Purification,Characterization, and Chemical Modification of Neurotoxic Peptide from Daboia russelii Snake Venom of India

Comprehensive knowledge of venom composition is very important for effective management of snake envenomation and antivenom preparation. Daboia russelii venom from the eastern region of India is the most neurotoxic among the four venom samples investigated. From the eastern D. russelii venom sample, neurotoxic peptide has been purified by combined method of ion exchange gel permeation chromatography and reversed phase high performance liquid chromatography. Molecular weight of Daboia neurotoxin III (DNTx‐III) found to be 6,849 Da (as measured on matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometer), and N‐terminal amino acid sequences is I K C F I T P D U T S Q A. Approximate LD₅₀ dosage was 0.24 mg/kg body weight. It produced concentration‐ and time‐dependent inhibition of indirectly stimulated twitches of Rana hexadactyla sciatic nerve gastrocnemius muscle preparations. Chemical modification of DNTx‐III tryptophan residue(s) reduced the twitch height inhibition property of toxin, signifying the importance of tryptophan residues for the neurotoxic function. This type of neurotoxic peptide is unique to east Indian regional D. russelii venom. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:295‐304, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21486     

Predicting drug polypharmacology from cell morphology readouts using variational autoencoder latent space arithmetic

A variational autoencoder (VAE) is a machine learning algorithm, useful for generating a compressed and interpretable latent space. These representations have been generated from various biomedical data types and can be used to produce realistic-looking simulated data. However, standard vanilla VAEs suffer from entangled and uninformative latent spaces, which can be mitigated using other types of VAEs such as β-VAE and MMD-VAE. In this project, we evaluated the ability of VAEs to learn cell morphology characteristics derived from cell images. We trained and evaluated these three VAE variants—Vanilla VAE, β-VAE, and MMD-VAE—on cell morphology readouts and explored the generative capacity of each model to predict compound polypharmacology (the interactions of a drug with more than one target) using an approach called latent space arithmetic (LSA). To test the generalizability of the strategy, we also trained these VAEs using gene expression data of the same compound perturbations and found that gene expression provides complementary information. We found that the β-VAE and MMD-VAE disentangle morphology signals and reveal a more interpretable latent space. We reliably simulated morphology and gene expression readouts from certain compounds thereby predicting cell states perturbed with compounds of known polypharmacology. Inferring cell state for specific drug mechanisms could aid researchers in developing and identifying targeted therapeutics and categorizing off-target effects in the future.     

The role of circulating serotonin in the development of chronic obstructive pulmonary disease

Background

Cigarette smoking is a major risk factor in the development of age-related chronic obstructive pulmonary disease (COPD). The serotonin transporter (SERT) gene polymorphism has been reported to be associated with COPD, and the degree of cigarette smoking has been shown to be a significant mediator in this relationship. The interrelation between circulating serotonin (5-hydroxytyptamine, 5-HT), cigarette smoking and COPD is however largely unknown. The current study aimed at investigating the mediation effects of plasma 5-HT on cigarette smoking-induced COPD and the relation between plasma 5-HT levels and age.

Methods

The association between plasma 5-HT, age and COPD was analyzed in a total of 62 COPD patients (ever-smokers) and 117 control subjects (healthy non-smokers and ever-smokers). Plasma 5-HT levels were measured by enzyme-linked immuno assay (EIA).

Results

The elevated plasma 5-HT levels were significantly associated with increased odds for COPD (OR = 1.221, 95% CI = 1.123 to 1.319, p<0.0001). The effect remained significant after being adjusted for age and pack-years smoked (OR = 1.271, 95% CI = 1.134 to 1.408, p = 0.0003). Furthermore, plasma 5-HT was found to mediate the relation between pack-years smoked and COPD. A positive correlation (r = 0.303, p = 0.017) was found between plasma 5-HT levels and age in COPD, but not in the control subjects (r = −0.149, p = 0.108).

Conclusion

Our results suggest that cigarette smoke-induced COPD is partially mediated by the plasma levels of 5-HT, and that these become elevated with increased age in COPD. The elevated plasma 5-HT levels in COPD might contribute to the pathogenesis of this disease.     

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could détéct SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was détécted by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of détéction of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was détécted in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.     

The Origin of the White Roman Goose

In order to avoid interference from nuclear copies of mitochondrial DNA (numts), mtDNA of the white Roman goose (domestic goose) was extracted from liver mitochondria. The mtDNA control region was amplified using a long PCR strategy and then sequenced. Neighbor-joining, maximum parsimony, and maximum-likelihood approaches were implemented using the 1,177 bp mtDNA control region sequences to compute the phylogenetic relationships of the domestic goose with other geese. The resulting identity values for the white Roman geese were 99.1% (1,166/1,177) with western graylag geese and 98.8% (1,163/1,177) with eastern graylag geese. In molecular phylogenetic trees, the white Roman goose was grouped in the graylag lineage, indicating that the white Roman goose came from the graylag goose (Anser anser). Thus, the scientific name of the white Roman goose should be Anser anser ‘White Roman.’     

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol