Local Anesthetic Systemic Toxicity in Cardiac Surgery Patient with Erector Spinae Plane Catheter: A Case Report.

Regional anesthesia nerve blocks are increasingly used for patients undergoing cardiac surgery as part of multimodal pain management. Though rare, local anesthetic systemic toxicity (LAST) is a severe complication that requires vigilant monitoring. We present a case of a postcardiac surgery patient who developed LAST multiple days after surgery from lidocaine via an erector spinae plane catheter. This episode was determined to be a result of impaired lidocaine metabolism from liver shock caused by worsening pulmonary hypertension. Even under continuous monitoring, patients with cardiac or liver dysfunction are at increased risk of complications from local anesthetics.

Isolation of midgut escape mutants of two American genotype dengue 2 viruses from Aedes aegypti.

BACKGROUND: Several studies have shown that American genotype dengue 2 viruses (DENV2) have reduced viral fitness in the mosquito vector, Aedes aegypti, compared to other DENV2 genotypes. Diminished replication efficiency or inability to efficiently traverse membrane barriers encompassing organs such as the midgut or salivary glands are considered major factors negatively impacting viral fitness in the mosquito. RESULTS: We analyzed the vector competence of Ae. aegypti for two American DENV2 strains, QR94 and PR159 originating from Mexico and Puerto-Rico, respectively. Both strains infected mosquito midguts following acquisition of infectious bloodmeals. However, DENV2-QR94 and DENV2-PR159 poorly disseminated from the midgut at 7 or 14 days post-bloodmeal (pbm). We detected one virus isolate, EM33, among 31 DENV2-QR94 infected mosquitoes, and one isolate, EM41, among 121 DENV2-PR159 infected mosquitoes, generating high virus titers in mosquito carcasses at 7 days pbm. In oral challenge experiments, EM33 and EM41 showed midgut dissemination rates of 40-50%. Replication efficiency of EM41 in secondary mosquito tissue was similar to that of a dissemination-competent control strain, whereas the replication efficiency of EM33 was significantly lower than that of the control virus. The genome sequence of DENV2-QR94 encoded seven unique amino acids (aa), which were not found in 100 of the most closely related DENV2 strains. EM33 had one additional aa change, E202K, in the E protein. DENV2-PR159 encoded four unique aa residues, one of them E202K, whereas EM41 had two additional aa substitutions, Q77E in the E protein and E93D in NS3. CONCLUSIONS: Our results indicate that the midgut of Ae. aegypti acts as a selective sieve for DENV2 in which genetically distinct, dissemination-competent virus variants are rapidly selected from the viral quasispecies to be transmitted to vertebrates.

Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress.

Type 2 diabetes mellitus (T2DM) results from pancreatic beta cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the beta cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany beta cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory beta cell mass expansion in response to diet-induced insulin resistance through its roles in promoting beta cell survival and compensatory hypertrophy. Pdx1-deficient beta cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances beta cell susceptibility to ER stress-associated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of beta cell compensation for insulin resistance at least in part by impairing critical functions of the ER.

The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti.

BACKGROUND: The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity. RESULTS: As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced approximately 50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes. CONCLUSIONS: We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.

Interspecific transfer of Wolbachia into the mosquito disease vector Aedes albopictus.

Intracellular Wolbachia bacteria are obligate, maternally inherited endosymbionts found frequently in insects and other invertebrates. The evolutionary success of Wolbachia is due in part to an ability to manipulate reproduction. In mosquitoes and many other insects, Wolbachia causes a form of sterility known as cytoplasmic incompatibility (CI). Wolbachia-induced CI has attracted interest as a potential agent for affecting medically important disease vectors. However, application of the approach has been restricted by an absence of appropriate, naturally occurring Wolbachia infections. Here, we report the interspecific transfer of Wolbachia infection into a medically important mosquito. Using embryonic microinjection, Wolbachia is transferred from Drosophila simulans into the invasive pest and disease vector: Aedes albopictus (Asian tiger mosquito). The resulting infection is stably maintained and displays a unique pattern of bidirectional CI in crosses with naturally infected mosquitoes. Laboratory population cage experiments examine a strategy in which releases of Wolbachia-infected males are used to suppress mosquito egg hatch. We discuss the results in relation to developing appropriate Wolbachia-infected mosquito strains for population replacement and population suppression strategies.

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection.

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.

Rectal gland of Bactrocera papayae: ultrastructure, anatomy, and sequestration of autofluorescent compounds upon methyl eugenol consumption by the male fruit fly.

