ROS-Sp1 axis is involved in thermochemotherapy-enhanced sensitivity of pancreatic cancer cells to gemcitabine.

Gemcitabine (GEM)-based chemotherapy represents the first choice for locally unresectable advanced pancreatic cancer, while the benefit is limited due to acquired chemoresistance or drug delivery insufficiency. Hyperthermia treatment potentially improves the clinical efficacy of GEM. However, the underlying mechanism is incompletely revealed. Our study aims to investigate the effect and involved mechanism of thermochemotherapy on cell survival. Pancreatic cancer cells PANC-1 and ASPC-1 were either treated with GEM or thermochemotherapy, then cell viability, apoptosis, migration, invasion, reactive oxygen species (ROS) production, and Sp1 expression were evaluated. The results showed that GEM dose and time-dependently affected cell viability, and 30 µM GEM achieved favorable effect in suppressing cancer cell growth. Meanwhile, hyperthermia preconditioning (43°C for 60 min) 24 h before GEM treatment yielded superior effect than other treatment sequence. GEM caused significant cell apoptosis and proapoptotic genes of both cancer cells, and thermochemotherapy further promoted apoptosis and genes transcription, accompanied by excessive ROS production. Thermochemotherapy was superior to GEM in suppressing cell migration and invasion of pancreatic cancer cells. Meanwhile, GEM significantly reduced Sp1 mRNA and protein and its downstream gene Cox-2, and thermochemotherapy further suppressed their expressions. ROS elimination with N-acetyl-l-cysteine notably neutralizes the suppressive effect of GEM and thermochemotherapy on cell growth and expressions of Sp1 and Cox-2. In conclusion, thermochemotherapy inhibited pancreatic cell viability, migration and invasion, and promoted cell apoptosis by inducing excessive ROS production, which subsequently suppressed Sp1 expression and the downstream Cox-2.

Pseudomonas aeruginosa MutL promotes large chromosomal deletions through non-homologous end joining to prevent bacteriophage predation.

Pseudomonas aeruginosa is an opportunistic pathogen with a relatively large genome, and has been shown to routinely lose genomic fragments during environmental selection. However, the underlying molecular mechanisms that promote chromosomal deletion are still poorly understood. In a recent study, we showed that by deleting a large chromosomal fragment containing two closely situated genes, hmgA and galU, P. aeruginosa was able to form 'brown mutants', bacteriophage (phage) resistant mutants with a brown color phenotype. In this study, we show that the brown mutants occur at a frequency of 227 ± 87 × 10-8 and contain a deletion ranging from ∼200 to ∼620 kb. By screening P. aeruginosa transposon mutants, we identified mutL gene whose mutation constrained the emergence of phage-resistant brown mutants. Moreover, the P. aeruginosa MutL (PaMutL) nicking activity can result in DNA double strand break (DSB), which is then repaired by non-homologous end joining (NHEJ), leading to chromosomal deletions. Thus, we reported a noncanonical function of PaMutL that promotes chromosomal deletions through NHEJ to prevent phage predation.

The Interaction of N-Acetylcysteine and Serum Transferrin Promotes Bacterial Biofilm Formation.

BACKGROUND/AIMS: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. METHODS: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. RESULTS: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. CONCLUSIONS: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Surface Display of Heterologous ß-Galactosidase in Food-Grade Recombinant Lactococcus lactis.

ß-Galactosidase is an essential enzyme for the metabolism of lactose in human beings and has an important role in the treatment of lactose intolerance (LI). ß-Galactosidase expressed by intestinal microflora, such as lactic acid bacteria (LAB), also alleviates LI. A promising approach to LI management is to exploit a food-grade LAB delivery system that can inhabit the human intestine and overproduce ß-galactosidase. In this study, we constructed a food-grade ß-galactosidase surface display delivery system and then integrated into the chromosome of Lactococcus lactis (L. lactis) NZ9000 using recombination. Western blot and immunofluorescence analyses confirmed that ß-galactosidase was expressed on the cell surface of recombinant L. lactis stain NZ-SDL. The whole-cell biocatalyst exhibits Vmax and Km values of 121.38 ± 7.17 UONPG/g and 65.36 ± 5.54 mM, based on ONPG hydrolysis. The optimum temperature for enzyme activity is 37 °C and the optimum pH is 5.0. Activity of the whole-cell biocatalyst is promoted by Mg2+, Ca2+, and K+, but inhibited by Zn2+, Fe2+, and Fe3+. The system has a thermal stability similar to purified ß-galactosidase but better pH stability, and is also more stable in artificial intestinal juice. Oral administration and intraperitoneal injections of NZ-SDL in mice cause no detectable health effects. In conclusion, we have successfully constructed a food-grade gene expression system in L. lactis that displays ß-galactosidase on the cell surface. This system exhibits good enzyme activity and stability in vitro, and is safe in vivo. It is therefore a promising candidate for use in LI management.

