Generation of a High-Affinity Nanobody Against CD147 for Tumor Targeting and Therapeutic Efficacy Through Conjugating Doxorubicin.

CD147, a glycosylated transmembrane protein in the immunoglobulin superfamily, is overexpressed on the surfaces of various tumor cells and promotes cancer cell proliferation, invasion, and metastasis. Nanobodies, characterized by small sizes, high affinities and specificities, and low immunogenicities, are promising diagnostic and therapeutic tools. However, there are few reports on nanobodies that specifically target CD147. In this work, a specific anti-CD147 nanobody has been successfully identified using phage display technology. The tumor target and antitumor effects have also been detected in different CD147-positive tumors in in vitro and in vivo assays, respectively. Meanwhile, it has a synergistic effect for inhibiting 4T1-bearing mice through conjugating doxorubicin. It may afford new strategies for cancer therapies.

Evaluation of Toxicity, Bacteriostatic, Analgesic, Anti-Inflammatory, and Antipyretic Activities of Huangqin-Honghua-Pugongying-Jinyinhua Extract.

The extensive use of antibiotics has caused the global spread of multidrug-resistant bacteria and genes, seriously reducing antibiotic efficacy and threatening animal and human health. As an alternative, traditional Chinese veterinary medicine (TCVM) was used in this study for its lack of drug resistance and low toxicity. Huangqin-honghua-pugongying-jinyinhua extract (HHPJE), a novel TCVM, consists of the extracts of Huangqin (Scutellaria baicalensis), Honghua (Carthami Flos), Pugongying (Taraxacum) and Jinyinhua (Lonicerae Japonicae Flos), and was developed to treat bovine mastitis. In this study, we evaluated the toxicity, bacteriostatic, analgesic, anti-inflammatory, and antipyretic activities of HHPJE. Our results show that HHPJE did not show any acute oral toxicity and can be considered safe for oral administration. Additionally, HHPJE possessed a dose-dependent antibacterial effect on Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Streptococcus dysgalactiae. HHPJE (60, 30 and 15 g/kg) can reduce the abdominal pain by 44.83 ± 7.69%, 43.15 ± 9.50% and 26.14 ± 4.17%, respectively. The percentages of anti-inflammation inhibition (60, 30 and 15 g/kg) were 35.34 ± 2.17%, 22.29 ± 2.74% and 12.06 ± 3.61%, respectively. The inhibition rates (60, 30 and 15 g/kg) of antipyretic activity were 82.05%, 65.71% and 52.80%, respectively. The evaluation of pharmacodynamics and toxicity indicate that HHPJE possesses significant bacteriostatic, analgesic, anti-inflammatory and antipyretic potential, and also that it is safe for acute oral toxicity, which means it has potential value for treating bovine mastitis in future and alleviating clinical symptoms with no drug resistance or side effects.

Prevalence and characteristics of extended-spectrum ß-lactamases-producing Escherichia coli from broiler chickens at different day-age.

Commensal Escherichia coli from the poultries have been considered as reservoirs of extended-spectrum ß-lactamases (ESBL)-encoding genes. Between May 2018 and March 2019, a total of 340 E. coli isolates were obtained from apparently healthy broiler chickens from 20 to 40 D old, distributed in 17 small-scale commercial farms. Finally, 45 isolates (8 from 20-day-old broiler chickens, 14 from 30-day-old ones, and 23 from 40-day-old ones) were identified as ESBL producers, which were further investigated to shed light on the virulence gene profiles, phylogenetic groups, and multilocus sequence types and to detect the ESBL plasmid-mediated quinolone resistance determinant (PMQR) genes as well as the mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC. Molecular analysis showed that phylogenic group A and B1 accounted for 66.7% of the ESBL producers. The overall occurrence of virulence genes ranged from 5.1% (cva) to 86.7% (papC). Twenty (44.4%) ESBL producers were considered as biofilm producers with moderate or heavy biofilm formation. The most predominant specific CTX-M subtype was blaCTX-M-14 (n = 19), followed by blaCTX-M-9 (n = 17), blaCTX-M-55 (n = 9), blaCTX-M-15 (n = 6), blaCTX-M-1 (n = 5), and blaCTX-M-65 (n = 4). Additionally, PMQR genes were identified in 86.7% of ESBL producers, qnrS (n = 21) was the most dominant PMQR gene, followed by the aac(6')-Ib-cr (n = 15), qnrB (n = 12), and qnrA (n = 9), and all of them co-expressed with ß-lactamase genes. All PMQR-positive isolates harbored simultaneously at least 1 mutation in the QRDR of gyrA and parC. Forty-five ESBL producers were assigned to 33 sequence types, and the most frequent sequence types (STs) was ST10 (n = 5) and followed by ST95 (n = 3). Additionally, ST302, ST88, ST410, ST187, and ST23 were represented by 2 ESBL producers, respectively, and the remaining ones exhibited diverse ST. Moreover, the prevalence of ESBL producers, the biofilm-forming ability, and the occurrence of the QRDR mutations among the E. coli isolates were characterized by gradually increased with advancing age of broiler chickens.