Sexually mature males of Bactrocera papayae are strongly attracted to and consume methyl eugenol (ME). Upon consumption, ME is biotransformed to two phenylpropanoids, 2-allyl-4,5-dimethoxyphenol (DMP) and (E)-coniferyl alcohol (CF), that are transported in the hemolymph, sequestered and stored in the rectal glands, and subsequently released as sex and aggregation pheromones during courtship. To date, very little work on the ultrastructure and anatomy of the rectal gland has been done, and the accumulation of phenylpropanoids in the rectal glands of males has not been observed visually. Our objectives are to describe the anatomy and fine structures of the rectal glands of males and females and to observe the accumulation of autofluorescent compounds in the rectal glands of males. The rectal glands of males and females have four rectal papillae with each papilla attached to a rectal pad. The rectal pads protrude from the rectal gland as the only surfaces of the gland that are not surrounded by muscles. The rectal papillae of ME-fed males had oil droplets and autofluorescent compounds that were absent from those of ME-deprived males. The autofluorescent compounds accumulated in the rectal sac, which is an evagination that is not found in rectal glands of females. The accumulation of these compounds increased with time and reached maximum at a day post-ME feeding and decreased thereafter. This trend is similar to the accumulation pattern of phenylpropanoids, CF and DMP in the rectal gland.

Oral gingival delivery systems from chitosan blends with hydrophilic polymers.

Chitosan blends with hydrophilic polymers including polyvinylalcohol (PVA), polyethyleneoxide (PEO) and polyvinylpyrrolidone (PVP), were investigated as candidates for oral gingival delivery systems. The bioavailabilty conferred by the chitosan blend delivery systems, as concluded from dog studies, was shown to be comparable to that based on chitosan alone, especially for those blends involving high molecular weight hydrophilic polymers. Results from differential scanning calorimetry and dynamic mechanical thermal analysis, Fourier transform infrared spectroscopy and tensile testing, indicated that the chitosan/PEO and chitosan/PVP blends showed evidence of miscibility in all blend ratios studied, while the chitosan/PVA blend only showed evidence of interaction for the (50:50) and (80:20) blends, but not for the (20:80) blend. However, even a phase separated system may show interesting and exploitable properties, as evidenced by the tensile testing data for the high molecular weight PVA blend (20:80). The study also indicated that chitosan blends were superior in other properties compared to chitosan alone. These included improved comfort and reduced irritation, ease of processing, improved film quality, improved flexibility, and enhanced dissolution. Blends of chitosan with different hydrophilic polymers could thus be promising candidates for formulation in oral mucosal delivery systems.

Evaluation of solid dispersion particles prepared with SEDS.

Formation of solid solution particles in the Solution Enhanced Dispersion by Supercritical fluids (SEDS) process from a model drug and two different types of carriers, mannitol and Eudragit E100 was evaluated. The crystal properties of samples and molecular interactions were investigated with DSC and FTIR, respectively. The effect of co-crystallisation of drug and mannitol on dissolution rate was studied. Even if a true one-phase solid dispersion was not obtained, the crystal structure of both drug and mannitol was mutually affected by the presence of the other. The drug was not in highly crystalline form in the co-precipitates. The interactions between the drug and mannitol could also be identified as hydrogen bonding between the amine or hydroxyl groups of the drug and the hydroxyl groups of mannitol. These interactions and changes in the crystal structure are probably directly related to the increase in the dissolution rate observed. A true solid solution was obtained when the drug was co-processed with Eudragit E100. A clear interaction between the acid hydroxyl group of the drug and the basic carbonyl group on the Eudragit E100 was observed. SEDS was shown to be an effective process for forming intimate blends and solid solutions for the drug and two different types of carriers.

Evaluation of controlled-release polar lipid microparticles.

The aim of the present study was to prepare controlled-release tablets of poorly-soluble drug, felodipine, and various erodable lipophilic excipients. Spray chilling was used to formulate the drug and the excipients into solid dispersion microparticles, which were then compressed. The microparticles were characterised by Fourier transform infrared spectroscopy, hot-stage microscopy, scanning electron microscopy, and image analysis. The amine and the carbonyl groups of felodipine formed hydrogen bonds with the carriers. The shape of the particles was spherical with the median particle diameter ranging from 25 to 35 microm. Surprisingly, the degree of crystallinity in felodipine and the ease of tablet disintegration played a more significant role on the felodipine dissolution rate than the matrix lipophilicity. Felodipine release rate was slowest from the least lipophilic tablets.

Evaluation of polar lipid-hydrophilic polymer microparticles.

The aim of the present study was to prepare controlled-release tablets of poorly-soluble drug, felodipine. Spray chilling was used to formulate the drug, the polar lipids and the hydrophilic polymers into solid dispersion microparticles, which were then compressed. The microparticles were characterised by Fourier transform infrared and Raman spectroscopies, X-ray powder diffraction, hot-stage microscopy, scanning electron microscopy, and image analysis. The crystallinity of felodipine had decreased in all the samples, and the amount of crystalline felodipine varied depending on the composition of the solid dispersion. The particles were spherical with the median particle diameter ranging from 20 to 35 microm. The addition of hydrophilic polymer into the matrix widened the particle size distribution and increased the amount of agglomerates. Most promising dissolution patterns were obtained from tablets containing glycerides; e.g. from Precirol ATO 5/Pluronic F127 tablets the release was of zero order.

