The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems

Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis – a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact‐dependent growth inhibition (CDI) systems of γ‐proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA‐CT/cdiI coding regions, which are predicted to encode CdiA‐CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA‐CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA‐CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA‐CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.     

The early onset dystonia protein torsinA interacts with kinesin light chain 1

Early onset dystonia is a movement disorder caused by loss of a glutamic acid residue (Glu(302/303)) in the carboxyl-terminal portion of the AAA+ protein, torsinA. We identified the light chain subunit (KLC1) of kinesin-I as an interacting partner for torsinA, with binding occurring between the tetratricopeptide repeat domain of KLC1 and the carboxyl-terminal region of torsinA. Coimmunoprecipitation analysis demonstrated that wild-type torsinA and kinesin-I form a complex in vivo. In cultured cortical neurons, both proteins co-localized along processes with enrichment at growth cones. Wild-type torsinA expressed in CAD cells co-localized with endogenous KLC1 at the distal end of processes, whereas mutant torsinA remained confined to the cell body. Subcellular fractionation of adult rat brain revealed torsinA and KLC associated with cofractionating membranes, and both proteins were co-immunoprecipitated after cross-linking cytoplasmically oriented proteins on isolated rat brain membranes. These studies suggest that wild-type torsinA undergoes anterograde transport along microtubules mediated by kinesin and may act as a molecular chaperone regulating kinesin activity and/or cargo binding.     

Heterozygote advantage in the American chestnut, Castanea dentata (Fagaceae)

The American chestnut (Castanea dentata; Fagaceae) was a dominant canopy tree in the Appalachian Mountains of North America. Since the introduction of the chestnut blight fungus (Cryphonectria parasitica; Valsaceae) in America, the American chestnut has been reduced to a predominantly clonal, understory species. Our objective was to determine whether the ecological changes and absence of new recruits have influenced the population genetics of American chestnut. Leaf samples were collected from four populations in southwestern Virginia. Electrophoretic data from five polymorphic loci were used to determine the genetic diversity and population structure of the populations and subpopulations. Growth data and infection status were recorded for one of the populations to determine their relationship with heterozygosity. F statistics revealed a significant amount of differentiation among subpopulations and an excess of heterozygotes within subpopulations. Heterozygous individuals also had higher rates of vegetative growth. The superior performance and excess of heterozygotes suggests that selection favors heterozygous individuals. The prolonged absence of sexual reproduction in C. dentata has allowed subtle fitness differences to accumulate to the extent that they have had significant effects on the genetics of chestnut populations.     

Tagging gene and protein names in biomedical text

MOTIVATION: The MEDLINE database of biomedical abstracts contains scientific knowledge about thousands of interacting genes and proteins. Automated text processing can aid in the comprehension and synthesis of this valuable information. The fundamental task of identifying gene and protein names is a necessary first step towards making full use of the information encoded in biomedical text. This remains a challenging task due to the irregularities and ambiguities in gene and protein nomenclature. We propose to approach the detection of gene and protein names in scientific abstracts as part-of-speech tagging, the most basic form of linguistic corpus annotation. RESULTS: We present a method for tagging gene and protein names in biomedical text using a combination of statistical and knowledge-based strategies. This method incorporates automatically generated rules from a transformation-based part-of-speech tagger, and manually generated rules from morphological clues, low frequency trigrams, indicator terms, suffixes and part-of-speech information. Results of an experiment on a test corpus of 56K MEDLINE documents demonstrate that our method to extract gene and protein names can be applied to large sets of MEDLINE abstracts, without the need for special conditions or human experts to predetermine relevant subsets. AVAILABILITY: The programs are available on request from the authors.     