Arctigenin Attenuates Tumor Metastasis Through Inhibiting Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma via Suppressing GSK3ß-Dependent Wnt/ß-Catenin Signaling Pathway In Vivo and In Vitro.

Arctigenin (ARG) has been reported to be a bioactive lignan from Arctium lappa exerting various activities including anti-cancer and immune-regulation. The present study aimed to investigate the anti-metastasis activity and mechanism of ARG against hepatocellular carcinoma in vitro and in vivo. The results showed that ARG exhibited a significant cytotoxicity on Hep G2 and SMMC 7721 cells (but not on normal liver cells LO2). In addition, the migration and invasion of Hep G2 and SMMC 7721 cells were also remarkably repressed. Furthermore, ARG attenuated Wnt/ß-catenin signaling activation, resulting in the down-regulation of ß-catenin target genes including c-Myc, cyclin D1, MMP-9, and ZO-1. Noticeably, ARG attenuated the activation of Wnt/ß-catenin through a GSK3ß-dependent pathway. Besides, we also found that ARG potentially inhibited epithelial-mesenchymal transition by up-regulating the epithelial and down-regulating the mesenchymal marker proteins. In vivo, intraperitoneal injection of ARG not only significantly inhibited the growth of subcutaneous transplanted tumor but also dramatically alleviated the tumor metastasis in liver. Our data demonstrated that ARG exerted anti-epithelial-mesenchymal transition and anti-metastasis activities against hepatocellular carcinoma, which might make it a candidate as a preventive agent for cancer metastasis.

Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Multidrug Resistant Escherichia coli From Swine in Northwest China.

Objectives: The aim of the present study was to explore the prevalence and molecular characterization of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli collected from pig farms in Northwest China. Methods: Between May 2015 and June 2017, a total of 456 E. coli isolates were collected from fecal samples of healthy and diarrheal pigs in Northwest China to screen the ESBL producers. The ß-lactamases, plasmid-mediated quinolone resistance (PMQR) genes and virulence genes among ESBL producers were corroborated by PCR and sequencing. Finally, ESBL producers were further grouped according to phylogenetic background and genetic relatedness. Results: Forty-four (9.6%) out of the 456 E. coli isolates were identified as ESBL-producing isolates. All ESBL producers exhibited multidrug resistance (MDR) phenotype, and more than 90% of the ESBL producers were resistant to amoxicillin, amoxicillin-clavulanic acid, oxytetracycline, enrofloxacin and sulfamethoxazole/trimethoprim. All ESBL producers harbored at least one type of ß-lactamase, with blaCTX-M, blaTEM, blaSHV, blaOXA-48, and blaKPC-2 being detected in forty, thirty, seven, four, two and one isolates, respectively. Sequencing revealed the most common blaCTX-M subtype was blaCTX-M-14 (n = 24), followed by blaCTX-M-15 (n = 14), blaCTX-M-64 (n = 11), blaCTX-M-9 (n = 10) and blaCTX-M-123 (n = 9). qnrS (n = 23) was the predominant PMQR gene, and all PMQR genes were detected in co-existence with ß-lactamase genes. estA (n = 18) and F4 (n = 18) were the most prevalent enterotoxin and fimbrial adhesin, respectively, and 27 different virotypes were found with respect to the association of enterotoxins and fimbrial adhesins. Twenty-four different sequence types (STs) were identified among 44 ESBL producers, and clones ST405, ST10 and ST648 were strongly present in more than one-third (34.1%) of ESBL producers. Conclusion: All ESBL-producing E. coli isolates exhibited MDR phenotype, and showed high prevalence of ß-lactamase and PMQR genes. Especially, one isolate harbored ESBL genes blaTEM, blaSHV, blaCTX-M-9, blaCTX-M-14, blaCTX-M-64, and carbapenemase gene blaOXA-48 and blaKPC-2, as well as PMQR genes qnrS, qnrB, qnrD, qepA and aac(6')-Ib-cr.

