagen, type XVIII, alpha 1) and Col17a1 (collagen type XVII, alpha 1). In summary, these data provided novel identifying biomarkers for two cell types, which can provide a resource of DEGs for further investigations.

Magnetic Multiarm Scaffold for the One-Step Purification of Epitope-Specific Neutralizing Antibodies.

Epitope-specific neutralizing antibodies (EsAbs) are of prime importance in the diagnosis and treatment of various serious diseases. However, obtaining EsAbs by the monoclonal antibody technique involves time-consuming and sophisticated multistep procedures, and the epitopes of the resulting antibodies are often not explicit. It is also very challenging to isolate EsAbs from numerous kinds of total immunoglobulins because of nonspecific adsorption and low separation efficiency. Herein, a magnetic core@multiarm shell-epitope (M@A-E) bioconjugate was fabricated to enrich and isolate EsAbs from immune serums. This robust multiarm scaffold exhibits outstanding binding capacity and good resistance to nontarget adsorption and serves as a reservoir for the release and reloading of EsAbs for repeatable applications. The EsAbs yield per milligram of the M@A-E was about 30 µg, which was approximately twice that of commercially available beads (16 µg). After 10 cycles of loading and release in glycine buffer (0.1 M, pH 2.5), the M@A-E bioconjugates still showed relatively high specificity and capture capacity (20 µg) superior to the same amount of new, unused conventional ones. This strategy provides a promising platform for enriching and isolating substantial quantities of EsAbs, which have great potential for applications in the detection and treatment of critical illness.

Astilbin Inhibits the Activity of Sortase A from Streptococcus mutans.

Streptococcus mutans (S. mutans) is the primary etiological agent of dental caries. The S. mutans enzyme sortase A (SrtA) is responsible for anchoring bacterial cell wall surface proteins involved in host cell attachment and biofilm formation. Thus, SrtA is an attractive target for inhibiting dental caries caused by S. mutans-associated acid fermentation. In this study, we observed that astilbin, a flavanone compound extracted from Rhizoma Smilacis Glabrae, has potent inhibitory activity against the S. mutans SrtA, with an IC50 of 7.5 µg/mL. In addition, astilbin was proven to reduce the formation of biofilm while without affecting the growth of S. mutans. The results of a molecular dynamics simulation and a mutation analysis revealed that the Arg213, Leu111, and Leu116 of SrtA are important for the interaction between SrtA and astilbin. The results of this study demonstrate the potential of using astilbin as a nonbactericidal agent to modulate pathogenicity of S. mutans by inhibiting the activity of SrtA.

Structural Characters and Pharmacological Activity of Protopanaxadiol-Type Saponins and Protopanaxatriol-Type Saponins from Ginseng.

Ginseng has a long history of drug application in China, which can treat various diseases and achieve significant efficacy. Ginsenosides have always been deemed important ingredients for pharmacological activities. Based on the structural characteristics of steroidal saponins, ginsenosides are mainly divided into protopanaxadiol-type saponins (PDS, mainly including Rb1, Rb2, Rd, Rc, Rh2, CK, and PPD) and protopanaxatriol-type saponins (PTS, mainly including Re, R1, Rg1, Rh1, Rf, and PPT). The structure differences between PDS and PTS result in the differences of pharmacological activities. This paper provides an overview of PDS and PTS, mainly focusing on their chemical profile, pharmacokinetics, hydrolytic metabolism, and pharmacological activities including antioxidant, antifatigue, antiaging, immunodulation, antitumor, cardiovascular protection, neuroprotection, and antidiabetes. It is intended to contribute to an in-depth study of the relationship between PDS and PTS.

Gubi Zhitong formula alleviates osteoarthritis in vitro and in vivo via regulating BNIP3L-mediated mitophagy.

