Elucidating The Role of MifS-MifR Two-Component System in Regulating Pseudomonas aeruginosa Pathogenicity

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections

Squash Smear Technique in Rapid Intraoperative Diagnosis of Brain Tumors

The role of rapid intraoperative diagnosis is crucial to neurosurgeons to not only define surgical approach, but also to ensure that minimum injury is caused to normal structures surrounding the lesion. Intraoperative cytology has been shown to be an important diagnostic modality. Squash smears are gaining popularity with increasing use of stereotactic biopsies which give limited tissue yield. The study was designed to assess the accuracy of intraoperative squash smear in the rapid intraoperative diagnosis of brain tumors. 100 consecutive cases of CNS tumors in which the tissue was removed at craniotomy or burr-hole biopsy were studied. The age range of the cases varied from 1 to 80 years. The diagnostic accuracy of squash smears was found to be 91%.In all the cases, clinicoradiological correlation was done with smear diagnosis. Smears were stained with 1% toluidine blue/H&E. In all the cases, results were compared with the paraffin section prepared from tissue remaining after the squash smears were made. Immunohistochemistry/Immunocytochemistry wasdone as needed

N-alkylamides : from plant to brain

Background: Plant N-alkylamides (NAAs) are bio-active compounds with a broad functional spectrum. In order to reach their pharmacodynamic targets, they have to overcome several barriers of the body in the absorption phase. The permeability kinetics of spilanthol (a diene NAA) and pellitorine (a triene NAA) across these barriers (i.e. skin, oral/gut mucosa, blood-brain barrier) were investigated. Methods: The skin and oral mucosa permeability were investigated using human skin and pig mucosa in an ex vivo in vitro Franz diffusion cell set-up. The gut absorption characteristics were examined using the in vitro Caco-2 cell monolayer test system. The initial blood-brain barrier transport kinetics were investigated in an in vivo mice model using multiple time regression and efflux experiments. Quantification of both NAAs was conducted using HPLC-UV and bio-analytical UPLC-MS methods. Results: We demonstrated that spilanthol and pellitorine are able to penetrate the skin after topical administration. It is likely that spilanthol and pellitorine can pass the endothelial gut as they easily pass the Caco-2 cells in the monolayer model. It has been shown that spilanthol also crosses the oral mucosa as well as the blood-brain barrier. Conclusion: It was demonstrated that NAAs pass various physiological barriers i.e. the skin, oral and gut mucosa, and after having reached the systemic circulation, also the blood-brain barrier. As such, NAAs are cosmenutriceuticals which can be active in the brain

Squash Smear Technique in Rapid Intraoperative Diagnosis of Non-neoplastic and Cystic Lesions of CNS

In neurosurgical practice, a rapid intraoperative diagnosis helps the neurosurgeon to monitor and modify the approach at surgery. Smears and frozen sections are the two rapid tissue preparations that may be used by neuropathologists for giving opinion on intraoperative biopsy specimens of suspected lesions. The smear technique, which could be either squash/crush smears or impression smears, has been applied in neurosurgical units worldwide. This method plays a very important role in the analysis of sample from craniotomies and the small specimens obtained from stereotactic biopsies.Squash smear technique is a very rapid technique and the study was designed to assess the accuracy of squash smear in the rapid intraoperative diagnosis of non-neoplastic and cystic lesions of CNS and to document the cytomorphology of these lesions. A total of 23 cases were studied. The tissue was removedat craniotomy or by burr hole biopsy.In all the cases clinicoradiological correlation was done with smear diagnosis. Smears were stained with 1% Toluidine blue and H&E. In all cases, results were compared with the paraffin section prepared from the tissue remaining after the squash smears. Immunohistochemistry was done in one case. Special stainswere used as required.Amongst non-neoplastic lesions, tuberculous lesions comprised the maximum number of cases (n=9), two cases of PML, one case of fungal lesion, one case of non-specific abscess, one case of infarct, only reactive changes in one and normal cortex and white matter in one. In cystic lesions of the CNS, there was one case of Rathke’s Cleft cyst, four cases of Epidermoid cyst and two cases of arachnoid cyst. Cases of PML and Rathke’s cleft cyst could not be identified on smears. In all the cases, smear diagnosis was compared with histopathological diagnosis

Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for alpha-Ketoglutarate Utilization

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (sigma(54)). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a beta-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO Delta mifS, PAO Delta mifR and PAO Delta mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO Delta mifS and PAO Delta mifR revealed that these mutants were unable to utilize C-5-dicarboxylate alpha-ketoglutarate (alpha-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except alpha-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in alpha-KG transport. The inability of the mutants to utilize alpha-KG was rescued by expressing PA5530, encoding C-5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, alpha-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing a-KG and regulating its transport and subsequentmetabolism

Melt-cast films significantly enhance triamcinolone acetonide delivery to the deeper ocular tissues

© 2019 The Author(s). Background: Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies. Methods: We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors. Results: In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8 + T cells). Conclusions: This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction

EVALUATION OF PUNICA GRANATUM FRUIT PEELS EXTRACTS FOR ITS FREE RADICAL SCAVENGING AND ANTI-INFLAMMATORY ACTIVITY

Objective: To evaluate free radical scavenging and anti-inflammatory activity of petroleum ether, ethyl acetate, aqueous, methanol:water and methanol extracts of Punica granatum fruit peels (PGFP) (Family: Lythraceae) by in vitro methods.Methods: The free radical scavenging effect was studied using 1,1â€Diphenylâ€2â€Picrylhydrazyl (DPPH) and nitric oxide radical scavenging assay. Antiâ€inflammatory activity was evaluated by HRBC membrane stabilization assay.Results: All the extracts of PGFP exhibited significant free radical scavenging effect. The methanol extract exhibited maximum significant DPPH and nitric oxide radical scavenging activity with IC50 value of 24.43 and 45.56µg/ml and maximum stabilization (86.96%) of HRBC membrane at 80 µg/ml among all the extracts of PGFP.Conclusion: Methanol as an extraction solvent was found to be the best in obtaining the extract of PGFP rich in radical scavenging and anti-inflammatory phytoconsituents.Â

Quantitative in vitro and in vivo evaluation of intestinal and blood-brain barrier transport kinetics of the plant N-alkylamide pellitorine

Objective. To evaluate the gut mucosa and blood-brain barrier (BBB) pharmacokinetic permeability properties of the plant N-alkylamide pellitorine. Methods. Pure pellitorine and an Anacyclus pyrethrum extract were used to investigate the permeation of pellitorine through (1) a Caco-2 cell monolayer, (2) the rat gut after oral administration, and (3) the BBB in mice after intravenous and intracerebroventricular administration. A validated bioanalytical UPLC-MS2 method was used to quantify pellitorine. Results. Pellitorine was able to cross the Caco-2 cell monolayer from the apical-to-basolateral and from the basolateral-to-apical side with apparent permeability coefficients between 0.6 center dot 10(-5) and 4.8 center dot 10(-5) cm/h and between 0.3 center dot 10(-5) and 5.8 center dot 10(-5) cm/h, respectively. In rats, a serum elimination rate constant of 0.3 h(-1) was obtained. Intravenous injection of pellitorine in mice resulted in a rapid and high permeation of pellitorine through the BBB with a unidirectional influx rate constant of 153 mu L/(g center dot min). In particular, 97% of pellitorine reached the brain tissue, while only 3% remained in the brain capillaries. An efflux transfer constant of 0.05 min(-1) was obtained. Conclusion. Pellitorine shows a good gut permeation and rapidly permeates the BBB once in the blood, indicating a possible role in the treatment of central nervous system diseases