Phage Abp1 Rescues Human Cells and Mice from Infection by Pan-Drug Resistant Acinetobacter Baumannii.

BACKGROUND/AIMS: As an "ESKAPE" pathogen, Acinetobacter baumannii is one of the leading causes of drug-resistant infections in humans. Phage therapy may be a useful strategy in treating infections caused by drug-resistant A. baumannii. Among 21 phage strains that were isolated and described earlier, we investigated the therapeutic efficacy of Abp1 because of its relatively wide host range. METHODS: Phage stability assays were used to evaluate thermal and pH stability of Abp1. Abp1 was co-cultured with A. baumannii (AB1) over a range of multiplicities of infection to determine its bactericidal efficacy. HeLa or THP-1 cells were used in the cytotoxicity and protection assays. Finally, the therapeutic effects of Abp1 on local and systemic A. baumannii infection in mice were determined. RESULTS: We found that Abp1 exhibits high thermal and pH stability and has a low frequency of lysogeny. Bacteriophage resistance also occurs at a very low frequency (3.51±0.46×10-8), and Abp1 can lyse almost all host cells at a MOI as low as 0.1. Abp1 has no detectable cytotoxicity to HeLa or THP-1 cells as determined by LDH release assay. Abp1 can rescue HeLa cells from A. baumannii infection, even if introduced 2 hours post infection. In both local and systemic A. baumannii infection mouse models, Abp1 treatment exhibits good therapeutic effects. CONCLUSION: Abp1 is an excellent candidate for phage therapy against drug-resistant A. baumannii infections.

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

BACKGROUND: Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. RESULTS: Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (ß-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. CONCLUSIONS: By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

Activation of AMPK restricts coxsackievirus B3 replication by inhibiting lipid accumulation.

Coxsackievirus B3 (CVB3) is the major pathogen of human viral myocarditis. CVB3 has been found to manipulate and modify the cellular lipid metabolism for viral replication. The cellular AMP-activated protein kinase (AMPK) is a key regulator of multiple metabolic pathways, including lipid metabolism. Here we explore the potential roles AMPK plays in CVB3 infection. We found that AMPK is activated by the viral replication during CVB3 infection in Hela cells and primary myocardial cells. RNA interference mediated inhibition of AMPK could increase the CVB3 replication in cells, indicating that AMPK contributed to restricting the viral replication. Next, we showed that CVB3 replication could be inhibited by several different pharmacological AMPK activators including metformin, A769662 and AICAR. And the constitutively active AMPK mutant (CA-AMPK) could also inhibit the CVB3 replication. Furthermore, we found that CVB3 infection increased the cellular lipid levels and showed that the AMPK agonist AICAR both restricted CVB3 replication and reduced lipid accumulation through inhibiting the lipid synthesis associated gene expression. We further found that CVB3 infection would also induce AMPK activated in vivo. The AMPK agonist metformin, which has been widely used in diabetes therapy, could decrease the viral replication and further protect the mice from myocardial histological and functional changes in CVB3 induced myocarditis, and improve the survival rate of infected mice. Lastly, it was demonstrated that the AICAR-mediated restriction of viral replication could be rescued partially by exogenous palmitate, the first product of fatty acid biosynthesis, demonstrating that AMPK activation restricted CVB3 infection through its inhibition of lipid synthesis. Taken together, these data in the present study suggest a model in which AMPK is activated by CVB3 infection and restricts viral replication by inhibiting the cellular lipid accumulation, and inform a potential novel therapeutic strategy for CVB3-associated diseases.