BACKGROUND: Osteoarthritis (OA) is characterized by degeneration of articular cartilage, leading to joint pain and dysfunction. Gubi Zhitong formula (GBZTF), a traditional Chinese medicine formula, has been used in the clinical treatment of OA for decades, demonstrating definite efficacy. However, its mechanism of action remains unclear, hindering its further application. METHODS: The ingredients of GBZTF were analyzed and performed with liquid chromatography-mass spectrometry (LC-MS). 6 weeks old SD rats were underwent running exercise (25 m/min, 80 min, 0°) to construct OA model with cartilage wear and tear. It was estimated by Micro-CT, Gait Analysis, Histological Stain. RNA-seq technology was performed with OA Rats' cartilage, and primary chondrocytes induced by IL-1ß (mimics OA chondrocytes) were utilized to evaluated and investigated the mechanism of how GBZTF protected OA cartilage from being damaged with some functional experiments. RESULTS: A total of 1006 compounds were identified under positive and negative ion modes by LC-MS. Then, we assessed the function of GBZTF through in vitro and vivo. It was found GBZTF could significantly up-regulate OA rats' limb coordination and weight-bearing capacity, and reduce the surface and sub-chondral bone erosions of OA joints, and protect cartilage from being destroyed by inflammatory factors (iNOS, IL-6, IL-1ß, TNF- α, MMP13, ADAMTS5), and promote OA chondrocytes proliferation and increase the S phage of cell cycle. In terms of mechanism, RNA-seq analysis of cartilage tissues revealed 1,778 and 3,824 differentially expressed genes (DEGs) in model vs control group and GBZTF vs model group, respectively. The mitophagy pathway was most significantly enriched in these DEGs. Further results of subunits of OA chondrocytes confirmed that GBZTF could alleviate OA-associated inflammation and cartilage damage through modulation BCL2 interacting protein 3-like (BNIP3L)-mediated mitophagy. CONCLUSION: The therapeutic effectiveness of GBZTF on OA were first time verified in vivo and vitro through functional experiments and RNA-seq, which provides convincing evidence to support the molecular mechanisms of GBZTF as a promising therapeutic decoction for OA.

Integrated Network Pharmacology and Experimental Validation Approach to Investigate the Mechanisms of Radix Rehmanniae Praeparata - Angelica Sinensis - Radix Achyranthis Bidentatae in Treating Knee Osteoarthritis.

Background: Knee osteoarthritis (KOA) is a persistent degenerative condition characterized by the deterioration of cartilage. The Chinese herbal formula Radix Rehmanniae Praeparata- Angelica Sinensis-Radix Achyranthis Bidentatae (RAR) has often been used in effective prescriptions for KOA as the main functional drug, but its underlying mechanism remains unclear. Therefore, network pharmacology and verification experiments were employed to investigate the impact and mode of action of RAR in the treatment of KOA. Methods: The destabilization of the medial meniscus model (DMM) was utilized to assess the anti-KOA effect of RAR by using gait analysis, micro-computed tomography (Micro-CT), and histology. Primary chondrocytes were extracted from the rib cartilage of a newborn mouse. The protective effects of RAR on OA cells were evaluated using a CCK-8 assay. The antioxidative effect of RAR was determined by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) production. Furthermore, network pharmacology and molecular docking were utilized to propose possible RAR targets for KOA, which were further verified through experiments. Results: In vivo, RAR significantly ameliorated DMM-induced KOA characteristics, such as subchondral bone sclerosis, cartilage deterioration, gait abnormalities, and the degree of knee swelling. In vitro, RAR stimulated chondrocyte proliferation and the expression of Col2a1, Comp, and Acan. Moreover, RAR treatment significantly reduced ROS accumulation in an OA cell model induced by IL-1ß and increased the activity of antioxidant enzymes (SOD and GSH). Network pharmacology analysis combined with molecular docking showed that Mapk1 might be a key therapeutic target. Subsequent research showed that RAR could downregulate Mapk1 mRNA levels in IL-1ß-induced chondrocytes and DMM-induced rats. Conclusion: RAR inhibited extracellular matrix (ECM) degradation and oxidative stress response via the MAPK signaling pathway in KOA, and Mapk1 may be a core target.

Function and regulation of nuclear factor 1 X-type on chondrocyte proliferation and differentiation.

Nuclear factor 1 X-type (Nfix) is a transcription factor related to mental and physical development. However, very few studies have reported the effects of Nfix on cartilage. This study aims to reveal the influence of Nfix on the proliferation and differentiation of chondrocytes, and to explore its potential action mechanism. We isolated primary chondrocytes from the costal cartilage of newborn C57BL/6 mice and with Nfix overexpression or silencing treatment. We used Alcian blue staining and found that Nfix overexpression significantly promoted ECM synthesis in chondrocytes while silencing inhibited ECM synthesis. Using RNA-seq technology to study the expression pattern of Nfix in primary chondrocytes. We found that Nfix overexpression significantly up-regulated genes that are related to chondrocyte proliferation and extracellular matrix (ECM) synthesis and significantly down-regulated genes related to chondrocyte differentiation and ECM degradation. Nfix silencing, however, significantly up-regulated genes associated with cartilage catabolism and significantly down-regulated genes associated with cartilage growth promotion. Furthermore, Nfix exerted a positive regulatory effect on Sox9, and we propose that Nfix may promote chondrocyte proliferation and inhibit differentiation by stimulating Sox9 and its downstream genes. Our findings suggest that Nfix may be a potential target for the regulation of chondrocyte proliferation and differentiation.

