Application of Recombinant Lactic Acid Bacteria (LAB) Live Vector Oral Vaccine in the Prevention of F4+ Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets.

Characterization of TLR1 and expression profiling of TLR signaling pathway related genes in response to Aeromonas hydrophila challenge in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂).

Toll-like receptor 1 (TLR1) mediates the innate immune response to a variety of microbes through recognizing cell wall components (such as bacterial lipoproteins) in mammals. However, the detailed molecular mechanism of TLR1 involved in pathogen immunity in the representative hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂) has not been well studied. In the present study, we identified the TLR1 gene from the hybrid yellow catfish, and further comparative synteny data from multiple species confirmed that the TLR1 gene is highly conserved in teleosts. Phylogenetic analysis revealed distinguishable TLR1s in diverse taxa, suggesting consistence in evolution of the TLR1 proteins with various species. Structural prediction indicated that the three-dimensional structures of TLR1 proteins are relatively conserved among different taxa. Positive selection analysis showed that purifying selection dominated the evolutionary process of TLR1s and TLR1-TIR domain in both vertebrates and invertebrates. Expression pattern analysis based on the tissue distribution showed that TLR1 mainly transcribed in the gonad, gallbladder and kidney, and the mRNA levels of TLR1 in kidney were remarkably up-regulated after Aeromonas hydrophila stimulation, indicating that TLR1 participates in the inflammatory responses to exogenous pathogen infection in hybrid yellow catfish. Homologous sequence alignment and chromosomal location indicated that the TLR signaling pathway is very conserved in the hybrid yellow catfish. The expression patterns of TLR signaling pathway related genes (TLR1- TLR2 - MyD88 - FADD - Caspase 8) were consistent after pathogen stimulation, revealing that the TLR signaling pathway is triggered and activated after A. hydrophila infection. Our findings will lay a solid foundation for better understanding the immune roles of TLR1 in teleosts, as well as provide basic data for developing strategies to control disease outbreak in hybrid yellow catfish.

Deterministic processes dominate microbial community assembly in artificially bred Schizothorax wangchiachii juveniles after being released into wild.

Fish artificial breeding and release is an important method to restore wild populations of endemic fish species around the world. Schizothorax wangchiachii (SW) is an endemic fish in the upper Yangtze River and is one of the most important species for the artificial breeding and release program implemented in the Yalong River drainage system in China. It is unclear how artificially bred SW adapts to the changeable wild environment post-release, after being in a controlled and very different artificial environment. Thus, the gut samples were collected and analyzed for food composition and microbial 16S rRNA in artificially bred SW juveniles at day 0 (before release), 5, 10, 15, 20, 25, and 30 after release to the lower reaches of the Yalong River. The results indicated that SW began to ingest periphytic algae from the natural habitat before day 5, and this feeding habit is gradually stabilized at day 15. Prior to release, Fusobacteria are the dominant bacteria in the gut microbiota of SW, while Proteobacteria and Cyanobacteria generally are the dominant bacteria after release. The results of microbial assembly mechanisms illustrated that deterministic processes played a more prominent role than stochastic processes in the gut microbial community of artificially bred SW juveniles after releasing into the wild. Overall, the present study integrates the macroscopic and microscopic methods to provide an insight into the food and gut microbial reorganization in the released SW. This study will be an important research direction to explore the ecological adaptability of artificially bred fish after releasing into the wild.

Host Species Influence the Gut Microbiota of Endemic Cold-Water Fish in Upper Yangtze River.

The fish gut microbiome plays an important role in nutrition absorption and energy metabolism. Studying the gut microbes of cold-water fish is important to understand the dietary adaptation strategies in extreme environments. In this study, the gut samples of Schizothorax wangchiachii (SW, herbivorous), Schizothorax kozlovi (SK, omnivorous), and Percocypris pingi (PP, carnivorous) in the upper Yangtze River were collected, and we sequenced 16S rRNA amplicon to study the potential relationship between gut microbes and host species. The results showed that gut microbial composition and diversity were significantly different between the three cold-water fishes. These fishes had different key taxa in their gut microbes, including bacteria involved in the breakdown of food (e.g., Cetobacterium, Aeromonas, and Clostridium sensu stricto 10). The highest alpha diversity indices (e.g., Chao 1 index) were identified in the herbivore (SW), followed by the carnivore (PP), and the lowest in the omnivore (SK). Non-metric multidimensional scaling (NMDS) results revealed that the gut microbial community of these species was different between host species. The neutral community model (NCM) showed that the microbial community structure of SW was shaped by stochastic processes, and the highest species dispersal was found in SW, followed by PP, and the lowest in SK. The results of niche breadth agreed with these findings. Our results demonstrated that host species influenced the gut microbiome composition, diversity, and microbial community assembly processes of the three cold-water fishes. These findings implied that the variation of gut microbiome composition and function plays a key role in digesting and absorbing nutrients from different foods in cold-water fish.

Differential Refractive Index Sensor Based on Coupled Plasmon Waveguide Resonance in the C-Band.

We proposed a differential fiber-optic refractive index sensor based on coupled plasmon waveguide resonance (CPWR) in the C-band. The sensor head is a BK7 prism coated with ITO/Au/ITO/TiO2 film. CPWR is excited on the film by the S-polarized components of an incident light. The narrow absorption peak of CPWR makes it possible to realize dual-wavelength differential intensity (DI) interrogation by using only one incident point. To implement DI interrogation, we used a DWDM component to sample the lights with central wavelengths of 1529.55 and 1561.42 nm from the lights reflected back by the sensor head. The intensities of the dual-wavelength lights varied oppositely within the measurement range of refractive index, thus, a steep slope was produced as the refractive index of the sample increased. The experimental results show that the sensitivity is 32.15/RIUs within the measurement range from 1.3584 to 1.3689 and the resolution reaches 9.3 × 10-6 RIUs. Benefiting from the single incident point scheme, the proposed sensor would be easier to calibrate in bio-chemical sensing applications. Moreover, this sensing method is expected to be applied to retro-reflecting SPR sensors with tapered fiber tip to achieve better resolution than wavelength interrogation.

Cleavage of E-cadherin by porcine respiratory bacterial pathogens facilitates airway epithelial barrier disruption and bacterial paracellular transmigration.

Airway epithelial cells are the first line of defense against respiratory pathogens. Porcine bacterial pathogens, such as Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Glaesserella (Haemophilus) parasuis, and Pasteurella multocida, breach this barrier to lead to local or systematic infections. Here, we demonstrated that respiratory bacterial pathogen infection disrupted the airway epithelial intercellular junction protein, E-cadherin, thus contributing to impaired epithelial cell integrity. E-cadherin knocking-out in newborn pig tracheal cells via CRISPR/Cas9 editing technology confirmed that E-cadherin was sufficient to suppress the paracellular transmigration of these porcine respiratory bacterial pathogens, including G. parasuis, A. pleuropneumoniae, P. multocida, and B. bronchiseptica. The E-cadherin ectodomain cleavage by these pathogens was probably attributed to bacterial HtrA/DegQ protease, but not host HtrA1, MMP7 and ADAM10, and the prominent proteolytic activity was further confirmed by a serine-to-alanine substitution mutation in the active center of HtrA/DegQ protein. Moreover, deletion of the htrA gene in G. parasuis led to severe defects in E-cadherin ectodomain cleavage, cell adherence and paracellular transmigration in vitro, as well as bacterial breaking through the tracheal epithelial cells, systemic invasion and dissemination in vivo. This common pathogenic mechanism shared by other porcine respiratory bacterial pathogens explains how these bacterial pathogens destroy the airway epithelial cell barriers and proliferate in respiratory mucosal surface or other systemic tissues.

Case Report: A Novel Heterozygous ZP3 Deletion Associated With Empty Follicle Syndrome and Abnormal Follicular Development.