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

BACKGROUND: Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. RESULTS: After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. CONCLUSIONS: This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

Accurate prediction of bacterial type IV secreted effectors using amino acid composition and PSSM profiles.

MOTIVATION: Various human pathogens secret effector proteins into hosts cells via the type IV secretion system (T4SS). These proteins play important roles in the interaction between bacteria and hosts. Computational methods for T4SS effector prediction have been developed for screening experimental targets in several isolated bacterial species; however, widely applicable prediction approaches are still unavailable RESULTS: In this work, four types of distinctive features, namely, amino acid composition, dipeptide composition, .position-specific scoring matrix composition and auto covariance transformation of position-specific scoring matrix, were calculated from primary sequences. A classifier, T4EffPred, was developed using the support vector machine with these features and their different combinations for effector prediction. Various theoretical tests were performed in a newly established dataset, and the results were measured with four indexes. We demonstrated that T4EffPred can discriminate IVA and IVB effectors in benchmark datasets with positive rates of 76.7% and 89.7%, respectively. The overall accuracy of 95.9% shows that the present method is accurate for distinguishing the T4SS effector in unidentified sequences. A classifier ensemble was designed to synthesize all single classifiers. Notable performance improvement was observed using this ensemble system in benchmark tests. To demonstrate the model's application, a genome-scale prediction of effectors was performed in Bartonella henselae, an important zoonotic pathogen. A number of putative candidates were distinguished. AVAILABILITY: A web server implementing the prediction method and the source code are both available at http://bioinfo.tmmu.edu.cn/T4EffPred.

A functional peptidoglycan hydrolase characterized from T4SS in 89K pathogenicity island of epidemic Streptococcus suis serotype 2.

BACKGROUND: Streptococcus suis serotype 2 (S. suis 2) has evolved efficient mechanisms to cause streptococcal toxic shock syndrome (STSS), which is a new emerging infectious disease linked to S. suis. We have previously reported that a type IV secretion system (T4SS) harbored by the specific 89K pathogenicity island (PAI) of S. suis 2 contributes to the development of STSS and mediates horizontal transfer of 89K. However, the 89K T4SS machinery assembly in vivo and in vitro is poorly understood, and the component acting directly to digest the bacterial cell wall needs to be identified. RESULTS: The virB1-89K gene product encoded in the 89K PAI is the only one that shows similarity to the Agrobacterium VirB1 component and contains a conserved CHAP domain that may function in peptidoglycan hydrolysis, which makes it a plausible candidate acting as a hydrolase against the peptidoglycan cell wall to allow the assembly of the T4SS apparatus. In the current study, the CHAP domain of VirB1-89K from S. suis 89K PAI was cloned and over-expressed in Escherichia coli, and its peptidoglycan-degrading activity in vitro was determined. The results indicated that the VirB1-89K CHAP domain can degrade the peptidoglycan layer of bacteria. Deletion of virB1-89K reduces significantly, but does not abolish, the virulence of S. suis in a mouse model. CONCLUSIONS: The experimental results presented here suggested that VirB1-89K facilitates the assembly of 89K T4SS apparatus by catalyzing the degradation of the peptidoglycan cell wall, thus contributing to the pathogenesis of S. suis 2 infection.

Antibacterial properties of Acinetobacter baumannii phage Abp1 endolysin (PlyAB1).

BACKGROUND: Acinetobacter baumannii has emerged as one of the most important hospital-acquired pathogens in the world, because of its resistance to almost all available antibiotic drugs. Endolysins from phages are attracting increasing interest as potential antimicrobial agents, especially for drug-resistant bacteria. We previously isolated and characterized Abp1, a virulent phage targeting the multidrug-resistant A. baumannii strain, AB1. METHODS: To evaluate the antimicrobial potential of endolysin from the Abp1 phage, the endolysin gene plyAB1 was cloned and over-expressed in Escherichia coli, and the lytic activity of the recombinant protein (PlyAB1) was tested by turbidity assessment and bacteria counting assays. RESULTS: PlyAB1 exhibits a marked lytic activity against A. baumannii AB1, as shown by a decrease in the number of live bacteria following treatment with the enzyme. Moreover, PlyAB1 displayed a highly specific lytic effect against all of the 48 hospital-derived pandrug-resistant A. baumannii isolates that were tested. These isolates were shown to belong to different ST clones by multilocus sequence typing. CONCLUSIONS: The results presented here show that PlyAB1 has potential as an antibiotic against drug-resistant A. baumannii.

