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Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus)
Microbial Cell Factories volume 18, Article number: 142 (2019)
Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV.
Glycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 109 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 104–105 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 102–103 L. lactis cells could still be recovered.
HIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.
Hirame novirhabdovirus (HIRRV), belonging to the genus Novirhabdovirus of Rhabdoviridae family, was first isolated from cultured flounder (Paralichthys olivaceus) and ayu (Plecoglossus altivelis) in Japan in 1984 . Since then, it has been reported to infect several species of marine fish such as stone flounder (Kareius bicoloratus) , black seabream (Acanthopagrus schlegeli)  and spotted sea bass (Lateolabrax maculatus) , as well as two freshwater hosts, grayling (Thymallus thymallus) and brown trout (Salmo trutta) . With significant morbidity and heavy economic losses, HIRRV has become a vital menace for aquaculture especially in Asia and Europe. Vaccination has become a rather important part of aquaculture, since it is considered one of the most effective disease control strategies . With regard to rhabdovirus, viral glycoprotein is a spike protein responsible for virus attachment to cell receptors, and also determines the serological properties of rhabdovirus [7, 8]. So far, several vaccines against HIRRV, including subunit vaccine  and DNA vaccine [10, 11], were all developed based on the viral glycoprotein, which could induce a good protective immunity against HIRRV infection. Moreover, the recombinant viral hemorrhagic septicemia virus (VHSV) harboring HIRRV-G gene instead of VHSV-G gene could produce high serum neutralization titer against HIRRV but not VHSV in flounder . These researches suggested that the glycoprotein of HIRRV was an attractive and promising vaccine candidate.
Oral vaccination has the advantages of being painless, less expensive and convenient to administration , which is particularly important and needed for aquatic animals, especially for fish fries. Oral vaccine can trigger both mucosal and systemic immune responses, which may enhance vaccine efficacy and minimize vaccine side effects by avoiding direct contact to the systemic circulation [14, 15]. Lactic acid bacteria (LAB), most of which are generally regarded as safe (GRAS), have long been used in food fermentation and preservation . For nearly three decades, they have been studied as live vehicles for the delivery of antigens to mucosal sites in human, mice and chickens [17,18,19,20]. In the recent years, several LAB live vector vaccines were developed against pathogens in aquatic animals, such as infectious pancreatic necrosis virus (IPNV) in rainbow trout , Streptococcus iniae and Edwardsiella tarda in flounder [22, 23] and Aeromonas veronii in common carp , and showed significant immune protective effects. Therefore, it will be a promising prospect to apply lactococcal expression systems for disease prevention and control in aquaculture.
In this study, we first designed an expression cassette in L. lactis NZ9000 using pNZ8148 vector, and with HIRRV-G gene insertion, a recombinant L. lactis strain expressing G protein was constructed. After oral immunization, specific antibody responses in serum and gut mucus were analyzed in flounder model. Furthermore, the immune protective efficacies including virus load and relative percent survival (RPS) were investigated after HIRRV injection. In addition, we explored the survival and adhesion of the recombinant L. lactis in flounder intestine.
Design and construction of expression cassette in L. lactis
The signal peptide of Usp45 (SPUsp45) and the C terminus of AcmA (cA, containing three LysM motifs), as integral components for the protein secretion and surface exposure, were cloned into pNZ8148 vector, yielding pSLC. And, multiple cloning site (MCS) sequence was artificially integrated behind the cA domain, providing a pathway for insertion of the target genes. In this way, HIRRV-G gene was successfully inserted, generating pSLC-G. Under the control of PnisA, the open reading frame (ORF) of the constructed expression cassette was 798 bp (pSLC, Fig. 1a) or 2250 bp (pSLC-G, Fig. 1b), which was confirmed by PCR and nucleotide sequencing.
After electrotransformation, the recombinant L. lactis NZ9000, named Ll:pSLC and Ll:pSLC-G, were induced by nisin and then subjected to SDS-PAGE and western blotting analysis. SDS-PAGE analysis exhibited enhancement of protein bands in bacterial cell lysate samples with molecular weights of approximately 28 kDa (Ll:pSLC; Fig. 1c, lane 2) and 81 kDa (Fig. 1c, lane 3) compared to the sample before induction of Ll:pSLC-G (Fig. 1c, lane 1), which was consistent with the expectations. Nevertheless, no corresponding protein bands appeared in all supernatant samples (Fig. 1c, lanes 4–6). The 81 kDa protein band in lane 3 was submitted for mass spectrometry (MS) analysis, and the result showed that it matched 8 peptides in G protein of HIRRV with 15.4% coverage of amino acid sequences (Additional file 1: Figure S1).
Characteristics of recombinant L. lactis expressing G protein
HIRRV-G protein was expressed in E. coli Transetta with pET-28a system and mouse anti-rG polyclonal antibodies (Pab) was prepared. The results of western blotting showed that the anti-rG Pab could specifically recognize the recombinant G protein expressed by L. lactis (Fig. 1c, lane 9), while no stained bands appeared in the cell lysate samples of non-induced L. lactis and induced Ll:pSLC (Fig. 1c, lanes 7 and 8). Meanwhile, the immunofluorescence assay (IFA) was further preformed to detect whether the HIRRV-G protein could display on the bacterial cell surface, and the result showed that specific green fluorescence was observed on the surface of Ll:pSLC-G after induction (Fig. 2a). Moreover, the flow cytometry (FCM) analysis showed that the proportion of positive bacteria was more than 85% after induction for 3 h (Fig. 2f). Based on these results, we can conclude that the HIRRV-G protein was successfully expressed and displayed on the cell surface of the L. lactis NZ9000.
Colonization ability of recombinant L. lactis
In order to detect the colonization of recombinant L. lactis in flounder intestine, the homogenates of foregut, midgut and hindgut were separately cultured on agar plates for 1–2 days. Typical colony characteristics, such as round, globular, white, with a smooth surface and entire edge, could be observed on the plates from the Ll:pSLC-G group, while no colonies were grown on plates from PBS (phosphate buffered saline) group. The result of colony PCR confirmed that the cultured colonies were L. lactis NZ9000 harboring the pSLC-G.
Based on plate counts, we calculated the number of recombinant L. lactis attached to different regions of the intestinal mucosa of flounder (Fig. 3). On the first day after oral feeding for 1 week, the calculated number of cells was approximately 104–105 cfu per gram homogenate of flounder intestine. Adhesion was most prominent in midgut and the number of cells was up to 1.8 × 105 cfu per gram tissue. Then, the viable L. lactis gradually decreased with time. By the 14th day, 103–104 cfu per gram tissue could still be recovered from foregut, midgut and hindgut, accounting for 12.2%, 18.8% and 16.7% of the cell number on the first day, respectively. On day 21, the amount of L. lactis that remained adhered to the intestine tract was 102–103 cfu, which is less than 2% of the cell number on the first day.
Double immunofluorescence staining was used to detect the recombinant G protein on the cell surface of L. lactis NZ9000 located in flounder intestine. The results showed that L. lactis-positive red signals were mainly localized at the bottom of gut mucus layer, near the intestinal epithelial cell layer, and some in epithelial cell layer (Fig. 4c). According to the merged picture, most of the red signals could be co-localized with G protein-positive green signals (Fig. 4a), indicating that quite a large proportion of L. lactis could displayed G protein. No positive signals were observed in negative control staining.
Antibody response against HIRRV in flounder
The levels of specific anti-HIRRV IgM in serum and gut mucus of the immunized flounder were determined by ELISA (Fig. 5). It showed the similar dynamic trends in serum and gut mucus samples. Compared with the controls, significantly higher level of specific IgM was elicited in Ll:pSLC-G group (p < 0.05). And after booster immunization, higher levels of antibodies were also observed in the flounder vaccinated with Ll:pSLC-G. By contrast, there was no significant difference among the three control groups, where flounders were fed with commercial diet pellets coated with Ll:pSLC, L. lactis NZ9000 (Ll) or PBS, respectively. Besides, in Ll:pSLC-G group, significant high levels of IgM were detected in gut mucus rather than serum at week 2, indicating that antibodies were elicited earlier in gut mucus.
Immune protective efficacies against HIRRV
The virus copies in spleens of challenged flounders on day 1 and 3 post-challenge were examined by qPCR, and the results were shown in Fig. 6a. In all experimental groups, the virus copies increased on day 3 compared to day 1. Compared with the controls, the fish in Ll:pSLC-G group had a significantly lower viral load on the 1st day post-challenge (p < 0.05), and extremely significant difference was detected on the 3rd day (p < 0.01). There was no statistical significant difference in virus copies among the three control groups (p > 0.05), but relatively low mean number of viral copies were detected in fish vaccinated with L. lactis NZ9000 and Ll:pSLC on the 3rd day.
The cumulative mortality of vaccinated fish after challenging with HIRRV were monitored (Fig. 6b). In PBS group, the fish began to die on 4th day post-challenge, and died quickly on 5th to 8th day; the cumulative mortality tended to be stable on day 10 at 93.3%. As for two negative groups, group Ll and Ll:pSLC, the death both occurred on day 4 to day 11, and the cumulative mortalities were 76.7% and 73.3%, respectively. In contrast, the fish vaccinated with Ll:pSLC-G showed a significantly lower mortality of 36.7% compared with the controls. The RPS of the groups Ll:pSLC-G, Ll:pSLC and Ll were 60.7%, 21.4% and 17.9%, respectively. The infected fish exhibited typical signs of HIRRV infection, such as focal hemorrhage of the skeletal muscle and fins.
With the extensive researches and long-term application, LAB have been proposed as mucosal delivery vehicles because of its safety and the capacity to survive in digestive tract. Due to the known complete genome, mature genetic tools and easiest genetic manipulability, L. lactis has been widely used for the development of candidate vaccines . Till now, many different heterologous antigens have been efficiently produced in L. lactis [18, 26,27,28]. Among them, the nisin-controlled gene expression (NICE) system remains one of the most promising and widely-used inducible systems based on nisA promoter, whose activity depends on extracellular concentration of nisin, a small peptide produced and secreted by several strains of L. lactis . After genetic recombination, we constructed two recombinant plasmids, pSLC and pSLC-G, which were then successfully transformed into L. lactis NZ9000 and induced by adding nisin. The expressed proteins could be clearly detected in SDS-PAGE and Western blotting, and the recombinant HIRRV-G protein was confirmed by MS, which indicated that the engineered G gene of HIRRV was accurately transcribed and translated into the target protein in this L. lactis expression system.
It is noticed that the locations of heterologous proteins produced in L. lactis can be intracellular, extracellular, and cell wall anchored. Displaying proteins on bacterial cell wall allows higher expression levels of protein and a better antigen exposure to the immune system . Some studies demonstrated that surface-displayed strains could elicit a better immune response [30, 31]. Therefore, we chose to construct a surface display system of L. lactis for vaccine development. To date, the signal peptide of Usp45 (an unknown secreted protein of 45 kDa in L. lactis) is still the most generally used signal peptide in various expression system . An interesting and valuable strategy is that synthetic LEISSTCDA peptide closely following the SPUsp45 may improve the secretion efficiency in L. lactis . The cA domain of the AcmA is a very useful scaffold that can achieve cell surface display of heterologous proteins on various LABs . Based on these expression elements, many proteins have been successfully expressed on the cell surface of bacteria [34,35,36]. In this work, according to the results of IFA and FCM analysis, the majority of cells were found to express HIRRV-G protein on the cell surface of recombinant L. lactis after induction. Thus, we can draw a conclusion that the HIRRV-G protein is successfully displayed on the surface of L. lactis, which laid the foundation for further application and effect evaluation of oral vaccine.
Rhabdoviruses consist of a helically wound ribonucleocapsid surrounded by a lipid bilayer, where glycoprotein projects outside in the form of non-covalently-bound . As a spike protein, viral G protein is considered to be a crucial antigenic determinant in rhabdovirus and almost the only protein that elicits neutralization antibody, which has been sufficiently proved in infectious hematopoietic necrosis virus (IHNV) , another virus in the same genus. In previous studies, HIRRV-G based subunit vaccine could bring out considerable protective effects against HIRRV injection , and DNA vaccine constructed based on HIRRV-G gene even showed a relatively higher protection level . In this work, the live vector vaccine of recombinant L. lactis expressing G protein exhibited a good protection of 60.7% against HIRRV infection, which is slightly lower than the effects of subunit and DNA vaccine. The small gap of the RPS value among different vaccine types might be mainly due to the difference of the amount of uptaken antigen, stimulus intensities and vaccination site. On another hand, oral vaccination offers the advantage of being easy to administer a large number of fish at one time, and it is less likely to cause immunological stress and mortality during vaccination . More importantly, LAB as a functional feed additive may significantly increase weight gains and feed efficiencies .
Being the first barrier against the pathogens infections, mucosal immune system plays a critical role in pathogenic defense and induction of immunity. Many studies showed that oral live vector vaccine could simultaneously trigger the mucosal immune and systemic immune response of vaccinated animals [40,41,42]. A similar phenomenon was observed in this work: the specific IgM antibody against HIRRV were both detected in serum and gut mucus of flounders orally vaccinated with Ll:pSLC-G. Interestingly, the significant increase of antibody level was detected earlier in mucus, which indicated that mucosal immune response was quickly evoked and produced mucosal antibodies post oral vaccination. The similar result was also found in immersion vaccination in our previous work . Taken together, oral vaccine can effectively trigger the mucosal and systemic immune responses, whereas mucosal immunity seems to make a quicker response.
The colonization experiment of probiotics in animals showed that colonizing bacteria can persist for different time durations in gut. In mouse model, several studies showed that LAB could exist in gut for a few days [44, 45], while some discovered that some of LAB species could not colonize the digestive tracts of man and animals, but can survive passage through the gut [28, 46]. In rainbow trout, approximately half proportions of lactobacillus were detected in the intestine at day 7 compared to the first day . In the present work, we found that a portion of L. lactis NZ9000 has the ability to survive and adhere to the intestine in flounder and can last for at least 3 weeks. It is probably that the difference of colonizing duration may be associated with characteristics of bacteria, quantity of external supplement and the existing gastrointestinal microbiota. In addition, considerable colonization could also be achieved by continuous feeding with a high dose of probiotic bacteria for a period of time [47, 48]. Therefore, continuous or intermittent enhanced inoculation is necessary for strong and persistent immunity.
Antigen dose and vaccination scheme are crucial issues for each vaccine system , and have a considerable impact on immune response of vaccinated host. Our previous studies have demonstrated that the immune response is correlated to the antigen dose in injection vaccination and immersion vaccination [43, 49]. In oral vaccination, researches showed that a booster vaccination could induce a higher level of immune response in flounder and common carp [23, 41]. Therefore, a booster vaccination was conducted in this work, and the result showed that higher levels of specific IgM were produced compared to the primary immunization, indicating a prime-boost strategy is required for this oral vaccine inoculation. In order to achieve better efficacy, the immune dosage and feeding scheme need to be further optimized and explored.
A surface-displayed system of L. lactis was constructed and the G protein of HIRRV was successfully expressed. Flounders fed with recombinant Lactococcus expressing G protein were triggered mucosal and systemic immune response, and exhibited a considerable protective effect against HIRRV. Hence, the genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine for flounder against HIRRV infection.
Bacterial strains, virus and fish
Bacterial strains and plasmids used in this study are listed in Additional file 1: Table S1. E. coli strains were grown in Luria–Bertani broth at 37 °C with vigorous shaking, while L. lactis strains were grown in GM17 (M17 medium supplemented with 0.5% glucose) broth at 30 °C without agitation. Agar (1.5%) was added to the medium for the plates. When necessary, ampicillin (100 μg/ml), kanamycin (50 μg/ml) and chloramphenicol (10 μg/ml) were used for E. coli, and chloramphenicol (10 μg/ml) and nisin (50 ng/ml) were used for L. lactis.