Association between virulence profile and fluoroquinolone resistance in Escherichia coli isolated from dogs and cats in China.

INTRODUCTION: Escherichia coli is not only a commensal organism in humans and animals, but also a causative agent of diarrhea and extraintestinal infections. Information about the relationship between population structure, virulence gene profiles, and fluoroquinolone resistance of E. coli in dogs and cats in China is limited. METHODOLOGY: A total of 174 pathogenic and commensal E. coli isolates were evaluated in terms of phylogenetic group, virulence gene profile, sequence types (STs), and fluoroquinolone susceptibility. RESULTS: A total of 46.6% of isolates belonged to phylogenetic group B2. Isolates displayed high resistance to tetracycline (82.2%), amoxicillin/clavulanic acid (73.6%), gentamicin (62.1%), and enrofloxacin (60.9%). fimH (81.6%) was the most prevalent virulence gene, and 83.9% of isolates contained one or more investigated virulence genes. The majority of the investigated virulence genes were more prevalent in fluoroquinolone-susceptible isolates and pathogenic isolates. Multilocus sequence typing (MLST) showed that E. coli isolates analyzed were assigned to 65 STs. Among of them, pathogenic-resistant and pathogenic-susceptible isolates had 44 and 10 STs, respectively, while there were 8 and 3 STs in the commensal resistant and susceptible isolates, respectively. CONCLUSIONS: Phylogenetic group B2 was the dominant group, accounting for 46.6% of the isolates. Pathogenic isolates and fluoroquinolone-susceptible isolates possessed more virulence genes. Pathogenic isolates and fluoroquinolone-resistant isolates exhibited high population diversity, and pandemic clone ST131 appeared in 9.8% of isolates.

High Prevalence of ß-lactamase and Plasmid-Mediated Quinolone Resistance Genes in Extended-Spectrum Cephalosporin-Resistant Escherichia coli from Dogs in Shaanxi, China.

Objective: The aim of this study was to investigate the occurrence and molecular characterization of extended-spectrum ß-lactamases (ESBL), plasmid-mediated AmpC ß-lactamase (pAmpC) and carbapenemases as well as plasmid-mediated quinolone-resistant (PMQR) among extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli from dogs in Shaanxi province in China. Methods: A total of 40 ESC-R Escherichia coli selected from 165 Extraintestinal pathogenic E. coli (ExPEC) isolated from dogs were screened and characterized for the genes encoding for the ESBLs, pAmpC, carbapenemases and PMQR genes by PCR and sequencing. Phylogenetic groups, virulence gene profiles and multilocus sequence typing (MLST) were used to investigate the genetic background of the ESC-R E. coli isolates. Results: Among 40 ESC-R E. coli, the predominant ß-lactamase gene was blaCTX-Ms (n = 35), and followed by blaTEM-1 (n = 31), blaSHV-12 (n = 14), blaOXA-48 (n = 8), blaTEM-30 (n = 4), blaCMY-2 (n = 3) and blaDHA-1 (n = 2). The most common specific blaCTX-M gene subtype was blaCTX-M-15 (n = 31), and followed by blaCTX-M-123 (n = 14), blaCTX-M-1 (n = 10), blaCTX-M-14 (n = 10) and blaCTX-M-9 (n = 7). PMQR genes were detected in 32 (80%) isolates, and the predominant PMQR gene was aac(6')-Ib-cr (n = 26), followed by qnrS (n = 12), qnrD (n = 9), qnrB (n = 8), qepA (n = 4), and all PMQR genes were detected in co-existence with ß-lactamase genes. traT (n = 34) and fimH (n = 32) were the most prevalent virulence genes, and virulence genes fimH, iutA, fyuA, malX, iha, and sat were more prevalent in phylogenetic group B2. The 40 ESC-R isolates analyzed were assigned to 22 sequence types (STs), and the clonal lineages ST131 (n = 10) and ST10 (n = 9) were the predominant STs. Conclusion: High prevalence of ß-lantamases and PMQR genes were detected among ESC-R E. coli from companion animals. This is also the first description of the co-existence of six ß-lantamase genes and five PMQR genes in one E. coli isolate. Moreover, 10 ST131 clones harboring CTX-M-15 were detected.

