A versatile and tunable bio-patterning platform for the construction of various cell array biochips.

In this work, we report a versatile and tunable platform for the construction of various cell array biochips using a simple soft lithographic approach to pattern polydopamine (PDA) arrays via microcontact printing (µCP). Instead of direct polymerization of PDA on the polydimethylsiloxane (PDMS) tips, dopamine monomers were first printed on the substrate followed by a self-oxidative polymerization step facilitated by ammonia vapor to grow PDA in situ, which greatly reduced the reaction time and prevented the PDMS tips from damaging. The improved robustness and utility of the PDMS tips allows the formation of tunable PDA array chips with controllable PDA feature size and shape. As a result, single cell, multi-cells and cell line arrays can be constructed. The obtained cell array chips showed high single cell capture efficiency, providing a standardized single cell array analysis platform. Meanwhile, the adhered cells can maintain excellent viability and proliferation ability on the PDA chips. Moreover, a cytotoxicity sensor with single cell resolution was enabled on the single cell array chip. This work provides a promising cell array biochip platform for high-throughput cellular analysis and cell screening.

Dynamic weight routing and optical-code algorithm based on SDN.

Optical transport networks are evolving towards multi-granularity switching and software management but require that the architecture of the existing control plane be adapted to multi-service and network management requirements. So a dynamic weighting algorithm based on optical code resources and the matching degree between service request and channel is proposed for a multi-granularity optical switching system. In which, Software Defined Networking (SDN) controller collects network resource information in real time, dynamically changes the weight of each path and use it for path calculation, provides a more matched path on the premise of load balancing, and combines its channel matching weights for resource allocation. The simulation results show that the multi-granularity switching system with the proposed algorithm can reduce the generation of optical code fragments and improve resource utilization effectively. In the end of the paper, the optical code routing algorithms are simulated and implemented, and their performances are compared and analyzed. The simulation results show that the blocking rate of the matching algorithm on the NSFNET network is reduced by an average of 4% compared with the traditional algorithm, and the resource utilization of the optical code is improved by 9%.

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

Fluorescence-linked immunosorbent assay for detection of phenanthrene and its homolog.

A competitive fluorescence-linked immunosorbent assay (FLISA) was developed using rhodamine B isothiocyanate (RBITC) as the model fluorescent dye conjugate monoclonal antibody (McAb) for detection of Phe and its homolog (acenaphthene, fluorene, fluoranthene, pyrene and indeno [1,2,3-cd] pyrene) in water samples. The detection range of the assay for Phe was from 2.10 to 91.95 ng/mL. The limit of detection was 1.05 ng/mL, which was approximately 2-fold lower than that of traditional ic-ELISA. Compared with traditional ic-ELISA, more than 70 min was saved because of only one immunoreaction step was needed to accomplish the assay. The average recoveries of Phe and its homolog from domestic water, contaminated water and natural water were 100.7%, 100.8% and 101.2% respectively. The accuracy and precision of the developed FLISA were validated with GC-MS/MS. There were good correlation between the two methods from tap water, contaminated water and river water samples were 0.9994, 0.9935 and 0.9967, respectively. The results suggested that the proposed FLISA could be a potential alternative format for rapid, sensitive, and quantitative detection of Phe and its homolog in environmental water.

Long Non-Coding RNAs: Emerging and Versatile Regulators in Host-Virus Interactions.

Long non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules, which are involved in various biological processes, including chromatin modification, cell differentiation, pre-mRNA transcription and splicing, protein translation, etc. During the last decade, increasing evidence has suggested the involvement of lncRNAs in both immune and antiviral responses as positive or negative regulators. The immunity-associated lncRNAs modulate diverse and multilayered immune checkpoints, including activation or repression of innate immune signaling components, such as interleukin (IL)-8, IL-10, retinoic acid inducible gene I, toll-like receptors 1, 3, and 8, and interferon (IFN) regulatory factor 7, transcriptional regulation of various IFN-stimulated genes, and initiation of the cell apoptosis pathways. Additionally, some virus-encoded lncRNAs facilitate viral replication through individually or synergistically inhibiting the host antiviral responses or regulating multiple steps of the virus life cycle. Moreover, some viruses are reported to hijack host-encoded lncRNAs to establish persistent infections. Based on these amazing discoveries, lncRNAs are an emerging hotspot in host-virus interactions. In this review, we summarized the current findings of the host- or virus-encoded lncRNAs and the underlying mechanisms, discussed their impacts on immune responses and viral replication, and highlighted their critical roles in host-virus interactions.

Efficacy evaluation of the C-strain-based vaccines against the subgenotype 2.1d classical swine fever virus emerging in China.