The HIRRV CNPo2015 strain, isolated from cultured flounder in 2015 , was used in this study. The virus was propagated in epithelioma papulosum cyprini (EPC) cell cultures at 15 °C, supplemented with 4% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin G, and 100 mg/ml streptomycin (Gibco, USA). When virus titer was around 1 × 106.8 TCID50/100 μl, the cell cultures were harvested and centrifuged to remove cell debris. The supernatant was collected as virus supernatant for the following ELISA and challenge assay.
Specific pathogen-free flounders with body weights of 35 ± 5 g were obtained from an aquafarm in Rizhao, Shandong province of China. Before vaccination, the fish were maintained at 18–20 °C in tanks containing aerated sand-filtered seawater and fed daily with commercial diet (Great Seven, Qingdao, China) for 1 week.
HIRRV-G gene excluding the region of signal peptide was amplified from the virus genome with the primers G-F/G-R (Additional file 1: Table S1); the product was cloned into pET-28a vector and transformed into E. coli Transetta (DE3) (Transgen, Beijing, China). Following nucleotide sequencing, the confirmed clone was cultured in LB broth and induced by adding 1 mM isopropylthiogalactoside. Eight hours growth later, the cells were collected, sonicated and purified using His Trap™ HP Ni-Agarose (GE healthcare, Beijing, China) . After dialysis, the protein purity was checked by SDS-PAGE and the concentration was quantified by BCA protein assay kit (Sangon Biotech, Shanghai, China). The recombinant glycoprotein was used to immunize Balb/c mice to produce the mouse anti-rG polyclonal antibodies according to the previous immunization procedure . After immunization, antiserum was obtained from immunized mice, purified with protein G–agarose column (Pierce/Thermo Scientific) and characterized by western blotting.
Rabbit anti-L. lactis NZ9000 Pab was also prepared in this study. After cultivation, L. lactis cells were harvested and inactivated with PBS containing 0.5% formalin (v/v) for 72 h at 30 °C. Following reconfirmation by plate culture, inactivated L. lactis NZ9000 cells were washed with sterilized PBS, and the concentration was adjusted to 1.0 × 1010 cfu ml−1. New Zealand white rabbit was immunized with inactivated L. lactis four times, and the polyclonal antiserum was taken when the titer reached to 1:100,000–200,000. After purification, the concentration and purity of the IgG fraction were estimated using BCA protein assay kit and SDS-PAGE, respectively.
Mouse anti-flounder IgM monoclonal antibody (FIgM-Mab) was produced previously in our laboratory , and the dilution used in ELISA was 1: 1000.
Construction of bacterial expression cassette
pET-32a, an expression vector for E. coli, was utilized for assembly of the cloning fragments. pNZ8148, an E. coli-L. lactis shuttle vector, was used as the L. lactis expression vector after subcloning of entire cassettes.
The signal peptide of Usp45 and the C-terminal anchor domain of AcmA gene fragments were amplified from L. lactis MG1363 genomic DNA using two primer pairs SPUsp45-F/SPUsp45-R and cA-F/cA-R (Additional file 1: Table S1), respectively. The purified products were digested with restriction enzymes NcoI/BamHI (SPUsp45) and BamHI/HindIII (cA), respectively, and ligated into the pET-32a vector one by one using T4 DNA ligase to be joined together. It is noted that the LEISSTCDA nucleotide sequence has been incorporated into the reverse primer of Usp45, which has been reported to increase the secretion of the heterologous proteins in L. lactis . Besides, MCS sequences containing five restriction enzyme genes (SacI, SalI, EcoRI and HindIII in order of ORF) were integrated into the reverse primer of cA. The fused fragment containing SPusp45 and cA domain in pET-32a vector was named as SLC and is flanked by restriction enzymes NcoI and HindIII. Next, the connected fragment SLC was cleaved with NcoI/HindIII restriction enzymes and cloned into the same sites on pNZ8148 under the promoter of nisA, generating an expression cassette named pSLC. The recombinant vector was transformed into E. coli MC1061 competent cells, which were prepared by treatment with 0.1 M of cooled CaCl2 .
The amplified G gene mentioned above was inserted after cA domains in pSLC by digestion with restriction enzymes SacI and HindIII, yielding pSLC-G (Fig. 1b). The DNA sequence of G gene in pSLC-G vector was confirmed via nucleotide sequencing by Tsingke Bio (Qingdao, China).
Transformation of L. lactis cells and induction expression
The recombinant plasmids were transformed into L. lactis NZ9000 by electroporation as previously described  with certain modifications. For competent cell preparation, an overnight culture of L. lactis NZ9000 was inoculated 1:50 in fresh GM17 broth containing 1% glycine and 0.5 M sucrose and incubated at 30 °C. At early exponential phase (OD600 = 0.5), the culture was harvested by centrifugation (5000g, 4 °C and 10 min). The pellet was washed thrice with an ice-cold washing solution (5 mM KH2PO4, 2 mM MgCl2, 10% glycerol, 0.5 M sucrose), resuspended in 1/100 culture volume of washing solution and then electroporated immediately or stored in aliquots at − 80 °C. For electroporation, 40 μl of prepared competent cell was mixed with 1 μg of recombinant plasmid dissolved in double-distilled water, and transferred to an ice-cooled electroporation cuvette (0.2 mm). A single pulse was delivered by a Gene Pulser (Bio-Rad, USA) at 2.0 kV, 200 Ω for 4.0 ms. Immediately, the cell suspension was mixed with 900 μl of recovery medium (GM17 broth containing 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2) and incubated at 30 °C for 2 h. After that, 200 μl of suspension culture was spread on GM17 plate containing chloramphenicol and incubated for 2 days. Two constructed Lactococcus lactis were denoted Ll:pSLC and Ll:pSLC-G, respectively.
For induction, recombinant L. lactis cells were cultured overnight in GM17 broth with chloramphenicol and then inoculated 1:50 in fresh GM17 medium. When the OD600 of the culture reached 0.6, nisin was added and cultured for an additional 3 h. Then, the bacterial cells were collected by centrifugation (8000g, 4 °C and 5 min) for the following assays.
Western blotting and mass spectrometry analysis
After centrifugation of the induced bacterial culture, the supernatant and cells were processed separately [55, 56]. The supernatant was filtered through 0.2-μm-size filter and proteins were precipitated by adding ice-cold 80% (w/v) TCA (16% final concentration). After washing twice with precooled acetone, the resulting pellet was dried in a vacuum centrifuge and dissolved in 50 mM NaOH. On the other hand, the cell pellets were washed twice with TES buffer (50 mM Tris–HCl, 1 mM EDTA, 25% sucrose; pH 8), resuspended in 10% volume cultures of TES buffer containing lysozyme (1 mg/ml) and subsequently incubated at 37 °C for 30 min. Then, the cell lysates were treated with ultrasound generator (Sonics VC750, USA; 750 W, 20 kHZ) on ice for 15 min (on and off cycle of 5 s at 36% amplitude). Finally, all samples were added with equal volumes of 2× loading buffer and subjected to SDS-PAGE (10% polyacrylamide). One was stained with Coomassie brilliant bule R-250, and others were transferred to polyvinyldifluoride (PVDF) membrane (Merck Millipore, USA). The PVDF membrane was blocked overnight with 4% bovine serum albumin (BSA) in PBS at 4 °C. Mouse anti-rG Pab and alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (1:3000, Merck Millipore) were used as primary and secondary antibodies, respectively. Finally, the bands were stained with fresh substrate solution (100 mM NaCl, 100 mM Tris and 5 mM MgCl2; pH 9.5) containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates (NBT/BCIP, Sigma), and the reaction was stopped by washing with distilled water.
According to the positive band in the PVDF membrane, the relevant band in polyacrylamide gels was excised, reduced and alkylated by 10 mM dithiothreitol and 55 mM iodoacetamide for protein identification using MS by Shanghai Applied Protein Technology (Shanghai, China).
Immunofluorescence and FCM assays
For confirmation of the surface-displayed expression of recombinant Lactococcus lactis, IFA was performed as described . The induced cultures of bacteria were harvested and washed twice with PBS. Cell pellets were suspended in sterile PBS containing 4% BSA and mouse anti-rG Pab, and incubated for 2 h at 37 °C. Goat anti-mouse IgG-488 conjugate (Dylight, Abbkine, China) was used as secondary antibody at a dilution of 1:500. After that, cells were washed three times with PBS. A drop of the suspension was laid onto APES coated slides and observed with fluorescence microscope. In parallel, the cell suspensions were diluted to approximately 106 cells/ml and analyzed by Accuri C6 flow cytometer (BD, Accuuri™, Piscataway, NJ, USA). The primary antibody replaced with mouse negative serum was used as negative control.
Vaccination and sampling of flounder
For oral vaccination, the induced cultures of recombinant L. lactis were mixed evenly with commercial diet at the final concentration of 1.0 × 109 cfu/g diet. After air drying, the LAB-based feed pellets were enwrapped with sodium alginate (0.6%, w/w; food grade) and fish oil (1%, v/w) onto their surface and kept at 4 °C prior to feeding. Four groups were divided with 150 fish in each one. Experimental group of fish was fed with baits containing Ll:pSLC-G, while control group were fed with baits containing Ll:pSLC, Ll or PBS, respectively. Oral vaccination was performed at week 1 (first vaccination) and week 5 (booster vaccination) with a feeding rate of 1–2% body weight daily. Beyond those, the fish were fed regular pellet feed.
For the detection of specific antibodies, five fish from each group were randomly sacrificed to collect blood and gut mucus at weeks 0, 2, 4, 6, 8 since the beginning of feeding. Serum were separated from blood via overnight standing at 4 °C and centrifugation and then stored at − 80 °C until usage. Gut mucus was collected according to the method in previous studies [58, 59]. Briefly, gut mucus samples were collected by gently scraping the inner surface of hindgut with PBS containing 1 mM phenylmethylsulfonyl fluoride and 0.5% BSA. After vortexing and centrifugation, the supernatants were concentrated about tenfold with the same volume in all samples and stored at − 80 °C until usage.
To detect the colonization and proportion of recombinant L. lactis NZ9000 in fish intestine tract after one week of oral feeding, five fish in Ll:pSLC-G group and PBS group (negative control) were sacrificed on days 1, 7, 14 and 21 post feeding. Fish were starved for 24 h prior to collection and the sampling procedure was as follows. Firstly, scrub the ventral surface with 75% ethanol, extract the intestine and separate it into foregut, midgut and hindgut. Remove visible residual food particles, transfer the sections to sterile dishes and weigh. Then, wash twice with sterile PBS to rinse the passage bacteria, homogenize them and serially dilute in sterile PBS. After that, 150 µl of each sample was spread on GM17 agar plates with chloramphenicol and incubated at 30 °C for 1–2 days. When typical bacterial characteristics occurred, the bacterial colonies were counted and determined as log10 cfu/g tissues. Furthermore, 10 single colonies from each sample were randomly selected and subjected to colony PCR with the primers pNZ-F/pNZ-R and nisRK-F/nisRK-R (Additional file 1: Table S1), which were used to identify L. lactis NZ9000 and the expression cassette of recombinant plasmid, respectively. PCR products were confirmed by nucleotide sequencing.
On the other hand, the intestine samples on the third day post oral feeding were prepared for tissue cryosections according to previous description , and IFA was carried out. Briefly, after blocking, sections were incubated with rabbit anti-L. lactis NZ9000 Pab (1:2000) and mouse anti-rG Pab (1:800) for 1.5 h at 37 °C. Non-immune mouse and rabbit serum were used as negative control. After washing, goat anti-rabbit IgG-Cy3 conjugate (1:400, Merck Millipore) and goat anti-mouse IgG-488 conjugate (1:500, Dylight) were incubated for 1 h as secondary antibodies. After that, the sections were stained with DAPI for 10 min at room temperature and observed with fluorescence microscopy. All incubations were conducted in a moisture chamber in the dark, followed by three washes with PBST.
Detection of antibodies against HIRRV by ELISA
To detect the specific IgM against HIRRV in serum and gut mucus, 100 µl of prepared virus supernatant was coated into wells of microplates (96-wells, Costar) overnight at 4 °C. After three washes with PBST (PBS containing 0.05% Tween-20), all wells were blocked with 4% BSA at 37 °C for 2 h. Thereafter, serum or mucus samples (1:20 diluted in PBS) from five flounder were incubated for 1.5 h as primary antibodies. FIgM-Mab and alkaline phosphatase-conjugated goat anti-mouse IgG were sequentially added into each well and incubated for 1 h as the secondary and third antibodies, respectively. Following three washes, 100 µl 0.1% (w/v) p-nitrophenyl phosphate (pNPP, Sigma, USA) in 50 mM carbonate-bicarbonate buffer (pH 9.8) containing 0.5 mM MgCl2 was added to each well and incubated at room temperature for 20 min in the dark. The reaction was stopped with 50 µl per well of 2 M NaOH and absorbance was measured with an automatic ELISA reader at 405 nm. Myeloma culture supernatant instead of FIgM-Mab was used as the negative control, and each sample was accessed in three parallels.
Challenge and sample collection
After the last sampling at 8th week, all fish were transferred to tanks equipped with a refrigerating apparatus and adapted to 14 °C by gradual decrease of water temperature. One week later, all the fish of four experimental groups were intraperitoneally injected with 100 µl of prepared virus suspension (106.8 TCID50/fish) according to the lethal dose (LD50) in flounder . After challenge, thirty fish were randomly selected from each experimental group for monitoring mortality. Mortality was recorded daily for 14 d post-challenge and RPS was calculated using the following formula: (1−% mortality of vaccinated fish/% mortality of control fish) × 100. Another thirty fish were randomly selected from each experimental group for monitoring the HIRRV proliferation in the spleen of infected flounder. For sampling, three fish from each group were sacrificed on day 1 and day 3 post-injection, and the spleens were sampled and stored in Sample Protector (Takara) at − 80 °C until usage.
Detection of HIRRV load by quantitative PCR
The absolute fluorescence quantitative PCR standard curve has been established for quantifying the G gene copies . The fragment of HIRRV-G gene was cloned into the pMD19-T vector with the primers G-qF and G-qR (Additional file 1: Table S1) to construct standard plasmid. After purification and quantification, the copy number was calculated as described previously , and it was diluted in tenfold serial dilution series as templates for qPCR assay using Roche 480 real-time PCR system (LightCycler®480, USA). The 20 μl volume of the reaction mixture containing 10 μl of SYBR Green I Master, 1 μl of forward and reverse primers (10 μM), 1 μl of each diluted standard plasmid and 7 μl of RNase-free water, was subjected to the following procedure: 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s and 60 °C for 1 min. Thereafter, the threshold cycle (Ct) values were measured and standard curve equations (y = − 3.6984 x + 41.964, R2 = 0.9996) were produced by regression analysis of mean Ct (y) versus the log of standard plasmid copy numbers (x).
1 µg of total RNA from spleen samples was reverse transcribed into cDNA and then 2 µl of the products was used as the templates for qPCR. Following the manual above, the assay was performed in triplicate for each sample and non-infected samples were used as the negative control. After amplification, viral copies were determined by extrapolating Ct values from the standard curve and expressed as mean log10 copies/0.1 µg RNA.
The comparison of antibodies production and viral copies were performed with one-way analysis of variance (ANOVA) using SPSS 20.0 software; the results were expressed as mean ± SD and the significance level was defined as p < 0.05. The survival rates following HIRRV challenge were compared by Log-rank (Mantel-Cox) test using GraphPad Prism 7 software.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional file.
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This study was supported by the National Natural Science Foundation of China (31730101; 31872590; 31672685; 31672684; 31472295), Natural Science Foundation of Shandong Province (ZR2019MC029), National key Research and Development Program of China (2018YFD0900503), Fundamental Research Funds for the Central Universities (201822015) and Taishan Scholar Program of Shandong Province.
Ethics approval and consent to participate
This study was performed in strict accordance with the recommendations in the Guide for the Institutional Animal Care and Use Commission (IACUC). The protocols were approved by the Committee on the Ethics of Animal Experiments of the Ocean University of China (Permit Number: 20150101). All experiments were performed under MS-222 anesthesia, and every effort was made to minimize suffering.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1: Table S1. Bacterial strains, plasmids and primers used in this study. Figure S1. Mass spectrographic analysis of the G protein expressed by L. lactis NZ9000. (A) The amino acid sequence of HIRRV-G protein. Eight matched peptides were underlined and two of the best matched peptides were labeled in bold. (B) Fingerprints of the two best matched peptides. (C) The matched protein information of mass spectrometric analysis.
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Cite this article
Zhao, L., Tang, X., Sheng, X. et al. Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus). Microb Cell Fact 18, 142 (2019). https://doi.org/10.1186/s12934-019-1195-9
- Lactococcus lactis
- HIRRV glycoprotein
- Surface display
- Oral vaccine
- Immune protection

