Development of an efficient insecticide substrate and inhibitor screening system of insect P450s using fission yeast.

Metabolic resistance is one of the most frequent mechanisms of insecticide resistance, characterized by an increased expression of several important enzymes and transporters, especially cytochrome P450s (CYPs). Due to the large number of P450s in pests, determining the precise relationship between these enzymes and the insecticide substrates is a challenge. Herein, we developed a luminescence-based screening system for efficient identification of insecticide substrates and insect P450 inhibitors. We recombinantly expressed Bemisia tabaci CYP6CM1vQ (Bt CYP6CM1vQ) in the fission yeast Schizosaccharomyces pombe and subsequently permeabilized the yeast cells to convert them into "enzyme bags". We exploited these enzyme bags to screen the activity of twelve luciferin substrates and identified Luciferin-FEE as the optimal competing probe that was further used to characterize the metabolism of eight candidate commercial insecticides. Among them, Bt CYP6CM1vQ exhibited notable activity against pymetrozine and imidacloprid. Their binding modes were predicted by homology modeling and molecular docking, revealing the mechanisms of the metabolism. We also tested the inhibitory effect of eight known P450 inhibitors using our system and identified letrozole and 1-benzylimidazole as showing significant activity against Bt CYP6CM1vQ, with IC50 values of 23.74 µM and 1.30 µM, respectively. Their potential to be developed as an insecticide synergist was further proven by an in vitro toxicity assay using imidacloprid-resistant Bemisia tabaci. Overall, our luciferin-based enzyme bag method is capable of providing a robust and efficient screening of insect P450 substrates and, more importantly, inhibitors to overcome the resistance.

Mutual Influence of Human Cytochrome P450 Enzymes and UDP-Glucuronosyltransferases on Their Respective Activities in Recombinant Fission Yeast.

Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are the most important human drug metabolizing enzymes, but their mutual interactions are poorly understood. In this study, we recombinantly co-expressed of each one of the 19 human members of the UGT families 1 and 2 with either CYP2C9, CYP2D6, or CYP4Z1 in fission yeast. Using these strains, we monitored a total of 72 interactions: 57 cases where we tested the influence of UGT co-expression on CYP activity and 15 cases of the opposite approach. In the majority of cases (88%), UGT co-expression had a statistically significant (p < 0.05) effect on P450 activity (58% positive and 30% negative). Strong changes were observed in nine cases, including one case with an activity increase by a factor of 23 (CYP2C9 activity in the presence of UGT2A3) but also four cases with a complete loss of activity. When monitoring the effect of CYP co-expression on the activity of five UGTs, activity changes were generally not so pronounced and, if observed, always detrimental. UGT2B7 activity was not influenced by CYP co-expression, while the other UGTs were affected to varying degrees. These data suggest the notion that mutual influence of CYPs and UGTs on each other's activity is a widespread phenomenon.

Exploring the Chemical Space of Proluciferins as Probe Substrates for Human Cytochrome P450 Enzymes.

We report the synthesis of 21 new proluciferin compounds that bear a small aliphatic ether group connected to the 6' hydroxy function of firefly luciferin and either contain an acid or methyl ester function at the dihydrothiazole ring. Each of these compounds was found to be a substrate for some members of the human CYP1 and CYP3 families; a total of 92 new enzyme-substrate pairs were identified. In a screen of the whole human P450 complement (CYPome) with three selected proluciferin acid substrates, another 13 enzyme-substrate pairs were detected, which involve enzymes belonging to the CYP2, CYP4, CYP7, CYP21, and CYP27 families. All in all, we identified new probe substrates for members of seven out of 18 human CYP families.

A convenient test system for the identification of CYP4V2 inhibitors.