Identification of in-vivo induced genes of Streptococcus suis serotype 2 specially expressed in infected human.

Streptococcus suis (S. suis) serotype 2 usually cause infection in swine. Recently, two large-scale outbreaks in China with severe streptococcal toxic shock syndrome (STSS) and high mortality raised worldwide concern to human S. suis infection. To reveal the molecular pathogenesis of S. suis 2 during human infection, in-vivo induced antigen technology (IVIAT) was applied to identify the in-vivo induced genes (ivi genes) of S. suis 05ZYH33. The ivi genes are specifically expressed or up-regulated in-vivo and always associated with the in-vivo survival and pathogenicity of pathogens. In present study, convalescent sera from S. suis 05ZYH33 infected patients were pooled and fully adsorbed with in-vitro grown S. suis 05ZYH33 and Escherichia coli BL21 (DE3). Genomic expression library of 05ZYH33 was repeatedly screened with colony immunoblot assay using adsorbed sera. Finally, 19 genes were assessed as ivi genes of 05ZYH33. Fifteen of 19 genes encode proteins with biological functions in substance transport and metabolism, cell structure biogenesis, cell cycle control, replication, translation and other functions. The 4 remaining genes encode proteins with unknown functions. Of the 19 ivi genes, five (SSU05_0247, 0437, 1577, 1664 and 2144) encode proteins with no immunoreactivity to control sera from healthy individuals never exposed to 05ZYH33. The successful identification of ivi genes not only sheds light on understanding the pathogenesis of S. suis 05ZYH33 during its human infection, but also provides potential targets for the developments of new vaccines, therapeutic drugs and diagnostic reagents against human S. suis infection.

Proteome analysis of the two-component SalK/SalR system in Epidemic Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is a major swine pathogen of significant commercial importance worldwide and an emerging zoonotic agent of human streptococcal toxic shock-like syndrome. We previously reported that the Chinese highly pathogenic SS2 strains specifically harbor an 89K pathogenicity island, and the SalK/SalR two-component system encoded within this island mediates a piglet-lethal phenotype. However, its regulatory mechanism remains obscure. In the present study, we performed a proteome analysis to identify the target proteins regulated by the SalK/SalR system. This expression profiling analysis reveals 14 down-regulated proteins and 1 up-regulated protein. In partial accord with our previous microarray analysis, 9 out of the 14 down-regulated genes are also repressed at the transcriptional level. From the 9 matched genes, 5 were selected and further subjected to gene knockout and experimental infection of mice. The results show that the selected 5 genes are potential virulence factors and participate to various extents in the pathogenesis of SS2 infection, and may contribute to the attenuation of virulence mediated by the SalK/SalR system.

Characterization and genome sequencing of phage Abp1, a new phiKMV-like virus infecting multidrug-resistant Acinetobacter baumannii.

While screening for alternative antibiotics against multidrug-resistant Acinetobacter baumannii, we isolated a virulent A. baumannii bacteriophage Abp1. Transmission electron microscopy revealed that the phage had an icosahedral head with a short tail and should be classified as a member of the Podoviridae family. SDS-PAGE showed that Abp1 contained at least one major and nine minor proteins. In a single-step growth test, we demonstrated that Abp1 had a latent period of 10 min and a burst size of 350. Abp1 also had a relatively narrow host range. The entire genome was sequenced, and the final assembly yielded a 42,185 bp, linear, double-stranded DNA molecule with a G+C content of 39.15 % and containing 54 putative genes. Among these genes, 26 were functionally known, leaving 28 unknown putative genes. Abp1 is a new member of the phiKMV-like virus subgroup of the T7 group; its genome sequence is very similar to that of the A. baumannii phage phiAB1.

[Construction and virulence evaluation of the virB1-89K gene knockout mutant of type IV-like secretion system of Streptococcus suis serotype 2].