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The TSX sputtered in two of the last three trading sessions of last month. Nonetheless, the index remains in record territory after finishing at 21,037.10 on October 29, 2021. Volatility is ever present, despite the 5% improvement from a weak September and the 20.67% year-to-date gain.
Based on forecasts by the Bank of Canada, the annual inflation rate would be close to 5% by year-end 2021. However, it should hit its target of 2% by the end of 2022. Also, the central bank might hike interest rates sooner than later because of the ongoing supply chain disruptions and higher energy prices.
If a market pullback worries you, consider moving to safer grounds. Emera (TSX:EMA), WELL Health Technologies (TSX:WELL), and Medical Facilities (TSX:DR) are companies with low-risk, high-growth profiles. Their respective businesses should be stable in 2022 and beyond.
Emera, which is in the utility sector, is resilient as ever amid the pandemic environment. The $14.93 billion multinational energy holding company has seven regulated companies under its umbrella. The diversified portfolio and its highly regulated assets serve 2.5 million customers in six countries.
The Halifax-based company provides essential gas and electricity services under franchises, and, therefore, the business is enduring. Likewise, cash flows are stable because revenues come from cost-of-service, rate-regulated arrangements. In the first half of 2021, net income declined 56% to $256 million versus the same period in 2020.
However, management said that without the market-to-market (MTM) losses, the top and bottom lines should have been higher. Still, Emera projects its rate base to grow between 7.5% and 8.5% through 2023. It should enable the company to raise its dividend by 4-5% through 2022. The share price is $57.58, while the dividend yield is 4.6% if you invest today.
WELL Health is an attractive investment prospect, given the focus on people’s health these days. The $1.35 billion owner and operator of outpatient health clinics seek to advance the digital modernization of Canada’s healthcare system. It also provides electronic medical records (EMR) services to over 2,800 medical clinics.
WELL commits to meet the unprecedented demand for mental health services. Management reported a 72% organic increase in mental health visits in its primary care clinics in Q3 fiscal 2021. Hamed Shahbazi, WELL’s founder and CEO, said, “The adoption of virtual mental health platforms will be a permanent and growing fixture of the healthcare system.”
Now is an excellent opportunity to snag the healthcare stock ($6.58 per share) while it’s trading at a discount (-18.26% year to date). You’d be investing in a well-managed company in a fast-growing industry.
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Medical Facilities has similarities with WELL Health, although the operations are in the United States. The $287.42 million company operates high-quality surgical facilities with physicians as its investors and managers at the same time. The unique business model and management philosophy focus on the efficiency and productivity of the facilities.
The company’s specialty surgical hospitals and ambulatory surgery centres are the next-best alternatives to traditionally run hospitals. Medical Facilities derives 100% of its revenue from facility fees charged to patients or their insurers. At $9.24 per share (+34.84% year to date), the stock pays a decent 3.02% dividend.
Rough sailing ahead
November 2021 could be rough sailing for the stock market due to the rising inflation risks. However, investors can prepare and counter the risks by moving to safe assets.