Classical swine fever (CSF) is a devastating infectious disease of pigs caused by classical swine fever virus (CSFV). The disease has been controlled following extensive vaccination with the lapinized attenuated vaccine C-strain for decades in China. However, frequent CSF outbreaks occurred recently in a large number of C-strain-vaccinated pig farms in China and a new subgenotype 2.1d of CSFV has been reported to be responsible for the outbreaks. Here we analyzed the molecular variations and antigenic differences among the C-strain-based commercial vaccines of different origins from different manufacturers in China, and reevaluated the vaccines against the emerging subgenotype 2.1d strain of CSFV. The results showed that the C-strain adapted to the continuous ST cell line (CST) contain a unique M290K variation on the E2 protein, compared to those of primary BT cells (CBT) or rabbit origin (CRT) and the traditional C-strain sequences available in the GenBank database. Serum neutralization test revealed the antigenic differences between CST and CBT or CRT. Notably, the neutralizing titers of porcine anti-C-strain sera against the CSFV isolate of subgenotype 2.1d were significantly lower than those against C-strain or Shimen strain. The C-strain-vaccinated, subgenotype 2.1d HLJZZ2014 strain-challenged pigs did not show any clinical signs and all survived. However, these pigs displayed mild pathological and histological lesions, and the CSFV viral RNA was detected in the various tissue and blood samples. Taken together, the C-strain-based vaccines of different origins showed molecular variations and antigenic differences, and could provide clinical but not pathological and virological protection against the subgenotype 2.1d CSFV emerging in China. Further investigation is needed to comprehensively assess the efficacy of C-strain of different doses against the subgenotype 2.1d CSFV.

Development of an updated PCR assay for detection of African swine fever virus.

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

A triplex real-time PCR for differential detection of classical, variant and Bartha-K61 vaccine strains of pseudorabies virus.

Pseudorabies (PR), also known as Aujeszky's disease, is an economically important infectious disease of pigs and other animals caused by pseudorabies virus (PRV). Since late 2011, increasing numbers of PR outbreaks have been reported on many Bartha-K61-vaccinated pig farms in China, and emerging PRV variants that differ from classical PRV strains genetically and antigenically have been confirmed to be responsible for the outbreaks. Accordingly, there is a need to differentiate diverse PRV strains co-circulating in the field. Here, we developed and evaluated a triplex real-time PCR for differential detection of wild-type PRV (classical and variant strains) and gE/gI gene-deleted vaccine strains based on three differently labeled TaqMan probes. The detection limits of the assay were 0.5 TCID50 for classical strains, 0.2 TCID50 for variant strains and 0.05 TCID50 for vaccine strains. The sensitivity was also determined to be 50, 50 and 5 copies for the TJ, SC and Bartha-K61 strain, respectively. The assay did not show cross-reactivity with several common porcine viruses. Reproducibility tests showed that the inter- and intra-assay coefficients of variation were less than 3 %. When testing a total of 234 clinical swine samples, the agreement between the triplex real-time PCR and virus isolation was 100 % (234/234) for classical strains, 99.5 % (233/234) for variant strains, and 100 % (234/234) for the Bartha-K61 vaccine strain. The results demonstrate that this method is sensitive and specific and will be useful for rapid detection and differentiation of diverse PRV strains.

microRNA-18b modulates insulin-like growth factor-1 expression in deer antler cell proliferation by directly targeting its 3' untranslated region.

Insulin-like growth factor-1 (IGF-1) is a multipromoter gene that has complex biological functions and plays an important role in Chinese sika deer antler cell differentiation and proliferation. microRNAs and their roles in deer antler growth have attracted much attention. In the present study, to investigate the effect of microRNAs on the regulation of IGF-1 during the rapid growth of antlers, miRNA GeneChip analysis and TargetScan Human software were used to screen microRNAs that bind to the 3' untranslated region (3'UTR) of IGF-1. The results indicated that a significantly differential expression of miR-18b was observed in cartilage and mesenchymal of antler tip tissue and the presence of miR-18b-binding sites within the IGF-1 3'UTR. A miR-18b mimic was then transfected into antler cartilage cells to overexpress miR-18b and the expression levels were quantified by real-time PCR. Real-time PCR showed that the expression level of miR-18b in transfected cells was significantly increased compared with the control group (p<0.01). Dual luciferase assays revealed that miR-18b decreased the fluorescence value of the luciferase reporter gene in the group transfected with the wild-type vector of IGF-1 3'UTR. In contrast, the relative luciferase activity in the group transfected with the mutant vector of IGF-1 3'UTR did not change obviously. MTT assays and cell cycle analyses confirmed that overexpression of the miR-18b mimic inhibited the proliferation of cartilage cells. In contrast, transfection of a miR-18b inhibitor increased the cell proliferation rate. Furthermore, Western blot analyses revealed that overexpression of miR-18b mimics downregulated the protein levels of IGF-1, while IGF-1 expression increased after transfection of miR-18b inhibitors. Taken together, our findings show that miR-18b is a potentially novel target in deer antler cell proliferation. miR-18b may modulate IGF-1 expression of sika deer antler.

A novel immunotoxin - rCCK8PE38 targeting of CCK-R overexpressed colon cancers.

