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BackgroundIn recent years, the genus Asaia (Rhodospirillales: Acetobacteraceae) has been isolated from different Anopheles species and presented as a promising tool to combat malaria. This bacterium has unique features such as presence in different organs of mosquitoes (midgut, salivary glands and reproductive organs) of female and male mosquitoes and vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for malaria vector control via paratransgenesis technology. Several studies have been performed on the microbiota of Anopheles mosquitoes (Diptera: Culicidae) in Iran and the Middle East to find a suitable candidate for controlling the malaria based on paratransgenesis approaches. The present study is the first report of isolation, biochemical and molecular characterization of the genus Asaia within five different Anopheles species which originated from different zoogeographical zones in the south, east, and north of Iran.MethodsMosquitoes originated from field-collected and laboratory-reared colonies of five Anopheles spp. Adult mosquitoes were anesthetized; their midguts were isolated by dissection, followed by grinding the midgut contents which were then cultured in enrichment broth media and later in CaCO3 agar plates separately. Morphological, biochemical and physiological characterization were carried out after the appearance of colonies. For molecular confirmation, selected colonies were cultured, their DNAs were extracted and PCR was performed on the 16S ribosomal RNA gene using specific newly designed primers.ResultsMorphological, biochemical, physiological and molecular results indicated that all isolates are members of the genus Asaia.ConclusionsContrary to previous opinions, our findings show that Asaia bacteria are present in both insectary-reared colonies and field-collected mosquitoes and can be isolated by simple and specific methods. Furthermore, with respect to the fact that we isolated Asaia within the different Anopheles specimens from distinct climatic and zoogeographical regions, it is promising and may be concluded that species of this genus can tolerate the complicated environmental conditions of the vector-borne diseases endemic regions. Therefore, it can be considered as a promising target in paratransgenesis and vector control programs. However, we suggest that introducing the new technologies such as next generation sequencing and robust in silico approaches may pave the way to find a unique biomarker for rapid and reliable differentiation of the Asaia species.

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Background: Outer membrane vesicles (OMVs) release from Gram-negative bacteria and are interesting alternatives that can replace those vaccines that contain naturally incorporated bacterial surface antigens, lipopolysaccharides (LPS) and outer membrane proteins (OMPs). Nanoparticles can be used to encapsulate vesicles for slow release and prevent macromolecular degradation. Objective: Therefore, encapsulation of OMVs of B. pertussis into sodium alginate nanoparticles was the main aim of the current study. Method: The OMVs of B. pertussis extracted and characterized by particle sizer, electron microscopy, SDSPAGE and Western blot assays. The extracted OMVs were encapsulated in sodium alginate nanoparticles (OMV-NP) using unique gelation process and injected into BALB/c mice. Immunogenicity indices such as different classes of antibodies and interleukins related to different T cell lines were evaluated in immunized mice by ELISA. The respiratory challenge was evaluated in the groups of mice. The existence of pertussis toxin (PTX), filamentous haemagglutinin (FHA) and PRN (pertactin) in B. pertussis OMVs was verified using SDS-PAGE and Western blot analysis. Results: TEM electron microscopy showed the size of these OMVs to be around 20-80 nm. The OMVs with appropriate quality were encapsulated into sodium alginate nanoparticles (OMV-NP), and the final size was about 500 nm after encapsulation. Immunity indices were significantly higher in the OMV-NP receiving group. In challenge tests, the OMV-NP vaccine was able to show the highest rate of lung clearance compared to the control groups (OMV and wPV) at the lowest injection dose. Conclusion: The results indicate the potential of OMV-NP as a novel vaccine delivery system.

A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, prove not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. As regards the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). The coding region of mature Col H gene was optimized according to the codon usage of E. coli using Gene Designer software (DNA 2.0). The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. Similarly, the higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. As has been reported elsewhere, the findings from this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli.In spite of that, experimental research is needed to confirm the improvement.PeerJ PrePrints | http://dx.doi.org/10.7287/peerj.preprints.754v1 | CC-BY 4.0 Open Access |

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A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, prove not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. As regards the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). The coding region of mature Col H gene was optimized according to the codon usage of E. coli using Gene Designer software (DNA 2.0). The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. Similarly, the higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. As has been reported elsewhere, the findings from this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. In spite of that, experimental research is needed to confirm the improvement.