BuShen JianGu Fang alleviates cartilage degeneration via regulating multiple genes and signaling pathways to activate NF-κB/Sox9 axis.

BACKGROUND: Osteoarthritis (OA) is an inflammatory response in chondrocytes, causing extracellular matrix (ECM) degradation and cartilage destruction, affecting millions of people worldwide. Chinese herbal formulae BuShen JianGu Fang (BSJGF) has been clinically applied for treating OA-related syndromes, but the underlying mechanism still unclear. METHODS: The components of BSJGF were analyzed by liquid chromatography-mass spectrometry (LC-MS). To make a traumatic OA model, the anterior cruciate ligament of 6-8-week-old male SD rats were cut and then the 0.4 mm metal was used to destroy the knee joint cartilage. OA severity was assessed by histological and Micro-CT. Mouse primary chondrocytes were utilized to investigate the mechanism of BSJGF alleviate osteoarthritis, which was examined by RNA-seq technology combined with a series of functional experiments. RESULTS: A total 619 components were identified by LC-MS. In vivo, BSJGF treatment result in a higher articular cartilage tissue area compared to IL-1ß group. Treatment also significantly increased Tb.Th, BV/TV and BMD of subchondral bone (SCB), which implied a protective effect on maintaining the stabilization of SCB microstructure. In vitro results indicated BSJGF promoted chondrocyte proliferation, increased the expression level of cartilage-specific genes (Sox9, Col2a1, Acan) and synthesized acidic polysaccharide, while inhibiting the release of catabolic enzymes and production of reactive oxygen species (ROS) induced by IL-1ß. Transcriptome analysis showed that there were 1471 and 4904 differential genes between IL-1ß group and blank group, BSJGF group and IL-1ß group, respectively, including matrix synthesis related genes (Col2a1, H19, Acan etc.), inflammation related genes (Comp, Pcsk6, Fgfr3 etc.) and oxidative stress related genes (Gm26917, Bcat1, Sod1 etc.). Furthermore, KEGG analysis and validation results showed that BSJGF reduces OA-mediated inflammation and cartilage damaged due to modulation of NF-κB/Sox9 signaling axis. CONCLUSION: The innovation of the present study was the elucidation of the alleviating cartilage degradation effect of BSJGF in vivo and in vitro and discovery of its mechanism through RNA-seq combined with function experiments, which provides a biological rationale for the clinical application of BSJGF for OA treatment.

One case of wrist cyst rupture with nerve and blood vessel compression.

INTRODUCTION AND IMPORTANCE: Tendon sheath cysts are mostly located around the joint capsule and tendon sheath, which often occur in the wrist, ankle, and wrist back (O'Valle et al., 2014; Nguyen et al., 2004 [1, 2]). The palmar side of the wrist is relatively rare, which is often associated with the wrist. Ultrasound and MRI can detect and diagnose early. CASE PRESENTATION: In this case report, we discussed an elderly woman with palmar carpal tendon sheath cyst and ruptured. She communicated with the outside world through the skin sinus, and at the same time pressed the radial artery and the median nerve to produce obvious clinical symptoms. CLINICAL DISCUSSION: Because of its deep position and close relationship with the surrounding important nerves and vessels, the operation was relatively difficult. In particular, in this case, tendon sheath cyst ruptured to form sinus, so there were some difficulties. CONCLUSION: Pathological diagnosis was tendon sheath cyst. The analysis of the relationship between the tumor and the surrounding tissue by preoperative MRI and other imaging examinations has important guiding significance for surgery.

Giant intermuscular lipoma of hip: A case report.

INTRODUCTION: Lipomas are one of the most common benign tumors, but deep tissue and huge lipomas are rare. PRESENTATION OF CASE: A 59-year-old middle-aged woman was admitted to hospital for right hip mass resection because of numbness and pain in her right lower limb for 6 months. Ultrasound and nuclear magnetic resonance imaging (MRI) showed irregular mass. On the assumption of malignancy, the patient underwent a right hip mass resection and returned to normal with no short-term or long-term response. HE (hematoxylin-eosin) staining of the tumor showed the characteristics of a benign tumor. DISCUSSION: The pathogenesis of myolipoma is not clear, and it may be related to brown adipose tissue. When lipoma is huge, it needs interventional treatment. Complete encapsulated myolipoma can usually be completely removed, with low recurrence possibility and good prognosis. CONCLUSION: Giant intramuscular lipoma of hip and its clinical manifestations are relatively rare. And the results of ultrasound imaging are similar to those of the surrounding normal adipose tissue. No significant difference was observed by naked eye. In particular, postoperative pathological examination is needed to make a definite diagnosis and differential diagnosis. Clinically, surgical treatment is often used, and the prognosis is good, but recurrence is possible. The following report discusses the experience of one of our patients, with literature review.