Purpose: Polymorphisms in the gene that codes for the human cytochrome P450 enzyme CYP4V2 are a cause of Bietti crystalline dystrophy (BCD). Therefore, inhibition of CYP4V2 activity may well be a cause of visual disability. However, monitoring the fatty acid hydroxylation reactions catalyzed by this enzyme is tedious and not well suited for inhibitor screening. Methods: We investigated the use of proluciferin compounds as probe substrates for efficient and convenient determination of CYP4V2 activity. Results: Ten proluciferins were tested for conversion by CYP4V2, and eight were found to be substrates of this enzyme. One point inhibitor assays were performed using luciferin 6' 3-furfuryl ether methyl ester (luciferin-3FEME) as the probe substrate and 12 test compounds. As expected, HET0016 had by far the strongest effect, while two other compounds (including osilodrostat) also displayed statistically significant inhibitory potency. The half maximal inhibitory concentration (IC50) for HET0016 was determined to be 179 nM. A recently identified potent inhibitor of human CYP4Z1 was found not to inhibit CYP4V2. To explore the selectivity of this compound between CYP4Z1 and CYP4V2, we developed a homology model of CYP4V2 and conducted docking experiments. Conclusions: We provide the first protocol for a robust and convenient CYP4V2 inhibitor assay that does not depend on fatty acid analysis but can be simply monitored with luminescence. Moreover, we demonstrate additional evidence for the concern that compounds with CYP-inhibitory properties may inhibit CYP4V2 activity and thus, possibly cause visual disability.

Evaluation of Inflammatory Acute Phase Protein Level and Different Leukocyte Counts in Chronic Periodontitis Normolipidemic Patients after Nonsurgical Periodontal Therapy.

AIM AND OBJECTIVE: To evaluate the effect of nonsurgical periodontal therapy on periodontal parameters, serum C-reactive protein (CRP) level, total leukocyte count (TLC), and differential leukocyte count (DLC) in normolipidemic patients with generalized chronic periodontitis. MATERIALS AND METHODS: A total of 60 subjects (38 males and 22 females) between 20 and 55 years of age were included in this study. Twenty subjects with generalized chronic gingivitis were assigned group I. Forty subjects with generalized chronic periodontitis were randomly divided into test groups, i.e., group II (n = 20) and control group, i.e., group III (n = 20). At baseline, clinical parameters (plaque and gingival indices, clinical attachment loss) were recorded and blood collected for lipid profile test, TLC, DLC, and CRP estimation. Patients with lipid values in the normal range continued the study. Groups I and II were provided nonsurgical periodontal therapy. Follow-up clinical examination and blood examination were done for CRP level, TLC, and DLC after 1 and 2 months. RESULTS: A significant improvement in the clinical parameters was evident following scaling and root planning in group II as compared to group III. A decrease in serum CRP and TLC count was also observed, but the difference was not significant. Moreover, a reduction was observed in neutrophils, monocytes, eosinophils post therapeutically in group II but the decrease was significant only for monocyte count. CONCLUSION: Based on the findings of the study, it can be concluded that nonsurgical periodontal therapy can reduce the inflammatory component. CLINICAL SIGNIFICANCE: Periodontal diseases comprise a wide range of inflammatory conditions affecting the supporting structures of teeth. Effect of nonsurgical periodontal therapy on chronic periodontitis can be evaluated by measuring the CRP and leukocyte concentration.

Conversion of five proluciferin esters by human cytochrome P450 enzymes.

BACKGROUND: Probe substrates are an important tool for activity monitoring of human drug metabolizing enzymes such as cytochromes P450 (CYPs). BRIEF METHODS: In the present study we have tested human CYPs for metabolization of five proluciferin ester substrates which had previously only been known to be hydroxylated by CYP26A1. MAJOR RESULTS: It was found that these substrates were converted by another 21 human CYPs, which belong to the CYP families 1 to 4, 7, and 26. Thus, 66 new pairs of enzyme and substrate were identified. Correlation analysis indicated the presence of three distinct sets of enzymes with high similarity in their activity profiles that encompass a total of 16 individual enzymes. CONCLUSIONS: Some of these newly identified correlations may serve as a starting point for further study of those human CYPs whose activities are not yet satisfactorily understood.

Discovery of a novel potent cytochrome P450 CYP4Z1 inhibitor.