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-l-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.Abbreviations OMT O-methyltransferase - bi-OMT bispecific O-methyltransferase - CAD cinnamyl alcohol dehydrogenase - Ptomt1 Populus tremuloides bi-OMT cDNA clone     

An analysis of Stein's model for stochastic neuronal excitation

Stein's model of stochastic neuronal excitation is a realistic, yet simplified, construction incorporating important measurable parameters from neurophysiology. One of the principle difficulties with the application of this model lies in solving the delay partial and ordinary differential equations that form the mathematical expression of the model. For the case of excitation only, we present some effective methods of calculating various aspects of the model including the interspike interval distribution.     

A Phase 2a randomized,single-center,double-blind,placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study.Trial registration: This study is registered with ClinicalTrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT04150250","term_id":"NCT04150250"}}NCT04150250.     

Position Three in Vasopressin Antagonist Tolerates Conformationally Restricted and Aromatic Amino Acid Substitutions: A Striking Contrast with Vasopressin Agonists

We report the solid-phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1–12) of the potent non-selective antagonist of the antidiuretic (V₂-receptor), vasopressor (V_1a-receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT-receptor) responses to oxytocin (OT), [1(-β mercapto-β,β-pentamethy lenepropionic acid)-2-O-ethyl-d -tyrosine 4-valine] arginine vasopressin [d(CH₂)₅D -Tyr(Et) ²VAVP] (A) and two analogues of ( B ) (peptides 13,14), the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid³ (Tic³) analogue of ( A ). Peptides 1–12 have the following substituents at position three in ( A ): (1) Pro; (2) Oic; (3) Atc; (4) D -Atc; (5) Aic; (6) D -Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr-NH₂ ⁹ analogue of ( B ): Peptide (14) is the D -Cys ⁶ analogue of ( B ). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V₂ and V_1a assays and in in vitro (no Mg²⁺) _n oxytocic assays. With the exception of the D -Phe³ peptide (No. 6), which exhibits very weak V₂ agonism (…0.0017 u/mg), none of the remaining 13 peptides exhibit any agonistic activities in these assays. In striking contrast to their deleterious effects on agonistic activities in AVP, the Pro³, Oic³, Tyr³, Trp³ and Hphe³ substitutions in ( A ) are very well tolerated, leading to excellent retention of V₂, V_1a and OT antagonistic potencies. All are more potent as V₂ antagonists than the Ile³ and Leu³ analogues of ( A ). The Tyr-NH₂⁹ and D -Cys⁶ substitutions in ( B ) are also well tolerated. The anti-V₂ pA₂ values of peptides 1–5 and 7–14 are as follows (1) 7.77±0.03; (2) 7.41± 0.05; (3) 6.86±0.02; (4) 5.66±0.09; (5) …5.2; (7) 7.25± 0.08; (8) 6.82±0.06; (9) 7.58±0.05; (10) 7.61±0.08; (11) 7.59±0.07; (12) 7.20±0.05; (13) 7.57±0.1; (14) 7.52± 0.06. All analogues antagonize the vasopressor responses to AVP, with anti-V _1a pA₂ values ranging from 5.62 to 7.64, and the in vitro responses to OT, with anti-OT pA₂ values ranging from 5.79 to 7.94. With an anti-V₂ potency of 7.77±0.03, the Pro³ analogue of ( A ) is surprisingly equipotent with ( A ), (anti-V₂ pA₂=7.81±0.07). These findings clearly indicate that position three in AVP V₂/V_1a antagonists, in contrast to position three in AVP agonists, is much more amenable to structural modification than had heretofore been anticipated. Furthermore, the surprising retention of V₂ antagonism exhibited by the Pro³, Oic³, Tyr³, Trp³ and Hphe³ analogues of ( A ), together with the excellent retention of V₂ antagonism by the Tyr-NH₂⁹ and D -Cys⁶ analogues of ( B ) are promising new leads to the design of potent and possibly orally active V₂ antagonists for use as pharmacological tools and/or as radioiodinatable ligands and for development as potential therapeutic agents for the treatment of the hyponatremia caused by the syndrome of the inappropriate secretion of the antidiuretic hormone (SIADH). © 1997 European Peptide Society and John Wiley & Sons, Ltd.