Deer antler extract promotes tibia fracture healing in mice by activating BMP-2/SMAD4 signaling pathway.

BACKGROUND: Deer antler is a traditional Chinese medicine with the function of tonifying kidney and strengthening bone, which is often used to treat orthopedic diseases. METHODS: Eight-week-old C57BL/6 mice were used as the fixation model of open tibial fracture with intramedullary nail. The mice were treated with deer antler extract (DAE) or PBS by oral gavage once daily. The tibial fracture samples were collected and performed to the tissue analysis, including X-ray, micro-CT, histology, qRT-PCR, immunohistochemistry. MC3T3-E1 cells were used to detect the effect of deer antler extract on ability of cell proliferation and migration by CCK-8 assay and cell scratch test. RESULTS: Imaging and micro-CT showed that DAE could promote the healing of tibial fracture in mice, and histological analysis showed that DAE could promote the transformation of cartilage callus to bone callus in fracture area. The results of qRT-PCR and immunohistochemistry showed that DAE could promote intrachondral ossification in fracture zone and the mechanism of promoting fracture healing may be related to the activation of BMP-2/SMAD4 signaling pathway. In the cytological experiment of DAE, it can be found that DAE promoted the proliferation of MC3T3-E1 cells and the migration of MC3T3-E1 cells at a certain concentration, which is also related to the promotion of fracture healing by DAE. CONCLUSION: DAE can promote fracture healing by activating BMP-2/SMAD4 signaling pathway. DAE has the potential to be used in clinic as an important means of promoting fracture healing.

Antiviral strategies targeting herpesviruses.

Herpesviruses, known as large DNA viruses, have a wide host range. In addition to human beings, cattle, and horses, even carp can be hosts for herpesvirus infection. Herpesviruses are pathogens possessing elaborate mechanisms that regulate host cell components for its replication, assembly and generating mature virus particles that can infect humans and most animals, usually causing multiple and lifelong infections. In addition, several human diseases, such as genital or mouth herpes, mononucleosis, and Burkitt lymphoma, are usually associated with herpesvirus infection. Blocking the steps of viral infection, such as entry, replication and assembly, may be an effective way for many different herpes viruses and their related diseases. Therefore, we aim to describe antiviral agents that are able to prevent herpesvirus entry, replication and assembly in host cells. We summarize antiviral strategies, including certain small molecular drugs, RNA interference and CRISPR/Cas9 system-based antiviral approaches, which represent promising approaches.

Tetrahedral Framework Nucleic Acid Delivered RNA Therapeutics Significantly Attenuate Pancreatic Cancer Progression via Inhibition of CTR1-Dependent Copper Absorption.

Copper is vital for various life processes, whereas severely toxic at excess level. Intracellular copper homeostasis is strictly controlled by a set of transporters and chaperones encoded by the copper homeostasis genes. Increasing evidence has shown that copper is usually overloaded in multiple malignancies, including pancreatic cancer, which has an extremely poor prognosis. Recently, silencing the SLC31A1 gene, which encodes a major transmembrane copper transporter (CTR1), has been demonstrated to be an effective means for reducing the malignant degree of pancreatic cancer by downregulating the cellular copper levels. Herein, we utilized tetrahedral framework nucleic acids (tFNAs) as vehicles to overcome the biological barriers for delivering small molecular RNAs and efficiently transferred two kinds of CTR1 mRNA-targeted RNA therapeutics, siCTR1 or miR-124, into PANC-1 cells. Both therapeutic tFNAs, termed t-siCTR1 and t-miR-124, prevented copper intake more effective than the free RNA therapeutics via efficiently suppressing the expression of CTR1, thereby significantly attenuating the progression of PANC-1 cells. In this study, therapeutic tFNAs are constructed to target metal ion transporters for the first time, which may provide an effective strategy for future treatment of other metal metabolism disorders.

Improved Antiviral Activity of Classical Swine Fever Virus-Targeted siRNA by Tetrahedral Framework Nucleic Acid-Enhanced Delivery.