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Alabama’s namesake ship, the the Ohio-class ballistic-missile submarine USS Alabama (SSBN 731) transited the Hood Canal on Friday, as the returned to its homeport at Naval Base Kitsap-Bangor following a routine strategic deterrent patrol.
Alabama is one of eight ballistic-missile submarines stationed at the base, providing the most survivable leg of the strategic deterrence triad —the nuclear weapons delivery of a strategic nuclear arsenal consisting of three components: land-based intercontinental ballistic missiles, strategic bombers, and submarine-launched ballistic missiles — for the United States.
According to the U.S. Navy, “nuclear deterrence is about influencing the decision not to use nuclear weapons or deterring coercion through the threat to use nuclear weapons.”
“The SSBN force represents the most survivable leg of the triad, “said Gen. Kehler in a 2013 speech. SSBNs that are underway in the Atlantic and Pacific Oceans remain undetected and increasingly comprise the largest concentration of our deployed warheads.”
Commissioned in 1985 and slated to remain in service through 2027, the USS Alabama operates as part of Submarine Squadron 17.
While stationed over 40 hours away from its name-state, the USS Alabama crew holds the Yellowhammer State in high regard. In 2015, the crew posted of the video giving a special shout-out to the University of Alabama football program.


Media professionals interested in reporting on university-related stories are encouraged to visit the media newsroom.
April 9, 2010
By Skyler Dillon
Throughout April and into May, the Center for Student Cultural Diversity will present more than 30 events celebrating the many cultures that compose the campus community. The Annual Intercultural Month (AIM) invites students, faculty and staff to engage with each other to learn more about the backgrounds of their colleagues, and to promote positive communication about the diversity of the University of Nevada, Reno campus.
“We want to expose students to thoughts and customs different than their own,” said Ellen Houston, counselor coordinator for the Center. “Students are encouraged to be respectful of the differences between them and their peers.”
The events will include competitions, graduation celebrations, workshops and lectures. One of the most anticipated events of the month is the third annual Stompin’ with the Pack Step Show, scheduled for 2 p.m. April 10 in the Virginia Street Gym. Organized by the Multicultural Greek Council, the show will feature five teams from across the west coast in a “step” competition. Stepping was developed from African tribal dances and uses foot-stomping, hand-clapping and finger-snapping to create rhythms and dance routines.
“It’s meant to really unify the students,” said Emma Garcia, a member of Lambda Phi Xi, part of the Multicultural Council. “It showcases the differences within the Greeks.”
Three University teams will compete, as well as two from California. An exhibition team from Oakland, Calif. will also perform. This year’s theme, “This is Ten,” will be incorporated into many of the routines, which according to Garcia convey an “oral history” of each organization.
“They tell the story of how we got to campus, what our organization is about, and where we’re going,” she said. “It’s highly competitive.”
Steppin’ with the Pack is open to the public, and Garcia hopes to draw 600 people this year.
“It’s the only show of its kind in Reno,” she said.
Tickets are $10 in advance and $12 at the door. Watch a video of the Nevada team at last year’s competition.
Highlighted events during AIM include:
For the complete calendar of events, visit the Center for Student Cultural Diversity Web site.
Skyler Dillon is a student writer in university media relations.