OBJECTIVE: To construct the virB1-89K gene knockout mutant and its complementary strain of Streptococcus suis serotype 2 (SS2) highly virulent strain 05ZYH33 and evaluate the role of virB1-89K in the pathogenesis of SS2. METHODS: The virB1-89K gene was knocked out by homologous recombination, then multiple-PCR and sequence analysis were used to identify the knockout strain deltavirB1-89K. The virB1-89K gene and its upstream promoter were cloned into the E. coli-S. suis shuttle vector pSET1, and the recombinant plasmid was electrotransformed into the deltavirB1-89K mutant to generate the complementary strain CvirB1-89K. The effects of virB1-89K deletion on the basic biological characteristics and virulence of SS2 were then determined in this study. RESULTS: The isogenic mutant deltavirB1-89K and its complementary strain CvirB1-89K were successfully constructed. No significant differences in biological characteristics were found among the three strains. However, the virulence of the deltavirB1-89K mutant was reduced to 30% of the wild-type level and functional complementation of virB1-89K restored its pathogenicity. CONCLUSION: The virB1-89K gene plays an important role in the pathogenesis of S. suis 2 infection.

GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2.

Pathogenicity islands (PAIs), a distinct type of genomic island (GI), play important roles in the rapid adaptation and increased virulence of pathogens. 89K is a newly identified PAI in epidemic Streptococcus suis isolates that are related to the two recent large-scale outbreaks of human infection in China. However, its mechanism of evolution and contribution to the epidemic spread of S. suis 2 remain unknown. In this study, the potential for mobilization of 89K was evaluated, and its putative transfer mechanism was investigated. We report that 89K can spontaneously excise to form an extrachromosomal circular product. The precise excision is mediated by an 89K-borne integrase through site-specific recombination, with help from an excisionase. The 89K excision intermediate acts as a substrate for lateral transfer to non-89K S. suis 2 recipients, where it reintegrates site-specifically into the target site. The conjugal transfer of 89K occurred via a GI type IV secretion system (T4SS) encoded in 89K, at a frequency of 10(-6) transconjugants per donor. This is the first demonstration of horizontal transfer of a Gram-positive PAI mediated by a GI-type T4SS. We propose that these genetic events are important in the emergence, pathogenesis and persistence of epidemic S. suis 2 strains.

Functional identification of the DNA packaging terminase from Pseudomonas aeruginosa phage PaP3.

Terminase proteins are responsible for DNA recognition and initiation of DNA packaging in phages. We previously reported the genomic sequence of a temperate Pseudomonas aeruginosa phage, PaP3, and determined its precise integration site in the host bacterial chromosome. In this study, we present a detailed functional identification of the DNA packaging terminase for phage PaP3. The purified large subunit p03 was demonstrated to possess ATPase and nuclease activities, as well as the ability to bind to specific DNA when it is unassembled. In addition, a small terminase subunit (p01) of a new type was found and shown to bind specifically to cos-containing DNA and stimulate the cos-cleavage and ATPase activities of p03. The results presented here suggest that PaP3 utilizes a typical cos site mechanism for DNA packaging and provide a first step towards understanding the molecular mechanism of the PaP3 DNA packaging reaction.

Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed in Escherichia coli.

Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni-NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.

Hybrid integrated photodetector with flat-top steep-edge spectral response.

Hybrid integrated photodetectors with flat-top steep-edge spectral responses that consist of an Si-based multicavity Fabry-Perot (F-P) filter and an InP-based p-i-n absorption structure (with a 0.2 µm In(0.53)Ga(0.47)As absorption layer), have been designed and fabricated. The performance of the hybrid integrated photodetectors is theoretically investigated by including key factors such as the thickness of each cavity, the pairs of each reflecting mirror, and the thickness of the benzocyclobutene bonding layer. The device is fabricated by bonding an Si-based multicavity F-P filter with an InP-based p-i-n absorption structure. A hybrid integrated photodetector with a peak quantum efficiency of 55% around 1549.2 nm, the -0.5 dB band of 0.43 nm, the 25 dB band of 1.06 nm, and 3 dB bandwidth more than 16 GHz, is simultaneously obtained. Based on multicavity F-P structure, this device has good flat-top steep-edge spectral response.