BACKGROUND: Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors. PURPOSE: Construct a new IT-targeting CCKR-overexpressing colon cancers. METHODS: To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice. RESULTS: The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18-26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8-3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice. CONCLUSIONS AND DISCUSSION: All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.

A monoclonal antibody based enzyme-linked immunosorbent assay for detection of phenylethanolamine A in tissue of swine.

Phenylethanolamine A (PEA) is a phenethanolamine member of the family of ß-adrenergic agonists compounds illegally used as feed additives for growth promotion. In this study, PEA was covalently linked to carrier protein cationized bovine serum albumin (cBSA) and egg albumin (OVA). A monoclonal antibody specific for PEA was produced and characteristics of monoclonal antibody (McAb) were studied. Based on the McAb, an enzyme-linked immunosorbent assay (ELISA) for detection of PEA was developed, which showed an IC50 value of 6.25 ng mL(-1) and a detection limit of 0.19 ng mL(-1). The average recovery of PEA from spiked samples was 103.4%, which demonstrated that the matrices of meat where PEA may be found do not interfere with the assay.

Identification of microRNA-18a as a novel regulator of the insulin-like growth factor-1 in the proliferation and regeneration of deer antler.

To investigate the effect of miR-18a on the regulation of the insulin-like growth factor (IGF-1) during growth of antlers in sika deer, miRNA Chip analysis, Target Scan and real-time PCR analysis were used to identify miRNAs that bind to the 3'-UTR of IGF-1. An miR-18a mimic was transfected into antler cartilage cells and the expression levels were quantified by real-time PCR. Dual luciferase assays revealed that miR-18a binds to the 3'-UTR of the IGF-1 gene thus indicating this to be a target gene regulated by miR-18a. MTT assays and cell-cycle analyses confirmed that miR-18a significantly inhibited proliferation of cartilage cells. In contrast, transfection of miR-18a inhibitors increased proliferation. Furthermore, Western blot analysis showed that over-expression of miR-18a down-regulated IGF-1 protein levels while IGF-1 expression was increased after transfection of miR-18a inhibitors. Thus, miR-1 down-regulated IGF-1 expression thus implicating miR-18a as an important regulator of antler proliferation.

MicroRNA let-7a and let-7f as novel regulatory factors of the sika deer (Cervus nippon) IGF-1R gene.

MicroRNAs and their roles in rapid antler growth and regeneration have attracted much attention. In the present study, we examined the effects of microRNAs let-7a and let-7f on antler cell proliferation. We used a luciferase reporter screen to demonstrate that insulin-like growth factor 1 receptor (IGF-1R) can be regulated by let-7a and let-7f. MTT assay confirmed that chondrocyte proliferation was inhibited by let-7a and let-7f mimics. In contrast, transfection of let-7a and let-7f inhibitors increased chondrocyte proliferation, indicating that inhibitors can competitively bind to endogenous miRNA, reducing the inhibitory effect of miRNA. Moreover, western blotting analysis further identified that let-7a and let-7f mimics suppressed IGF-1R expression, and that let-7a and let-7f inhibitors increased the expression level of IGF-1R. Taken together, our study demonstrates the important roles of let-7a and let-7f in antler proliferation and its potential application in antler development. let-7a and let-7f may represent novel regulatory factors of IGF-1R expression in deer antler.

MicroRNA­1 inhibits the proliferation of Chinese sika deer­derived cartilage cells by binding to the 3'-untranslated region of IGF­1.

Insulin­like growth factor­1 (IGF­1) is critical in the proliferation and regeneration of Chinese sika deer antler cells. The regulation of IGF­1 is complex and remains unclear. In the present study, miRNA GeneChip® and TargetScan Human software were used to identify microRNA­1 (miR­1), which binds to the 3'-untranslated region (3'UTR) of IGF­1. An miR­1 mimic was transfected into antler cartilage cells in order to induce the overexpression of miR­1. The expression levels of miR­1 in antler cartilage cells were determined by quantitative polymerase chain reaction (qPCR). A high­throughput luciferase reporter screen was used to demonstrate the potential regulation of IGF­1 by miR­1. miR­1 suppressed the luciferase activity of the pmiR­IGF­1 by ~50% compared with the negative control (NC). An MTT assay and cell cycle analyses confirmed that the overexpression of miR­1 significantly inhibited the proliferation of cartilage cells (P<0.05). Furthermore, western blot analysis revealed that overexpressed miR­1 downregulated the protein levels of IGF­1. The 3'UTR of IGF­1 was found to have an miR­1 combining site, which allowed the inhibition of IGF­1 protein expression, as demonstrated by a luciferase reporter assay, and miR­1 was shown to be an important and effective means of regulating IGF-1. In conclusion, miR­1 downregulated the protein expression of IGF­1 by directly targeting the 3'UTR of IGF­1. miR­1 may be crucial for inhibiting the proliferation of deer antler cartilage cells. Our findings provided the evidence for the first time that miR­1 directly regulates the expression of IGF­1 by directly targeting its 3'UTR.