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Sunday will represent the Monasterboice men's first ever Joe Ward defence
There are 2 games on the second weekend of the Louth Football Championships on Friday evening..
Both are in the Junior grade with Stabannon Parnells up against Glyde Rangers (Darver 7.30), and the John Mitchels facing St Nicholas at the Sean O'Mahonys pitch (8pm)
And this Sunday, Naomh Mairtin begin their Senior Championship title defence, with a game against one of the most impressive opening weekend winners, the Dreadnots..
The Clogherhead men saw-off the Geraldines with a ruthless performance, and Mairtins and County star Sam Mulroy feels they'll be a tough nut to crack


CLEVELAND, OH (WOIO) - Local law enforcement vehicles are in service with active recalls, putting you and your family at risk.
Basic recall information is always available, free of charge, on the National Highway Safety Administration website. What's the excuse for local law enforcement not knowing their vehicles have been recalled?
We found this unmarked law enforcement vehicle sitting right outside Cleveland City Hall with a potentially dangerous recall. A defective ignition switch on the Dodge Charger that could stall the car and turn off the air bags. A similar problem with General Motor vehicles killed more than 120 people.
The same vehicle also has bad airbag inflators on both the driver and passenger sides, which have been linked to 11 deaths in the U.S. A ruptured airbag could send metal shrapnel shooting through the car. That's what happened to Sara Baker, Her airbag exploded and sliced off half of her ear.
A Cuyahoga County Sheriff's vehicle spotted in downtown Cleveland also has an active recall for a faulty airbag inflator.
In Cleveland Heights, we found four city vehicles with unfixed recalls ranging from airbags that may not deploy to bad headlights. Problems that Vehicle Maintenance Supervisor Doug Ditto said are likely to remain unfixed for a while until the automakers come up with a solution.
"They send us these notices and they're saying they don't have the parts to repair them, but they don't give us any kind of time table on that, on like when are they going to get the parts. If it's something important like that, then the car's going to be down. It should not be in service" said Ditto.
In University Heights, two city-owned Ford Escapes with three recalls between the two of them for side doors that could fly open while driving and a problem that could short circuit the airbags and side curtains, possibly making them ineffective in a crash.
We tried to alert University Heights City Service Director Jeff Pokorny to the dangerous defects that do have fixes available, but he didn't want to hear it.
"Look, I've had enough with the camera, OK?" Pokorny said and grabbed our cameraman and slammed the door.
It's not just local government ignoring safety recalls. The General Services Administration is auctioning off recalled vehicles - to the public. We checked and found several cars currently for sale by the government, some with potentially dangerous defects and no warning to buyers.
That's why you should always do your due diligence and check for recalls before you buy.
To check for unfixed recalls on your own vehicle, you can use the free MyCarfax app -- search on iTunes Store or Google Play.for it -- or you can use the National Highway Traffic Safety Administration's website.


