PERI-ANESTHESIA ANAPHYLAXIS (PAA): WE STILL HAVE NOT STARTED POST-PAA TESTING FOR INCITING ANESTHESIA-RELATED ALLERGENS.

Anaphylaxis during anesthesia is uncommon. Diagnosis of peri-anesthesia anaphylaxis (PAA) requires anesthesia providers' vigilance for prompt diagnosis and treatment. In this case report, we present a challenging case with suspected PAA including its perioperative management, intensive care unit (ICU) course, and post-discharge follow-up. A 44-year-old female (body mass index = 26) presented for elective abdominal panniculectomy. Post-intubation, severe bronchospasm occurred that was non-responsive to nebulized albuterol and intravenous epinephrine. Continuous infusion of epinephrine was initiated. After aborting surgical procedure, the patient was transferred to ICU on continuous intravenous infusion of epinephrine. Venous blood sampling showed elevated troponin level. Echocardiography revealed ejection fraction of 25% suspicious of Takotsubo cardiomyopathy (mid cavitary variant). Tracheal extubation was only possible after three days. Subsequently, patient was discharged home with a cardiology follow-up appointment and a referral to an allergy specialist. Unfortunately at our institution (an academic university hospital in United States) along with neighboring institutions in near-by areas, the only allergy skin tests available are for local anesthetics and antibiotics, while neuromuscular blocking agents (NMBAs) cannot be tested (the suspected anaphylactic agent in our case was presumably rocuronium). In summary, PAA requires and responds to emergent diagnosis and immediate treatment; however there is still a long way to go to ensure post-PAA testing for inciting anesthesia-related allergens.

Design and synthesis of pyrazole-oxindole conjugates targeting tubulin polymerization as new anticancer agents.

A series of twenty one compounds with pyrazole and oxindole conjugates were synthesized by Knoevenagel condensation and investigated for their antiproliferative activity on different human cancer cell lines. The conjugates are comprised of a four ring scaffold; the structural isomers 12b and 12c possess chloro-substitution in the D ring. Among the congeners 12b, 12c, and 12d manifested significant cytotoxicity and inhibited tubulin assembly. Treatments with 12b, 12c and 12d resulted in accumulation of cells in G2/M phase, disruption of microtubule network, and increase in cyclin B1 protein. Zebrafish screening revealed that 12b, and 12d caused developmental defects. Docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.

CARM1 regulates astroglial lineage through transcriptional regulation of Nanog and posttranscriptional regulation by miR92a.

Coactivator-associated arginine methyltransferase (CARM1/PRMT4)-mediated transcriptional coactivation and arginine methylation is known to regulate various tissue-specific differentiation events. Although CARM1 is expressed in the neural crest region in early development, coinciding with early neuronal progenitor specification, the role of CARM1 in any neuronal developmental pathways has been unexplored. Using a specific small-molecule inhibitor of CARM1-mediated H3R17 methylation in human embryonic stem cell line, we find that H3R17 methylation contributes to the maintenance of the astroglial cell population. A network of regulation was observed on the miR92a promoter by which H3R17-responsive Nanog bound to the miR92a promoter decreased upon inhibition, resulting in an abnormal gene expression program influencing the glial lineage. This was also true in zebrafish, in which, with the help of CARM1 inhibitor and CARM1 morpholinos, we show that inhibition of H3R17 methylation results in defective glial cell morphology and a sensory defect in a subpopulation. A gain-of-function strategy in which mCARM1 was introduced in the morpholino-treated embryos exhibited recovery of the sensory defect phenotype. This study thus establishes the functional cooperation between arginine methylation and microRNA expression in the neuronal developmental process, with potential implications in sensory development pathways.

Pyrazole-oxadiazole conjugates: synthesis, antiproliferative activity and inhibition of tubulin polymerization.

A number of pyrazole-oxadiazole conjugates were synthesized and evaluated for their ability to function as antiproliferative agents on various human cancer cell lines. These conjugates are comprised of pyrazole and oxadiazole scaffolds closely attached to each other without any spacer as two structural classes. The Type I class has a trimethoxy substituent and the type II class has a 3,4-(methylenedioxy) substituent on their A rings. Among these conjugates 11a, 11d and 11f manifest potent cytotoxicity with IC50 values ranging from 1.5 µM to 11.2 µM and inhibit tubulin polymerization with IC50 values of 1.3 µM, 3.9 µM and 2.4 µM respectively. The cell cycle assay showed that treatment with these conjugates results in accumulation of cells in the G2/M phase and disrupts the microtubule network. Elucidation of zebrafish embryos revealed that the conjugates cause developmental defects. Molecular docking simulations determined the binding modes of these potent conjugates at the colchicine site of tubulin.

Synthesis and biological evaluation of imidazopyridine-oxindole conjugates as microtubule-targeting agents.

A library of imidazopyridine-oxindole conjugates was synthesised and investigated for anticancer activity against various human cancer cell lines. Some of the tested compounds, such as 10 a, 10 e, 10 f, and 10 k, exhibited promising antiproliferative activity with GI50 values ranging from 0.17 to 9.31 µM. Flow cytometric analysis showed that MCF-7 cells treated by these compounds arrested in the G2 /M phase of the cell cycle in a concentration-dependent manner. More particularly, compound 10 f displayed a remarkable inhibitory effect on tubulin polymerisation. All the compounds depolarised mitochondrial membrane potential and caused apoptosis. These results are further supported by the decreased phosphorylation of Akt at Ser473. Studies on embryonic development revealed that the lead compounds 10 f and 10 k caused delay in the development of zebra fish embryos. Docking of compound 10 f with tubulin protein suggested that the imidazo[1,2-a]pyridine moiety occupies the colchicine binding site of tubulin.

Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17.

Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).

Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation.

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.

Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant.

BACKGROUND: Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-kappaB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process. RESULTS: We show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking. CONCLUSIONS: Our results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

NF-kappaB mediates tumor necrosis factor alpha-induced expression of optineurin, a negative regulator of NF-kappaB.

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor kappaB (NF-kappaB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor alpha (TNFalpha). Mutation of a putative NF-kappaB-binding site present in the core promoter resulted in loss of basal as well as TNFalpha-induced activity. Overexpression of p65 subunit of NF-kappaB activated this promoter through NF-kappaB site. Oligonucleotides corresponding to this putative NF-kappaB-binding site showed binding to NF-kappaB. TNFalpha-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-kappaB (IkappaBalpha) super-repressor. Blocking of NF-kappaB activation resulted in inhibition of TNFalpha-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFalpha-induced NF-kappaB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFalpha-induced as well as basal NF-kappaB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-kappaB pathway in response to TNFalpha. In addition these results suggest that there is a negative feedback loop in which TNFalpha-induced NF-kappaB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-kappaB.