Human cytochrome P450 enzyme CYP4Z1 represents a promising target for the treatment of a multitude of malignancies including breast cancer. The most active known non-covalent inhibitor (1-benzylimidazole) only shows low micromolar affinity to CYP4Z1. We report a new, highly active inhibitor for CYP4Z1 showing confirmed binding in an enzymatic assay and an IC50 value of 63 ± 19 nM in stably transfected MCF-7 cells overexpressing CYP4Z1. The new inhibitor was identified by a systematically developed virtual screening protocol. Binding was rationalized using a carefully elaborated 3D pharmacophore hypothesis and thoroughly characterized using extensive molecular dynamics simulations and dynamic 3D pharmacophore (dynophore) analyses. This novel inhibitor represents a valuable pharmacological tool to accelerate characterization of the still understudied CYP4Z1 and might pave the way for a new treatment strategy in CYP4Z1-associated malignancies. The presented in silico model for predicting CYP4Z1 interaction provides novel mechanistic insights and revealed that the drug ozagrel interacts with CYP4Z1.

New luciferin-based probe substrates for human CYP26A1.

Activity of human CYP26A1 towards six proluciferin probe substrates and their ester derivatives was monitored. These included three monofluorobenzyl ether isomers and three five-membered heterocycles. Overall, luciferin substrates with a free acid group gave higher activities than the ester compounds. Also, luciferin derivatives with six-ring structures were better metabolized than those with five-rings. The best substrates identified in this study are Luciferin 6' 3-fluorobenzyl ether (Luciferin-3FBE) and its methyl ester (Luciferin-3FBEME). Taken together, we describe eleven new probe substrates for CYP26A1 and demonstrate for the first time that CYP26A1 does not only accept acid substrates but can also metabolize esters.

Assessment of patient and provider attitudes towards therapeutic drug monitoring to improve medication adherence in low-income patients with hypertension: a qualitative study.

OBJECTIVES: Previous studies have implicated therapeutic drug monitoring (TDM), by measuring serum or urine drug levels, as a highly reliable technique for detecting medication non-adherence but the attitudes of patients and physicians toward TDM have not been evaluated previously. Accordingly, we solicited input from patients with uncontrolled hypertension and their physicians about their views on TDM. DESIGN: Prospective analysis of responses to a set of questions during semistructured interviews. SETTING: Outpatient clinics in an integrated health system which provides care for a low-income, uninsured population. PARTICIPANTS: Patients with uncontrolled hypertension with either systolic blood pressure of at least 130 mm Hg or diastolic blood pressure of at least 80 mm Hg despite antihypertensive drugs and providers in the general cardiology and internal medicine clinics. PRIMARY AND SECONDARY OUTCOME MEASURES: Attitudes towards TDM and the potential impact on physician-patient relationship. RESULTS: We interviewed 11 patients and 10 providers and discussed the findings with 13 community advisory panel (CAP) members. Of the patients interviewed, 91% (10 of 11) and all 10 providers thought TDM was a good idea and should be used regularly to better understand the reasons for poorly controlled hypertension. However, 63% (7 of 11) of patients and 20% of providers expressed reservations that TDM could negatively impact the physician-patient relationship. Despite some concerns, the majority of patients, providers and CAP members believed that if test results are communicated without blaming patients, the potential benefits of TDM in identifying suboptimal adherence and eliciting barriers to adherence outweighed the risks. CONCLUSION: The idea of TDM is well accepted by patients and their providers. TDM information if delivered in a non-judgmental manner, to encourage an honest conversation between patients and physicians, has the potential to reduce patient-physician communication obstacles and to identify barriers to adherence which, when overcome, can improve health outcomes.

Effect of Intracanal Medicaments (Modified Triple Antibiotic Paste, Calcium Hydroxide, and Aloe Vera) on Microhardness of Root Dentine: An In Vitro Study.