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Our counseling team at the Barnes Center at The Arch offers you a strong support network of staff therapists who are dedicated to your well-being. This counseling is completely confidential, and just a phone call away. Even if it’s just managing the stress that can come with being a college student, we’ll help you face challenges and navigate through university life. The experienced and licensed counseling staff is available 24 hours a day for issues such as mental health and substance abuse.
The Office of Student Assistance is a central support hub for students and families. Professionals can help address crises and life trauma, psychological, physical or relationship issues, social adjustment, and more. Services include absence notifications, bias-related support, emergency loans, and more.

domain: health


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Take a look inside the historic Mackinac Island hotel where Mike Pence gave a speech this weekend — and then stirred outrage with his 8-car motorcade
|September 23, 2019||Posted by TravelKinds.Com under General||
- The Vice President of the United States, Mike Pence, gave a speech at the historic Grand Hotel on Mackinac Island this past weekend.
- Outrage erupted on the island, which banned automobiles in 1898, when a video showed Pence driving away from the hotel in an eight-car motorcade.
- Along with Pence, the hotel has been visited by other notable public figures throughout the years, including former President Gerald Ford.
- Visit Business Insider’s homepage for more stories.
Source : BusinessInsider.Com

domain: travel


Meet Umidigi Power 4G Phablet – Mighty Power in your Hand.
So far most big battery smartphones are thick and not comfortable to hold. UMIDIGI, the ‘Technology Fashion Pioneer’ as the company said, reshaped everyone’s perception of big battery phones and it made its new UMIDIGI Power series phone, which has both huge battery and pocket-friendly slim body. Let’s check out the specs of the new UMIDIGI Power.
The UMIDIGI Power features a 6.3-inch waterdrop full screen with FHD+ resolution(2340 x 1080) and in-cell LTPS display, protected by 2.5D curved glass. Thanks to the tiny waterdrop shape notch and narrow bezel, it reaches an incredible screen-to-body ratio of 92.7%. UMIDIGI Power is equipped with a high density 5150mAh big battery with 18W fast charge and NFC, and with the company’s industry-leading battery technology, the Power is just a 8.8mm thin pocket-friendly phone. The UMIDIGI Power is powered by the 12nm AI SoC Helio P35 with the 4GB RAM and 64GB onboard storage, with up to 256GB expansion via microSD. According to the other Helio P35 phone like the Mi Play, the 12nm Helio P35 SoC is ahead of the 14nm Snapdragon 625, delivering better performance and longer battery life.
On the back it’s got the dual camera 16MP + 5MP setup with F/1.8 aperture, while the front-side selfie camera contained within the waterdrop notch has a 16 megapixel sensor. Also, the UMIDIGI Power runs on stock Android 9.0 Pie out of the box.
Here are some new key features in Android 9 Pie
– Adaptive Battery
– Adaptive Brightness
– Gesture Navigation
– Notch Support
– App Actions
– New App Switcher
UMIDIGI Power Highlights
– 5150mAh Big Battery with Slim Design – Do More on a Single Charge. Equipped with a 5150mAh battery, one of the largest capacities found in any smartphone, and with curved edges as well as ultra-thin unibody design, the UMIDIGI Power features ultra-long endurance with power optimization and is easy to use in one hand. Also the 18W Fast charging replenishes the massive power swiftly. Do more and charge less.
– Cashless Life with Secure NFC. UMIDIGI Power supports Functional NFC where Google Pay and on-vehicle payment can be used.
– 16MP+5MP Dual Camera with F/1.8 Big Aperture – Capture Wonderful Moments. The improved 16MP+5MP dual camera setup with the flagship-grade f/1.8 big aperture together with the new software algorithm, work together to produce astonishing photos, allowing you to cherish your best memories in the quality they deserve.
– 16MP High Resolution Selfie. The front camera of the UMIDIGI Power has a 16MP high resolution sensor for superior photo quality. And with F/2.0 aperture and advanced algorithm, your every selfie is clear, beautiful and natural.
– 6.3″ FHD+ Waterdrop Full Screen – For Extensive View. The flagship 6.3-inch FHD+ waterdrop display, with 2340 x 1080 resolution, in-cell and LTPS technology, supporting a full screen of distinct clarity and vivid colour. The seamless design reaches remarkable 92.7% screen-to-body ratio, giving you a wide view to explore the world.
– Helio P35 AI Processor – 4GB RAM + 64GB ROM. The 12nm octa-core Helio P35 AI processor ensures a smooth operating experience with low power consumption and less heat emission. It acts like the powerful yet thrifty engine of your UMIDIGI Power, helping your phone deal with tasks smartly and swiftly.
– Stock Android 9 Pie – Latest Android Version Out of The Box. Powered by the latest android version Android 9.0, the UMIDIGI Power offers more features than ever on the fastest and smoothest Stock Android 9.0 without bloatware and ads.
– Global Bands & Dual 4G LTE – Connect to any Mobile Network of the World. UMIDIGI Power supports 33 global bands. Regardless of your place of residence or your destination for a journey, you can connect to the local mobile networks with your UMIDIGI Power anywhere in the world. It also supports 4G LTE in both slots simultaneously – enjoy faster data transmission, lower power consumption and more reliable connection from now.
1 x Umidigi Power 4G Phablet
1 x Power Adapter model HJ-FC010K7-EU
1 x Phone Case
1 x Type-C USB Cable
1 x Screen Protector (pre-applied)
1 x Multi-language User Manual
1 x SIM Needle / SIM Tray Ejector
1 x Umidigi ‘Thanks’ card
Photo gallery full album here. Official website here. Umidigi F1 4G Phablet unboxing here. Here you can read about the unboxing of UMIDIGI S2 Pro 4G 6GB RAM 128GB ROM Phablet. Unboxing UMIDIGI S2 Lite 4G Phablet here. Unboxing UMIDIGI A1 Pro here. Unboxing UMIDIGI A3 here.
About Umidigi At UMIDIGI we’re here for those that expect more from their smartphone than a run-of-the-mill experience. It is here, where the art of technology and life convene. We’re committed to focusing on premium products, differentiating innovation, achieving technological breakthroughs, bringing the latest technology to consumers and striving to realize dreams for people.