Effect of six fluoroquinolones on the expression of four efflux pumps in the multidrug resistant Escherichia coli isolates.

In this study, a total of 78 Escherichia coli clinical isolates were isolated from canines diagnosed with urinary tract infections. 23/78 isolates (29.5 %) showed multidrug resistance (MDR) phenotype, including the isolates both susceptible to fluoroquinolones (FQs) (FQ(S)-MDR, n = 12) and resistant to FQs (FQ(R)-MDR, n = 11). For these MDR isolates, mutations within quinolone-resistance determining region of gyrA and parC were determined by PCR amplification and DNA sequencing. The relative quantification of emrE, acrB, macB, and mdfA genes expression in MDR isolates was determined by quantitative real-time PCR before and after exposure to the FQs (10 µg/ml). The results showed that a temporary exposure to FQs could lead to various degrees of up or down-regulation on the expression of four efflux pumps in MDR isolates depending on the resistant phenotype and the activities of the FQs. Generally, the FQ(R)-MDR isolates showed more obvious changes in average expression levels of these transporters versus the FQ(S)-MDR isolates, with a largest increase in emrE, and followed by acrB, while the expression of macB and mdfA did not change as radically. Meanwhile, there is a reverse relationship between the expression changes and the activities of the FQs tested. The expression was higher in the isolates exposed to enrofloxacin, ciprofloxacin, and orbifloxacin, and followed by the marbofloxacin, gatifloxacin, and pradofloxacin, and the average expression levels of some efflux pumps even decreased as the isolates were exposed to gatifloxacin or pradofloxacin.

Trunk kidney of grass carp (Ctenopharyngodon idella) mediates immune responses against GCRV and viral/bacterial PAMPs in vivo and in vitro.

Trunk kidney is a vital organ for excretion in teleosts. There have been sporadic reports of processing pathogens for the immune function in trunk kidney. However, molecular processes of pathogen recognition receptors (PRRs) responding to virus and viral/bacterial pathogen-associated molecular patterns (PAMPs) are poorly elucidated in trunk kidney. In the present study, we investigated transcriptional profiles of twelve representative immune-related genes (TLRs (TLR3, TLR7 and TLR22); RLRs (RIG-I, MDA5 and LGP2); NLRs (NOD1 and NOD2); adapter molecules (MyD88 and IPS-1); effector molecule type I interferon (IFN-I) and immunoglobulin M (IgM)) in trunk kidney tissue of grass carp (Ctenopharyngodon idella) (designated as Ci) injection of grass carp reovirus (GCRV) utilizing quantitative real-time RT-PCR (qRT-PCR). Furthermore, mRNA expression patterns of these genes (IgM excepted) were examined post GCRV infection and polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) or peptidoglycan (PGN) stimulation in primary trunk kidney cells of grass carp. The relative values of CiTLR3, CiTLR22 and CiMyD88 were increased post GCRV challenge and viral/bacterial PAMPs stimulation. The mRNA transcriptions of CiTLR7 were obviously activated with GCRV challenge. Remarkably, the mRNA expressions of CiRIG-I, CiMDA5, CiLGP2 and CiIPS-1 were largely up-regulated with GCRV challenge and viral/bacterial PAMPs stimulation. Interestingly, the expression tendencies of CiNOD1 and CiNOD2 were differential not only in GCRV challenge and poly(I:C) stimulation, but also in LPS and PGN stimulation. It was demonstrated that CiIFN-I induced powerful anti-viral and anti-bacterial effects in trunk kidney. In addition, the expression of CiIgM was induced at 72 h post GCRV injection in vivo. Collectively, these results suggest that trunk kidney of grass carp serves as an important immune organ, and plays crucial roles in triggering anti-viral and anti-bacterial immune responses both in vivo and in vitro.