title: Negative feedback circuit for toll like receptor-8 activation in human embryonic Kidney 293 using outer membrane vesicle delivered bi-specific siRNA.
abstract: BACKGROUND
  TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/β molecule poses threat by developing tolerance in chronic infection cases and enhancing inflammatory response. Here we report a bi-specific siRNA based complex which differentially activates and silences the TLR8 and MYD88 respectively in a negatively regulated fashion.
  
  
  RESULTS
  Outer membrane vesicle from Escherichia coli used for siRNA delivery was observed more efficient when attached with invasive protein Ail along with OmpA (P<0.001) in HEK293-TLR8 cell line. siRNA complexed with p19 protein was efficient in activating TLR8, confirmed by the increment of INFβ molecules (P<0.001) in HEK293-TLR8 compared to its counterpart. Fusion of lipid bilayer of endosomal compartment was significant at pH 4.5 when fusogenic peptides (diINF-7) were incubated in membrane vesicle, thus facilitating the escape of siRNA complex to the host cytoplasm in order to silence MyD88 transcript (P<0.001).
  
  
  CONCLUSIONS
  We investigated the activation of TLR8 by bi-specific si-RNA for the production of INFβ. In the same setting we showed that bi-specific si-RNA was able to silence MyD88 transcript in a delayed manner. For the cases of auto immune disease and inflammation where over activation of endosomal TLRs poses serious threat, bi specific siRNA could be used as negative feedback controlled system.
authors.affiliation: Asian Institute of Technology & Management, Purbanchal University, Knowledge village, Khumaltar, Satdobato, Lalitpur, Nepal. adhikari_anurag13@yahoo.com.
authors.forename: Anurag
authors.initials: A
authors.lastname: Adhikari
authors.affiliation: Central Department of Biotechnology, Tribhuvan University, Kathmandu, Nepal. guptabirendra10@gmail.com.
authors.forename: Birendra Prasad
authors.initials: BP
authors.lastname: Gupta
authors.affiliation: Central Department of Biotechnology, Tribhuvan University, Kathmandu, Nepal. krishnamanandhar@gmail.com.
authors.forename: Krishna
authors.initials: K
authors.lastname: Das Manandhar
authors.affiliation: National Public Health Laboratory, Teku, Kathmandu, Nepal. mishrashravan75@gmail.com.
authors.forename: Shravan Kumar
authors.initials: SK
authors.lastname: Mishra
authors.affiliation: Asian Institute of Technology & Management, Purbanchal University, Knowledge village, Khumaltar, Satdobato, Lalitpur, Nepal. harisainju959@gmail.com.
authors.forename: Hari Krishna
authors.initials: HK
authors.lastname: Saiju
authors.affiliation: Padma Kanya Multiple Campus, Tribhuvan University, Kathmandu, Nepal. shresthar641@gmail.com.
authors.forename: Rajendra Maan
authors.initials: RM
authors.lastname: Shrestha
authors.affiliation: South Asian University, New Delhi, India. nawneetmishra6@gmail.com.
authors.forename: Nawneet
authors.initials: N
authors.lastname: Mishra
authors.affiliation: Asian Institute of Technology & Management, Purbanchal University, Knowledge village, Khumaltar, Satdobato, Lalitpur, Nepal. shishirsharma618@gmail.com.
authors.forename: Shishir
authors.initials: S
authors.lastname: Sharma
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D016923:Cell Death; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D025461:Feedback, Physiological; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016899:Interferon-beta / Q000378:metabolism; D008024:Ligands; D008561:Membrane Fusion; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism*; D051200:Toll-Like Receptor 8 / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D037521:Virulence Factors / Q000378:metabolism
chemical_list: C526543:Ail protein, Yersinia pestis; D001425:Bacterial Outer Membrane Proteins; D008024:Ligands; D053594:Myeloid Differentiation Factor 88; D034741:RNA, Small Interfering; C495349:TLR8 protein, human; D051200:Toll-Like Receptor 8; D053835:Unilamellar Liposomes; D014764:Viral Proteins; D037521:Virulence Factors; D016899:Interferon-beta