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Have you ever had one of those conversations where you feel like the other person is looking straight through you with a vacant stare? Not fun. Or what about that feeling that you know you could acheive something better if you teamed up with someone else, but for some reason you just don't make the move to do so?
Last week's North of England Education Conference (NEEC2013), hosted by Sheffield Hallam University and the University of Sheffield, shed some light on the questions above.
The theme of this year’s conference was Mind, Brain, Community: Inspiring Learners, Strengthening Resilience; it was packed with interesting sessions and had several high-quality speakers. Some who stood out as being particularly informative or inspiring were: Zoe Brownlie , Isaac Prilleltensky, Steven Edwards, Kenneth Gergen, and Lynda Ellis. But rather than rehash specific session presentations, I’d like cover just a couple of the themes that emerged over the course of the conference: attunement and collaboration.
Look in to my eyes:
Imagine having a conversation with someone who does not reply, nor nod understandingly, nor maintain eye contact. What does that feel like? We tried this exercise in a session run by Zoe Brownlie and again in a keynote by Marlo Winstead, so I can say from personal experience that it doesn’t feel great.
attunement is the interaction between people that shows a sense of connection and a degree of understanding
The exercise was to demonstrate what is meant by the word ‘attunement’. As mentioned briefly in a previous blog post, attunement is the interaction between people that shows a sense of connection and a degree of understanding. It is the reciprocal communication (not necessarily verbal), the ‘sensitive dance’ of attention and gesture (often subconscious mimicry) that happens between two people when they are in tune with each other.
The ‘still face exercise’ saw a whole auditorium full of people partner up, with one person a speaker and the other the listener. For the first 2 minutes the speaker spoke, and the listener listened as they would normally do. When prompted, the listener then had to maintain a still face – offering no real response to the speaker. What fell out of this was:
Overall, it was fascinating to see first-hand how our physical body – and even another person’s body - does not just influence our articulation and comprehension, but is part of it. This drove home for me the fact that our cognition is both embodied and embedded in a larger system of ourselves and others.
Attunement is not simply undivided attention; it is both more and less than that. It does not mean a parent giving in to every whim of a child. But it is the understanding of needs, and a response to those needs which ultimately help the other to regulate their emotions and arousal. It is bringing someone up when they need some stimulation, and bringing them down when they need calming.
Our physical body does not just influence our articulation and comprehension, but is part of it.
An understanding of attunement versus dissonance can be applied to innumerable areas in everyday life. [We’ll restrict this focus on attunement to the domain of educational practice, but it is not difficult to see the relevance of the topic to everyday peer-to-peer social interactions or manager/subordinate or client /supplier relationships at work.]
From birth, parents or caregivers teach their babies attunement (or dissonance) naturally. Where this is not done adequately, for whatever reason, the child may have trouble with self-regulation which may manifest in behavioural responses in, say, the classroom. So in the school setting, both a teacher’s awareness of the role of attunement (between caregiver and child) and the teacher’s own ability to attune him or herself with the students become significant. All of this suggests that a child’s interactions with others may have immediate effect on his (and their) articulation, comprehension, emotional self-regulation, and behaviour, and provides further support for the growing acceptance that school performance is not necessarily an indication of intelligence by a standard measure.
Keep your hands off my slice of pie:
Collaboration generally has a positive connotation; people seem to want it in their businesses, schools, and among their colleagues. It usually improves outputs and energises participants. The Report of the Academies Commission published earlier this month recommended greater intra- and inter-school collaboration, stating that “the evidence…suggests a more intensive drive to develop professional connections, collaborative activity and learning – both within and across schools – will generate fundamental change across the school system.”(p6) The push to collaborate seems to be everywhere.
So why aren’t we doing more of it?
This question was not directly addressed at the conference, but the general sense that there should be more collaboration certainly was. Some areas of the education system should be highlighted to help us understand what some of the barriers to doing so might be.
Firstly, assessment may be a barrier to collaboration. How does one evaluate collaboration? Should the assessment be done on the final output, or on the skills demonstrated by the students throughout the process? Can a teacher give the whole class the same grade?
Next, for some, and this will depend on assessment and incentives, collaboration holds a tension between competition and cooperation. If a student is evaluated and given an individual mark, rather than a mark for the group, or if teachers grade on a curve, then he might be compelled to both cooperate with his fellow students and also do a bit better than the others. Something like “I’ll collaborate to an extent, but keep the juicy ideas to myself.” On a school level, the rapid rate of recent academisation has provided many schools with increased autonomy. But with autonomy comes responsibility to develop best-practice and maintain high standards. Schools are being urged to collaborate and support each other, but the same tension present at the student level can also be found at the school level: why help the neighbouring school if it means that they will climb ahead of our school in the league tables?
Although not covered in the conference, some psychological barriers to collaboration have been covered in business literature. Organisational psychology points to in-group favouritism and the fixed-pie effect as potential barriers.
People mistakenly think the size of the 'pie' is fixed, and fear collaboration to be relinquishing a slice of their share of the rewards to their collaborator
In-group favouritism is a natural tendency to treat members of your group (however you identify that to be) more favourably than those outside the group. This has obvious evolutionary advantages, but is less helpful when it stymies inter-group cooperation.
And the fixed-pie effect is where people fail to recognize that by collaborating they can increase the potential rewards to be shared. Instead, they mistakenly think the size of the pie is fixed, and fear collaboration to be relinquishing a slice of their share of the pie to their collaborator/competitor.
I’d be interested to hear your experiences of collaboration and thoughts on the answer to the question: what is holding us back from better collaboration?

domain: psychology


Rockets 3 PM • Philadelphia 76ers vs. The 1987 World Hockey Championship is known for the Punch-up Piestany which saw Canada and the Soviet Union involved a bench-clearing brawl during the final game of the tournament. It rarely comes up NFL Jerseys 2017 conversation about much of anything these days. 1 on the depth chart was more a product of Detroit’s lack of other quality options at the position than anything , as Ameer Abdullah struggled his way out of the starting job and no other tailback proved capable of taking on a featured role. It is also not without its difficulties. Messier Baseball Jerseys Cheap won his first Hart Trophy as the NHL’s most valuable player 1990, the year of the Oilers’ most recent Cup championship, and won another one ’92 with the Rangers. While Resnick wouldn’t confirm what the health considerations were, Nash has suffered several reported concussions during his NHL career, most recently March as a member of the Boston Bruins. • The Union had no answer for Diomande, who now has a whopping seven goals the past four .
For this week’s edition — voted, as always, by ‘s panel of experts — we looked at X factor for each team, a player who could make or break his team’s first-round series. This nug from The Big Fish, : Cook was on pace for a whopping 340 touches before his ACL injury, a figure that would’ve put him the top five among all tailbacks. The Vikings signed two of the most-talked-about free agents on the market, but NFL Jerseys 2017 they aren’t the only team making moves. But even though the Bruins have been knocked out early May, here are a few things the fans can look forward to. his first two seasons, he combined to play and start only 13 NBA Basketball Jerseys . Every decision you make is critical.
Now that she has achieved the holy grail of Alaskan politics, legislation opening the Arctic National Wildlife Refuge to drilling, she can act on her statements that Trump is often wrong. Still, what was able to accomplish the NFL from his late 20s on — he threw for nearly 50 yards and almost 300 touchdowns — earned him a spot the Hall of Fame… His 15 sacks 2016 were the second most by a Falcon a single Cheap Atletico Madrid Jerseys since sacks became official nfl jerseys authentic statistic 1982. And it’s a big sacrifice — you got to leave your family, you got to leave what you’re doing, wherever you’re from. I think this event last year came at a point where I was pretty much the nike nfl jerseys size chart form of life, said Fleetwood. Analysis: Lopez ended a five-game rut which he totaled 26 points and shot 28 percent. As he continues to switch shoes at halftime NBA Basketball Jerseys of nearly every game, Clippers center Montrezl Harrell finds himself garnering attention yet again for his random rotation of coveted and vintage Air Jordan models. 2pm: BRITISH GP QUALIFYING LIVE! Among the changes , Cordy made his first start at left tackle since the season opener. Jets diehards aren’t alone.
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Returns exclude Bet Credits stake.


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Big Brother Naija star, Erica Nlewedim has bagged an endorment deal with a mobile phone brand, Partner Mobile Nigeria.
The reality star and actress took to her Instagram page to announce this news as she shared a photo of herself with a the company’s phone.
She captioned :
“Say hi to the new @partnermobileng ambassador”
View this post on Instagram

domain: entertainment


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West Lake Park
Fuzhou West Lake Park is located at the foot of Wolong Mountain in northwest Fuzhou, Fujiang Province. The west lake in Fuzhou was excavated in 282 A.D. by Yan Gao, an official of Jing Dynasty. In late Tang Dynasty, it became famous because of its wonderful scenery. During the age of Five Dynasties, an emperor built waterside pavilions around the lake and took it as his imperial garden. Later in Song Dynasty, it became a famous tourist attraction and a lot of poets paid a visit to it and wrote many beautiful poems for it. In 1828 in Qing Dynasty, Lin Zexu, the anti-opium patriot restored the bank around the lake. The lake was publicized as West Lake Park.
In the park across the lake, there is a long causeway. The original causeway was established in 1930 and it was 8 meters wide, 139 meters long. The restored one is largely widened. It is now 18 meters wide. Seen from a distance, the causeway looks like a silk ribbon floating over the water. Walking around it, you can enjoy the tender caress of willow branches swaying in the breeze. The lake is strewed with several small natural islets. Bridges connect any two islets. Kaihua Temple, the oldest building in the park, stands in the center of Kaihua Islet. It is said that it was built in 1037 in North Song Dynasty. Besides, in the park there is a zoo, a museum, and a pavilion named Opium-prohibition Pavilion, which was constructed in memory of Lin Zexu’s opium-prohibition movement.
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domain: history