AIM: To compare the effect of three different intracanal medicaments, namely, modified triple antibiotic paste (MTAP), calcium hydroxide (Ca(OH)2), and aloe vera, on the root dentine microhardness. MATERIALS AND METHODS: A total of 50 extracted mandibular bicuspids were prepared using ProTaper Next rotary files. The roots of the bicuspids were alienated to three groups (n = 10 each) and one control group (untreated; n = 20). In three groups, the root canals were filled with MTAP, Ca(OH)2, and aloe vera medicaments. After 21 days, medicaments were removed by Endo activator. Mean Knoop hardness numbers were calculated after treatment and compared with the untreated control group. Data were evaluated using the Student's t test (paired), ANOVA (one-way) followed, and the post hoc test. RESULTS: All treated groups except the aloe vera group had shown significant reduction (p < 0.05) in microhardness of the root dentin as compared with the untreated control group. The aloe vera group showed least reduction of microhardness and was statistically insignificant (p > 0.05). CONCLUSION: Aloe vera shows promising results in terms of fewer effects on microhardness of the root dentin compared to MTAP and Ca(OH)2. CLINICAL SIGNIFICANCE: Elimination of most of the bacterial infection from the root canal and very minimum to no effect on the microhardness of the dentin in the root part are the basics of success in any endodontic treatment. Further in vivo studies are required to compare the efficacy of these intracanal medicaments.

Evaluation of Physicochemical and Cooking Characteristics of Rice (Oryza sativa L.) Landraces of Lamjung and Tanahun Districts, Nepal.

After the Green Revolution, the increase in the choice of modern varieties at the expense of landraces has become a major cause of varietal loss. The preference, choice, and the economy of rice (Oryza sativa L.) largely depend on its physicochemical and cooking properties, which are found to be superior for landraces than modern varieties. In this study, we assessed and evaluated milled rice of 30 rice landraces on their physicochemical and cooking characteristics which aim to promote the revival of old landraces. Six parameters of physical properties, four parameters of chemical properties, and five parameters of cooking properties were evaluated based on the standard protocols. Significant variations (p < 0.05) were found in all the properties that were evaluated. The result showed that the highest milling recovery was found in Indrabeli (75.55%) whereas the lowest was found in Kalo Masino (66.98%) and bulk density ranged from 0.81 g/cm3 to 0.88 g/cm3 showing not much variability. Although most of them were of medium grain type, their 1000 kernel weight varied between 12.62 g and 25.65 g. From the observed chemical properties, Pahelo Anadi (9.73 ± 0.55 mm) showed the highest gel consistency and lowest apparent amylose content (7.23 ± 0.36%). Also, 13% of landraces possessed strong aroma while noble cooking properties were showed by Thakali Lahare Marsi with the highest elongation ratio (2.41 ± 0.05) and by Chiniya with the lowest gruel solid loss (0.033 ± 0.03%) and minimum optimum cooking time (23.45 ± 0.03 min). In the principal component analysis, the first four principal components retained 73.8% of the variance. The first and second principal components were mostly related with the physical and chemical characteristics while the third and fourth principal components were concerned with cooking characters. Superior characters possessed by rice landraces can be further assessed for the breeding programs so that the cultivation of these cherished rice landraces can be enhanced.

Rapid and convenient biotransformation procedure for human drug metabolizing enzymes using permeabilized fission yeast cells.

The development of convenient assays for the in vitro study of drug metabolizing enzymes (DMEs) such as cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) greatly facilitates metabolism studies of candidate drug compounds and other xenobiotics. We have developed and optimized an experimental approach that combines the advantages of recombinant expression in yeast with a microsomal-like biotransformation and thus allows for rapid and convenient enzymatic assays. Recombinant strains of the fission yeast Schizosaccharomyces pombe have previously been demonstrated to functionally express human CYPs and UGTs. Permeabilization of such cells with Triton X-100 results in the formation of enzyme bags, which can be used as biocatalysts. This protocol describes the preparation of such enzyme bags (3 h) and their application in enzyme activity assays (4 h) utilizing either pro-luminescent substrates and luminescence measurements or non-luminescent substrates and liquid chromatography coupled to mass spectrometry (LC-MS). Both applications provide practical tools for investigating CYP and UGT reactions in vitro without the need for additional sophisticated instrumentation or expertise.

