Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.

Methods

Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.

Results

Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD₁₀₀ of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.

Conclusions

Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.     

Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP^Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.     

Excited state intramolecular proton transfer in 3-hydroxychromone: a DFT-based computational study

Potential energy (PE) curves for intramolecular proton transfer in the ground (GSIPT) and intramolecular proton transfer in the excited (ESIPT) states of 3-hydroxychromone (3HC) have been studied using DFT-B3LYP/6-31G(d,p) and TD-DFT/6-31G(d,p) level of theory, respectively. Our calculations suggest the non-viability of GSIPT in 3HC. Excited states PE calculations show the existence of ESIPT process in 3HC. ESIPT in 3HC has also been explained in terms of HOMO and LUMO electron densities of the enol and keto tautomers of 3HC.     

164 Extracting dynamics information from multiple structures

Meaningful dynamics information can be extracted from multiple experimental structures of the same, or closely related, proteins or RNAs. The covariance matrix of atom positions is decomposable into its principal components, and in this way, it is possible to rank-order the changes in the set of structures, and to determine what the most significant changes are. Usually, only a few principal components dominate the motions of the structures, and these usually relate to the functional dynamics. This dynamics information provides strong evidence for the plasticity of protein and RNA structures, and also suggests that these structures almost always have a highly limited repertoire of motions. In some cases, such as HIV protease, the dominant motions are opening and closing over the active site. For myoglobin, the changes are much smaller, reflecting in part the small changes in sequence, but nonetheless they show characteristic details that depend on the species. Sets of structures can also be used to derive the effective microscopic forces that are forcing a given conformational transition.     

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.     

Factors influencing the distribution of leopard in a semiarid landscape of Western India

Previously, factors governing distribution of leopard (Panthera pardus fusca) in forest habitats of the Indian subcontinent were unknown. The present study assessed the influence of different ecogeographic variables determining the distribution of leopards in and around Sariska Tiger Reserve through MaxEnt habitat suitability model based on camera trapping method. Camera trapping was used to collect presence/absence information in the study area from December 2008 to June 2010. Information of 11 macrohabitat characteristics and variables (habitat types, prey species, Normalized Difference Vegetation Index (NDVI), elevation, livestock, village, water source, etc.) were collected along with leopard presence data. The probability of presence of leopards increased with decreasing distance to water and increasing encounter rate of peafowl (Pavo cristatus), chital (Axis axis), sambar (Rusa unicolor), and wild pig (Sus scrofa). It was found that the probability of presence of leopards increased with increasing area of Zizyphus mixed forest patches and NDVI. Results of this study showed that the probability of presence of leopards was higher in habitat types with intermediate cover, high wild prey base, and water sources. They also indicated that leopards are not always ‘generalists’ showing some degree of specialization, at least in their choice of habitat, and this information is useful for conserving leopard in human-dominated landscapes.     

Efficacy of l-proline administration on the early responses during cutaneous wound healing in rats

Proline (Pro) plays a versatile role in cell metabolism and physiology. Pro and hydroxypro are major imino acids present in collagen, an important connective tissue protein, essential for wound healing, which is a primary response to tissue injury. This study explains the role of l-pro on cutaneous wound healing in rats when administered both topically and orally. Open excision wounds were made on the back of rats, and 200 μl (200 mg) of pro was administered topically and orally once daily to the experimental rats until the wounds healed completely. The control wounds were left untreated. Granulation tissues formed were removed after day 4 and 8 of post excision wounding, and biochemical parameters such as total protein, collagen, hexosamine, and uronic acid were estimated. Levels of enzymatic and non-enzymatic antioxidants such as catalase, superoxide dismutase, glutathione peroxidase, ascorbic acid, and reduced glutathione were evaluated along with lipid peroxides in the granulation tissues. Tensile strength and period of epithelialization were also measured. It was observed that the treated wounds healed very fast as evidenced by augmented rates of epithelialization and wound contraction, which was also confirmed by histological examinations. The results strappingly authenticate the beneficial effects of the topical administration of l-proline in the acceleration of wound healing than the oral administration and control.     

Incidence and Prevalence of Tuberculosis among Household Contacts of Pulmonary Tuberculosis Patients in a Peri-Urban Population of South Delhi,India

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of mortality and morbidity across all age groups throughout the world, especially in developing countries.

Methodology/Principal Findings

In this study, we have included 432 open index cases with their 1608 household contacts in a prospective cohort study conducted from May 2007 to March 2009. The follow-up period was 2 years. All Index cases were diagnosed on the basis of suggestive signs and symptoms and sputum being AFB positive. Among the 432 index patients, 250 (57.9%) were males and 182 (42.1%) females; with mean age of 34±14.4 yr and 26±11.1 yr, respectively. Out of 1608 household contacts, 866 (53.9%) were males and 742 (46.1%) females; with mean age of 26.5±15.8 and 26.5±16.0 yr, respectively. Of the total 432 households, 304 (70.4%) had ≤4 members and 128 (29.6%) had ≥5 members. The median size of the family was four. Of the 1608 contacts, 1206 were able to provide sputum samples, of whom 83 (6.9%) were found MTB culture positive. Household contacts belonging to adult age group were predominantly (74, 89.2%) infected as compared to the children (9, 10.8%). On screening the contact relationship status with index patients, 52 (62.7%) were first-degree relatives, 18 (34.6%) second-degree relatives and 12 (14.5%) spouses who got infected from their respective index patients. Co-prevalent and incident tuberculosis was found in 52 (4.3%) and 31 (2.6%) contacts, respectively. In incident cases, the diagnosis could be made between 4 to 24 months of follow-up, after their baseline evaluation.

Conclusion

Active household contact investigation is a powerful tool to detect and treat tuberculosis at early stages and the only method to control TB in high-TB-burden countries.     

Interleukin 6 and/or Interleukin 17A Modulate the OPG/RANKL System of MC3T3-E1 Murine Osteoblast Cell Line

Cytokines such as interleukin-6 (IL-6) and IL-17 which act as key regulators of the immune response have been identified to have a potential role in the bone remodeling mechanism. Receptor activator of NF-κB ligand (RANKL) has been shown to regulate osteoclast differentiation and function while the osteoprotegerin (OPG) blocks the binding of RANKL and inhibits the differentiation of osteoclasts, thus favoring osteogenesis. Alkaline phosphatase (ALP) on the other hand works as early mineralization indicator in bone regulation. The current study aims to determine the potential role of IL-6 and IL-17A in regulating the OPG/RANKL system of the murine osteoblast cell line (MC3T3-E1). Gene expression analysis showed significant up-regulation of OPG and ALP by all the treated groups (rIL-6, rIL-17A and rIL-6 + rIL-17A). In contrast, treatment of cells with rIL-6 and/or rIL-17A showed down-regulation of RANKL expression. Interestingly, the osteoblast cells treated with combinations of rIL-6 + rIL17A showed marked increased in OPG/RANKL ratio. Similar pattern of protein expression was observed in the osteoblasts treated with rIL-6 and/or rIL-17A as detected by western blotting and ELISA. These findings suggest a new mechanism of regulation by these cytokines on the expression of OPG and RANKL, which could promote osteogenesis and diminish osteoclastogenesis.     

Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.