DNA self-assembled nanostructures have been considered as effective vehicles for biomolecule delivery because of their excellent biocompatibility, cellular permeability, noncytotoxicity, and small size. Here, we report an efficient antiviral strategy with self-assembled tetrahedral framework nucleic acids (tFNAs) delivering small interfering RNA (t-siRNA) to silence classical swine fever virus (CSFV) gene in porcine host cells. In this study, two previously reported siRNAs, C3 and C6, specifically targeting the CSFV genome were selected and modified on tFNAs, respectively, and termed t-C3 and t-C6. Results indicate that t-C3 and t-C6 can inhibit the viral proliferation of CSFV in kidney derived porcine cells, PK-15, effectively and that inhibition was markedly stronger than free siRNA-C3 or siRNA-C6 only. In addition, the DNA nanostructure also has high cargo-carrying capacity, allowing to deliver multiple functional groups. To improve the antiviral ability of tFNAs, a dual-targeting DNA nanostructure t-C3-C6 was constructed and used to silence the CSFV gene in porcine host cells. This study found that t-C3-C6 can inhibit the viral release and replication, exhibiting outstanding anti-CSFV capabilities. Therefore, these dual-targeting tFNAs have great potential in virus therapy. This strategy not only provides a novel method to inhibit CSFV replication in porcine cells but also verifies that tFNAs are effective tools for delivery of antiviral elements, which have great application potential.

Exploiting a conjugative CRISPR/Cas9 system to eliminate plasmid harbouring the mcr-1 gene from Escherichia coli.

The transfer of multi-drug-resistance plasmids by bacterial conjugation is largely responsible for the development of drug resistance in bacteria, and causes serious problems in the treatment of infectious diseases. Since the first discovery of plasmid-borne colistin resistance gene mcr-1 was reported in late 2016, this gene has been found in a great number of Escherichia coli and other Gram-negative pathogens separated from different types of sources worldwide. The elimination of plasmids carrying mcr-1 and restoration of polymyxin sensitivity has very important clinical significance because polymyxins are frequently used as last-resort antibiotics to treat extensively drug-resistant Gram-negative bacterial infections. A host-independent conjugative plasmid was constructed in this study, and an engineered CRISPR/Cas9 system was used to remove plasmid harbouring mcr-1 from bacteria. This study found that this conjugative plasmid can not only be used as a new tool to remove resistance plasmids and sensitize the recipient bacteria to antibiotics, but can also make the recipient cell acquire immunity against mcr-1. This strategy provides a novel method to counteract the ever-worsening spread of mcr-1 among bacterial pathogens.

Development of Whole-Porcine Monoclonal Antibodies with Potent Neutralization Activity against Classical Swine Fever Virus from Single B Cells.

Classical swine fever (CSF) is a highly contagious swine disease that causes devastating economic losses. However, there are few efficacious therapeutic antibodies against the CSF virus (CSFV). Accordingly, we isolated two whole-porcine anti-CSFV neutralizing antibodies (NAbs) directly from single B cells sorted using the conserved linear epitope of the CSFV E2 protein and goat anti-pig IgG. These mAbs, termed HK24 and HK44, can bind to the E2 protein by recognizing sites within the conserved linear epitope of E2. In addition, these two mAbs can detect virus infection with high specificity and possess potent neutralizing activity. HK24 and HK44 protect PK-15 cells from CSFV infections in vitro with potent IC50 values of 9.3 and 0.62 µg/mL, respectively. We anticipate that these antibodies can be used as diagnostic and antiviral agents for CSFV and that the method we describe here will accelerate the production of therapeutic antibodies for other viruses.

Biomimetic Octopus-like Particles for Ultraspecific Capture and Detection of Pathogens.

Infectious diseases caused by pathogenic bacteria (such as sepsis and meningitis) seriously threaten public health; therefore, rapid and accurate identification of the target bacteria is urgently needed to prevent and treat bacterial infections. Although technologies including plate-counting and polymerase chain reaction have been established to detect the pathogenic bacteria, they are either time-consuming or sophisticated. Herein, a biomimetic octopus-like structure integrating merits of multiarm and multivalent interaction is designed for ultraspecific capture and detection of pathogens. The flexible polymeric arms and multivalent ligands work together to mimic the arm-sucker coordination of an octopus to effectively grasp the target pathogens, leading to remarkably high capacity and specificity for the target capture (above 98%, 10 CFU mL-1) without a nonspecific absorption of background pathogens. The captured bacteria can be identified as a point of care by the surface-enhanced Raman spectroscopy method with a detection limit of 10 cells mL-1.