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Because the rise in respiratory sicknesses amongst younger folks outpaces hospitals in another components of the nation, Well being PEI says it is beginning to see indicators of that pattern right here.
Whereas total visits to the Island’s emergency division elevated solely barely this fall in comparison with final, there was a big bounce in visits by Islanders below the age of 18.
To this point in November, Prince County Hospital in Summerside has seen 34 p.c extra sufferers in that age group. There was a 17 p.c bounce at Charlottetown’s Queen Elizabeth Hospital.
PEI Well being suspects the truth that COVID-19, influenza and RSV are all circulating on the identical time is the primary motive for the rise.
“We all know that that is our first respiration season [since the start of the pandemic] that we do not have widespread masks in public. And influenza is right here sooner than standard,” mentioned Corinne Rowswell, chief working officer of Well being PEI.
‘It will worsen earlier than it will get higher’
At a press convention in Halifax Thursday, the top of pediatrics on the IWK Well being Heart – a Maritime specialty youngsters’s hospital – warned that the following few months might be difficult.
“Based mostly on what’s taking place now in Ontario and Quebec, within the youngsters’s hospitals there, they’ve the complete power of influenza on them. We simply began seeing it within the final week or so right here. So it should worsen earlier than it will get higher. That is our concern,” mentioned Dr Andrew Lynk.
Rowswell mentioned PEI has not seen a rise in admissions to both of its pediatric items at QEH or PCH.
However with workers and assets “stretched skinny” throughout the province, and lengthy affected person waits in emergency departments, he worries concerning the weeks forward.
“After all we fear, each time we’ve this wave probably coming our manner,” Rowswell mentioned. “The worst-case state of affairs is that we’ve steady service disruptions as a result of we’ve sick workers, or we’ve too many sufferers coming into our services.”
Meaning canceling scheduled procedures or surgical procedures, he mentioned, as is occurring at hospitals worst hit by the surge in respiratory sicknesses.
A name for masks is obligatory
This can be a pattern that has led some medical consultants in different areas to publicly name for the obligatory masks rule to be reinstated. Rowswell mentioned a number of PEI Well being workers have requested the identical right here.
“We’ve got heard from workers who really feel obligatory masks ought to be reinstated, however at this level, it has not been communicated from the top of the general public well being workplace that that might be referred to as for,” Rowswell mentioned.
PEI Chief Public Well being Officer Dr. Heather Morrison, encourages Islanders to put on masks, particularly after they have chilly or flu signs, or are recovering from an sickness.
He additionally pressured the significance of staying dwelling when you’re sick, and getting vaccinated in opposition to COVID-19 and influenza.
Rowswell mentioned if Islanders need to decrease the influence of this wave of respiratory illness on the well being care system, heeding that recommendation is important.
“I believe folks is likely to be complacent. They exit and about after they have signs as a result of it is not COVID-19,” he mentioned. “However it might be influenza that may be simply as harmful for sure people who find themselves immunocompromised or who’re very younger.”
Rowswell mentioned he was notably discouraged by the comparatively low uptake of vaccines amongst youngsters. Solely 10 p.c of Islanders below the age of 5 have obtained a dose of the COVID-19 vaccine.
“I strongly encourage our mother and father to verify their youngsters are vaccinated,” he mentioned. “The vaccine works, and it is not encouraging when you’ve gotten it and the uptake is not there.”

domain: news


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Aluminium Lifting gantry cranes / Portable Gantry Cranes
Aluminium lifting gantry cranes from Cranes Direct differ greatly from our steel mobile gantries. This is due to the fact that they are light they adjust in height and are foldable.
These portable gantry cranes may be moved whilst under load ONLY in certain circumstances (see below for more details). Cranes Direct can offer a range of quality aluminium gantry cranes. Our standard and most commonly used range having working load limits from 250kg up to 2000kg.
The brand new Rapide aluminium gantry is now available from Cranes Direct. The Rapide has even more benefits than a standard aluminium lifting gantry. First of all it is a much more compact folding system. The Rapide gantry can be handled by one person and comes with working load capacities up to 500kg. Additional benefits of dual rating enable it to be used to lift personnel. Furthermore some fall arrest situations can be dealt with using an aluminium gantry.
The Uses of an Aluminium Lifting Gantry
Aluminium gantry cranes have many uses in industrial and commercial sectors. Because they are not permanently fitted they can be a very cost effective alternative to a fitted overhead crane. Yet they can be more costly than a standard steel gantry.
Portable gantries are manufactured in aluminium. Because aluminium is corrosion resistant and therefore suitable for use in clean and/or refrigerated environments.
HVAC industries (heating, ventilation & air conditioning) regularly use aluminium gantry cranes for HVAC applications. Additionally they’re also commonly used in cemeteries for the removal of head stones and other more remote and difficult locations.
Other uses often include such things as animal rescue operations where a lifting frame maybe necessary. These mobile gantries are ideal for placement and retrieval of objects in low recessed or crevassed areas. Places such as man holes and canal locks because the gantry can span the hole/crevasse.
Other areas for use also include stately homes and castles. They maybe used for lifting and moving large ornamental statues etc. In fact portable aluminium gantry systems are used in numerous, locations for various lifting purposes. Sometimes they are used for quite off the wall lifting operations.
Portable gantry cranes are ideal when frequent assembly and disassembly is necessary. This may be to store out of the way where space is limited or for transportation to other locations.
Aluminium Gantry Crane Benefits
There are a few key benefits to using an aluminium lifting gantry crane. They are light in weight yet offer a strong and robust lifting framework. Because they can be folded up, they are easy to transport which makes them ideal for site work.
They are easy to assemble in just a few simple steps ready for work. They are just as easy to dismantle again ready for transportation to the next job. Because they have castors they are easy to move around the location where they are used. They also come with its own push travel trolley. This enables easy attachment of lifting slings and other lifting equipment. The trolley also enables the movement of the load along the top beam.
Another excellent benefit of these cranes is that they are made from high quality aluminium. Aluminium possesses excellent weather and corrosion resistance. There is no need to worry about rust on an aluminium gantry. This feature means that they are perfect for outdoor lifting applications.
Unlike standard steel mobile gantries, the aluminium gantry can be adjusted in height at small spaced increments. This makes the gantry even more versatile, suiting a wider range of lifting operations.
Aluminium gantries are easy to maintain. Checks should be carried out prior to each use ensuring all parts are sound and pins/bolts etc. are securely located. Periodic inspections should be undertaken to check for damaged parts. Look out for cracks, corrosion, dents, bent parts, missing parts, and wheel locks engage securely.
It is wise to keep your portable gantry cranes clean and periodically lubricate the castors. Cleaning an aluminium lifting gantry is as easy as washing a car. A bucket of soapy water and a brush or sponge will do the trick.
Moving under load
Cranes Direct always recommend that any portable lifting gantry should NOT be moved whilst it is holding a load. However in some circumstances it is possible to use an aluminium gantry crane to move a load. Moving the gantry whilst loaded should ONLY be done if the ground/surface of travel is level. The distance of travel should be minimal.
Furthermore there should be at least two people to move a loaded gantry. More if possible, as many hands make light work! This will ensure the gantry remains stable and the load does not swing. You should avoid excessive swinging of the load as this could topple the framework.
Peruse the aluminium lifting gantries for sale to see which option would best suit your lifting requirements. If you have any questions or just need a straight forward quotation then please contact us.
There are a few ways in which you can do this. You can call on 01384 76961, use the contact us page or the rapid enquiry button; a member of our sales team will be happy to help with your query.


text: Abstract: Among urban, primarily African American pregnant women, 74% were identified with Nugent score bacterial vaginosis (BV). All BV-associated bacteria were more prevalent among women with Nugent score BV. Bacterial vaginosis–associated bacteria 3 (BVAB3) had the highest positive predictive value, whereas Gardnerella vaginalis and Atopobium spp. had the highest sensitivity. Atopobium spp. levels had the most significant area under the curve.