Background: Empty follicle syndrome (EFS) is defined as the complete failure to retrieve oocytes after ovarian stimulation. Although several mutations in ZP1, ZP2, ZP3, and LHCGR have been identified as genetic causes of EFS, its pathogenesis is still not well-understood. Methods: Whole-exome sequencing (WES) was employed to identify the candidate pathogenic mutations, which were then verified by Sanger sequencing. A study in CHO-K1 cells was performed to analyze the effect of the mutation on protein expression. Additionally, immunohistochemistry (IHC) staining was used to examine follicular development and zona pellucida (ZP) assembly in the ovary of an EFS patient. Results: A novel heterozygous deletion in ZP3 (c.565_579del[p.Thr189_Gly193del]) was identified in the EFS patient. It was inherited dominantly and resulted in significant degradation of the ZP3 protein. Oocytes with degenerated cytoplasm and abnormal ZP assembly were observed in follicles up to the secondary stage, and many empty follicle-like structures were present. Conclusion: We identified a novel ZP3 mutation that expands the mutational spectrum associated with human EFS. We also showed the abnormal follicular development and ZP assembly of the EFS patient with the heterozygous ZP3 mutation, which provides new insights into the pathogenesis of EFS.

Highly sensitive differential fiber-optic SPR sensor in telecom band.

We proposed a differential fiber-optic SPR remote sensor with ultra-high sensitivity in telecom band. The working band of the sensor is designed as the C-band which is the low loss band of optical fiber communication aiming to improve the sensitivity and enable the capability of remote monitoring. The sensor head is a BK7 prism coated with Au/TiO2 films, enabling two channels for differential intensity interrogation. The intensities of the reflected lights through the channels vary oppositely within the measurement range of refractive index. Due to the sharp dip of angular resonant response in the C-band, the differential signal produces a steep slope as the refractive index of the sample varies, thus higher sensitivity is expected in a narrow measurement range. According to the results, the sensitivity is as high as 456 V/RIUs within the narrow measurement range of 1.3×10-2 RIUs and the resolution reaches to 6×10-6 RIUs. The measurement range can be tuned conveniently by adjusting the thickness of TiO2 film and can be expanded by increasing the number of sensing channels, which provides great convenience for the application of biosensor requiring high sensitivity.

A Polarization-Independent Fiber-Optic SPR Sensor.

Fiber-optic surface plasmon resonance (SPR) sensors possess the advantages of small size, flexible, allowing for a smaller sample volume, easy to be integrated, and high sensitivity. They have been intensively developed in recent decades. However, the polarizing nature of the surface plasmon waves (SPWs) always hinders the acquisition of SPR spectrum with high signal-noise ratio in wavelength modulation unless a polarizer is employed. The addition of polarizer complicates the system and reduces the degree of compactness. In this work, we propose and demonstrate a novel, polarization-independent fiber-optic SPR sensor based on a BK7 bi-prism with two incident planes orthogonal to each other. In the bi-prism, TM-polarized components of non-polarized incident lights excite SPWs on the first sensing channel, meanwhile the TE components and the remaining TM components are reflected, then the reflected TE components serve as TM components of incident lights for the second sensing channel to excite SPWs. Simulations show the proposed SPR structure permit us to completely eliminate the polarization dependence of the plasmon excitation. Experimental results agree well with the simulations. This kind of devices can be considered an excellent option for development of simple and compact SPR chemical sensors.

RBS Depth Profiling Analysis of (Ti, Al)N/MoN and CrN/MoN Multilayers.

(Ti, Al)N/MoN and CrN/MoN multilayered films were synthesized on Si (100) surface by multi-cathodic arc ion plating system with various bilayer periods. The elemental composition and depth profiling of the films were investigated by Rutherford backscattering spectroscopy (RBS) using 2.42 and 1.52 MeV Li2+ ion beams and different incident angles (0°, 15°, 37°, and 53°). The microstructures of (Ti, Al)N/MoN multilayered films were evaluated by X-ray diffraction. The multilayer periods and thickness of the multilayered films were characterized by scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM) and then compared with RBS results.

A preparation approach of exploring cluster ion implantation: from ultra-thin carbon film to graphene.

Based on the extensive application of 2 × 1.7MV Tandetron accelerator, a low-energy cluster chamber has been built to explore for synthesizing graphene. Raman spectrum and atomic force microscopy (AFM) show that an amorphous carbon film in nanometer was deposited on the silicon by C4 cluster implantation. And we replaced the substrate with Ni/SiO2/Si and measured the thickness of Ni film by Rutherford backscattering spectrometry (RBS). Combined with suitable anneal conditions, these samples implanted by various small carbon clusters were made to grow graphene. Results from Raman spectrum reveal that few-layer graphene were obtained and discuss whether I G/I 2D can contribute to explain the relationship between the number of graphene layers and cluster implantation dosage.