Screening of the whole human cytochrome P450 complement (CYPome) with enzyme bag cocktails.

We have previously introduced the use of permeabilized fission yeast cells (enzyme bags) that recombinantly express full-length CYPs for drug metabolism studies. Such enzyme bags are cells with pores that function as enzymes in situ. They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes. In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction. Moreover, we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity towards any given candidate substrate. An important aspect of this approach is the reduction of individual CYP test assays. If a cocktail containing many CYPs tests negative, it follows that all CYPs included in that cocktail need not be tested individually, thus saving time and resources. The new protocol was validated using two probe substrates.

Screening of the whole human cytochrome P450 complement (CYPome) with enzyme bag cocktails / 药物分析学报

We have previously introduced the use of permeabilized fission yeast cells (enzyme bags) that recom-binantly express full-length CYPs for drug metabolism studies. Such enzyme bags are cells with pores that function as enzymes in situ. They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes. In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction. Moreover, we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity to-wards any given candidate substrate. An important aspect of this approach is the reduction of individual CYP test assays. If a cocktail containing many CYPs tests negative, it follows that all CYPs included in that cocktail need not be tested individually, thus saving time and resources. The new protocol was validated using two probe substrates.

Arrhythmias and Adaptations of the Cardiac Conduction System in Former National Football League Players.

Background Habitual high-intensity endurance exercise is associated with increased atrial fibrillation (AF) risk and impaired cardiac conduction. It is unknown whether these observations extend to prior strength-type sports exposure. The primary aim of this study was to compare AF prevalence in former National Football League (NFL) athletes to population-based controls. The secondary aim was to characterize other conduction system parameters. Methods and Results This cross-sectional study compared former NFL athletes (n=460, age 56±12 years, black 47%) with population-based controls of similar age and racial composition from the cardiovascular cohort Dallas Heart Study-2 (n=925, age 54±9 years, black 53%). AF was present in 28 individuals (n=23 [5%] in the NFL group; n=5 [0.5%] in the control group). After controlling for other cardiovascular risk factors in multivariable regression analysis, former NFL participation remained associated with a 5.7 (95% CI: 2.1-15.9, P<0.001) higher odds ratio of AF. Older age, higher body mass index, and nonblack race were also independently associated with higher odds ratio of AF, while hypertension and diabetes mellitus were not. AF was previously undiagnosed in 15/23 of the former NFL players. Previously undiagnosed NFL players were rate controlled and asymptomatic, but 80% had a CHA2DS2-VASc score ≥1. Former NFL players also had an 8-fold higher prevalence of paced cardiac rhythms (2.0% versus 0.25%, P<0.01), compared with controls. Furthermore, former athletes had lower resting heart rates (62±11 versus 66±11 beats per minute, P<0.001), and a higher prevalence of first-degree atrioventricular block (18% versus 9%, P<0.001). Conclusions Former NFL participation was associated with an increased AF prevalence and slowed cardiac conduction when compared with a population-based control group. Former NFL athletes who screened positive for AF were generally rate controlled and asymptomatic, but 80% should have been considered for anticoagulation based on their stroke risk.

Functional expression and activity screening of all human cytochrome P450 enzymes in fission yeast.