Research resource: the pdx1 cistrome of pancreatic islets.

The homeodomain transcription factor pancreas duodenal homeobox 1 (Pdx1, also known as insulin promoter factor 1) is a master regulator of pancreas development, as mice or humans lacking Pdx1 function are a pancreatic. Importantly, heterozygous mutations in Pdx1 cause early and late onset forms of diabetes in humans. Despite these central roles in development and adult ß-cell function, we have only rudimentary knowledge of the transcriptome targets of Pdx1 that mediate these phenotypes. Therefore, we performed global location analysis of Pdx1 occupancy in pancreatic islets. We used evolutionary conservation of target genes to identify the most relevant Pdx1 targets by performing chromatin immunoprecipitation sequencing on both human and mouse islets. Remarkably, the conserved target set is highly enriched for genes annotated to function in endocrine system and metabolic disorders, various signaling pathways, and cell survival, providing a molecular explanation for many of the phenotypes resulting from Pdx1 deficiency.

Endoplasmic reticulum oxidoreductin-1-like ß (ERO1lß) regulates susceptibility to endoplasmic reticulum stress and is induced by insulin flux in ß-cells.

Hyperglycemia increases insulin flux through the endoplasmic reticulum (ER) of pancreatic ß-cells, and the unfolded protein response pathway is required to enhance insulin processing. Pancreatic and duodenal homeobox 1 (PDX1), a key pancreatic transcription factor, regulates insulin along with targets involved in insulin processing and secretion. Here we find that PDX1 is a direct transcriptional regulator of ER oxidoreductin-1-like ß (Ero1lß), which maintains the oxidative environment of the ER to facilitate disulfide bond formation. PDX1 deficiency reduced Ero1lß transcript levels in mouse islets and mouse insulinoma (MIN6) cells; moreover, PDX1 occupied the Ero1lß promoter in ß-cells. ERO1lß levels were induced by high glucose concentrations and by the reducing agent dithiothreitol, indicating potential roles in adaptation to increased oxidative protein folding load in the ß-cell ER. In MIN6 cells, small interfering RNA-mediated silencing of Ero1lß decreased insulin content and increased susceptibility to ER stress-induced apoptosis. These findings demonstrate roles for the PDX1 target ERO1lß in maintaining insulin content and regulating cell survival during ER stress.

Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation.

The canonical Wnt-beta-catenin signaling pathway is initiated by inducing phosphorylation of one of the Wnt receptors, low-density lipoprotein receptor-related protein 6 (LRP6), at threonine residue 1479 (Thr1479) and serine residue 1490 (Ser1490). By screening a human kinase small interfering RNA library, we identified phosphatidylinositol 4-kinase type II alpha and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled and dishevelled, the latter of which directly interacted with and activated PIP5KI. In turn, PtdIns (4,5)P2 regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a signaling mechanism for Wnt to regulate LRP6 phosphorylation.

Wolbachia establishment and invasion in an Aedes aegypti laboratory population.

A proposed strategy to aid in controlling the growing burden of vector-borne disease is population replacement, in which a natural vector population is replaced by a population with a reduced capacity for disease transmission. An important component of such a strategy is the drive system, which serves to spread a desired genotype into the targeted field population. Endosymbiotic Wolbachia bacteria are potential transgene drivers, but infections do not naturally occur in some important mosquito vectors, notably Aedes aegypti. In this work, stable infections of wAlbB Wolbachia were established in A. aegypti and caused high rates of cytoplasmic incompatibility (that is, elimination of egg hatch). Laboratory cage tests demonstrated the ability of wAlbB to spread into an A. aegypti population after seeding of an uninfected population with infected females, reaching infection fixation within seven generations.

Hagen's glands of the parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae): ultrastructure and the detection of entomopoxvirus and parasitism-specific proteins.

Hagen's glands of males of the parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae) secrete compounds that are involved in courtship and defense. Like the poison glands of female wasps, the Hagen's glands are secretory, membranous, and of ectodermal origin. The poison glands contain the symbiotic entomopoxvirus, DlEPV and the parasitism-specific protein, PSP 24. DlEPV proteins were detected in homogenates of male wasps. Our goal was to describe the ultrastructure of the Hagen's glands and determine whether they contain DlEPV virions and/or proteins as well as PSP 24. The Hagen's glands are bilateral and each consists of 12-16 tubules arranged in two clusters. In cross-section, a tubule has three zones that enclose a central cuticle-lined lumen. The outermost zone consists of aggregates ('islands') of small vesicles, interconnected by narrow ductules that lead to large cuticle-lined ducts, which transport electron-dense material to the lumen prior to its release from the gland. Large vesicles in Zone 2 and a thick layer of ribosomes and rough endoplasmic reticula in Zone 3 are the likely sites of storage and protein synthesis, respectively. While DlEPV virions were not seen in the Hagen's gland, DlEPV and PSP 24 proteins were present.