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19.

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

Understanding the mechanistic roles of 30Kc6 gene in apoptosis and specific productivity in antibody-producing Chinese hamster ovary cells.

Previously, we reported that the expression of Bombyx mori 30Kc6 gene in Chinese hamster ovary (CHO) cells increases recombinant protein production by both inhibiting apoptosis and enhancing specific productivity. In this study, in order to gain a thorough understanding of the roles of 30Kc6 gene in antibody production, the mechanisms modulating cell apoptosis and specific productivity were investigated. 30Kc6 gene was introduced into a CHO cell line producing a chimeric anti-human CD20 monoclonal antibody. The stable expression of 30Kc6 increased cell viability and productivity by 46.7% and 3.4-folds, respectively. It was observed that the Bax translocation from cytosol to mitochondria and the cytochrome c (cyt c) release from mitochondrial intermembrane space to cytosol were repressed, which resulted in a decrease in the activation of apoptosis executioner, caspase-3. On the other hand, 30Kc6 expression increased the specific productivity by 2.3-folds. However, at the transcription level, the relative levels of heavy and light chain mRNAs increased only by 8.3% and 8.7%, respectively, which was not accountable for the observed increment in the specific productivity. Instead, the mitochondrial membrane potential was maintained and the ATP generation was stimulated. A higher ATP level could activate the mammalian target of rapamycin (mTOR), which drives the translation initiation and elongation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). In the 30Kc6-expressing cells, both the 4EBP1 and S6K1 were phosphorylated at higher levels, which indicated that the increased specific productivity primarily resulted from the boost of translation process. Furthermore, it was also found that the specific uptake rates of glucose and glutamine were not affected by 30Kc6 expression, demonstrating that the enhanced ATP generation and consequently maintained mTOR activity were due to 30Kc6 expression but not the different metabolic uptake rates. In conclusion, 30Kc6 expression inhibited apoptosis by repressing the Bax translocation, which down-regulated the downstream cascade responses including cyt c release and caspase-3 activation. Also, 30Kc6 expression increased the specific productivity by enhancing the translation process.

A novel gas ionization sensor using Pd nanoparticle-capped ZnO.

A novel gas ionization sensor using Pd nanoparticle-capped ZnO (Pd/ZnO) nanorods as the anode is proposed. The Pd/ZnO nanorod-based sensors, compared with the bare ZnO nanorod, show lower breakdown voltage for the detected gases with good sensitivity and selectivity. Moreover, the sensors exhibit stable performance after more than 200 tests for both inert and active gases. The simple, low-cost, Pd/ZnO nanorod-based field-ionization gas sensors presented in this study have potential applications in the field of gas sensor devices.

Enhancement of recombinant human EPO production and sialylation in chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression.

Productivity and sialylation are two important factors for the production of recombinant glycoproteins in mammalian cell culture. In our previous study, we found that silkworm hemolymph increased the sialylation of recombinant secreted human placental alkaline phosphatase in the insect cells, promoted the transfer of sialic acids onto the glycoprotein oligosaccharides in an in vitro asialofetuin sialylation system, and enhanced recombinant protein production in the Chinese hamster ovary (CHO) cells. These beneficial effects were mainly due to the 30K proteins, which consist of five isoforms. Among the 30K proteins, 30Kc19 was determined to be the major component. In this study, the 30Kc19 gene was introduced into a CHO cell line producing recombinant human erythropoietin, and its effects on productivity and sialylation were investigated. The transient expression of 30Kc19 significantly improved the production and sialylation of EPO. A stable cell line containing 30Kc19 was also established to investigate the effect of 30Kc19 gene expression. The stable expression of 30Kc19 increased the production and sialylation by 102.6% and 87.1%, respectively. The enhanced productivity from 30Kc19 expression is believed to occur because the 30Kc19 protein suppresses the loss of mitochondrial membrane potential and consequently improves the generation of intracellular ATP. In addition, the positive effect of 30Kc19 expression on sialylation is believed to be due to its ability to maintain sialyltransferase activity. In conclusion, 30Kc19 expression is a novel approach to improve the production and sialylation of recombinant glycoproteins in CHO cells.