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

Dialytic interval and the timing of electrocardiographic screening for subcutaneous cardioverter-defibrillator placement in chronic hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients have a high risk of sudden death but limited vascular access and high complication rates from transvenous implantable cardioverter-defibrillators (ICDs). Subcutaneous ICDs (S-ICD) may be an alternative, but dynamic ECG changes may result in inappropriate shocks. This study aims to define the screen failure rate for S-ICD in patients pre- and post-HD. METHODS: ECG waveforms were obtained using electrodes mimicking the S-ICD sensing vectors in an unselected test group of chronic HD patients and a control group of ICD-eligible non-dialysis patients. Participants passed screening if their QRS and T-waves fit within the screening template in supine and standing positions in any lead. Test group participants were screened before and after HD and control group patients were screened at two separate time points. HD patients were stratified into the four following groups: (A) passed screening before and after HD, (B) failed screening before but passed after HD, (C) passed screening before but failed after HD, and (D) failed screening before and after HD. Patients in group A passed the screening for ICD implantation, and patients in groups B, C, and D failed the screening for ICD implantation. Control patients were similarly classified by pass/fail status at the two assessment points. RESULTS: Of the 76 patients enrolled, 51 were HD patients and 25 were controls. Of the 51 HD patients, 43 (84%) were in group A, four participants (8%) were in group B, one (2%) was in group C, and three participants (6%) were in group D. There were no differences in any of the clinical or demographic variables between the pass and fail test HD groups. None of the 25 controls failed the screening at either time point (p = 0.047 vs HD patients). CONCLUSIONS: Overall, HD patients were more likely to fail S-ICD screening compared to non-HD patients (16 v 0%, p = 0.047) and are more likely to do so prior to HD. Patients on HD should be screened at multiple time points around the dialytic interval to reduce the risk of inappropriate shocks.

Longitudinal associations between adiponectin and cardiac structure differ by hypertensive status: Coronary Artery Risk Development in Young Adults.

OBJECTIVE: We studied the longitudinal association between adiponectin and cardiac structure and function 10 years later stratified by hypertension status. METHODS: Multicenter longitudinal study of black and white men and women that began in 1985-1986, when participants were 18-30 years old. Adiponectin was measured at year 15(2000-2001). Echocardiograms were completed at year 25(2010-2011). Participants were stratified by the presence of hypertension. Risk factor-adjusted echocardiographic variables were compared across adiponectin quintiles. Linear and quadratic regression models were also derived for risk factor-adjusted echocardiographic variables. RESULTS: Relative to the lowest quintile of adiponectin, participants from the highest quintile had a 6% lower LV mass index (LVMi) among normotensives, and an 8% higher LVMi among hypertensives. Among normotensive participants, regression analysis demonstrated a linear inverse relationship between adiponectin and LV mass, LVMi, posterior wall thickness (PWT) and ventricular septal thickness (VST) (all p≤0.05). Among hypertensive participants, regression analysis demonstrated a U-shaped relationship between adiponectin and LV mass, LVMi, PWT and VST (p≤0.005 for all quadratic terms). CONCLUSIONS: Among normotensive participants, higher adiponectin may be a useful marker of less adverse future cardiac structure. Further study is required to see if adiponectin receptor agonists may provide a benefit among these individuals. Among hypertensive participants, further study is required to assess the prognostic and therapeutic use of adiponectin.

Does Hamulotomy during Palatoplasty Have Any Effect on Hearing Ability in Nonsyndromic Cleft Palate Patients? A Prospective, Single Blind, Comparative Study.

The primary goal of palatoplasty is to achieve a tension-free palatal closure ensuring no postoperative complications. Many surgeons fracture the pterygoid hamulus to minimize tension during palatoplasty. However, this maneuver gained criticism by some authors on the grounds that it may lead to Eustachian Tube dysfunction. Our study intended to figure out the relationship of hamulus fracture with the postoperative state of middle ear in cleft palate children. Fifty consecutive cleft palate patients with an age range of 10 months to 5 years were recruited. All the patients were assigned to either hamulotomy or nonhamulotomy group preoperatively. The patients were subjected to otoscopic examination and auditory function evaluation by brainstem evoked response audiometry (BERA) preoperatively and 1 month and 6 months postoperatively. Otoscopy revealed that the difference in the improvement of middle ear status in both groups was statistically insignificant. Moreover, there was no significant difference in the BERA outcomes of the fracture and nonfracture populations. Complication rate in both groups was also statistically not significant. It can be concluded that hamulotomy does not have any effect on the hearing ability in cleft palate population, so hamulotomy can be performed for tension-free closure during palatoplasty.

Negative feedback circuit for toll like receptor-8 activation in human embryonic Kidney 293 using outer membrane vesicle delivered bi-specific siRNA.

BACKGROUND: TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/ß 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 INFß 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 INFß. 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.