Design and synthesis of aminopropyl tetrahydroindole-based indolin-2-ones as selective and potent inhibitors of Src and Yes tyrosine kinase

A novel series of substituted 3-[3-(aminopropyl)-4,5,6,7-tetrahydro-1H-indol-2-ylmethylene]-1,3-dihydro-indole-2-ones was discovered as potent inhibitors of the non-receptor tyrosine kinase Src and Yes. A structure-activity relationship was developed in order to optimize their potency and selectivity. Syntheses of these compounds are also described herein.     

Keratinocyte growth factor-2 production in an ompT-deficient Escherichia coli K-12 mutant

A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110. When the protein was purified using a standard process, the first six N-terminal amino acids were rapidly and specifically removed from the protein. This cleavage resulted in a truncated KGF-2 species (S69-S208). To circumvent this problem, guanidine-HCl was used to inhibit the putative proteolytic activity. This modified process resulted in a massive loss of protein product due to precipitation, in addition to the cost and corrosiveness of guanidine-HCl. To develop an economically feasible, scaleable, and robust process for KGF-2 production, we were tasked with identifying the protease(s) responsible for the N-terminal degradation. Experimental evidence revealed that OmpT (outer membrane protein T) was the primary protease involved in the N-terminal cleavage of the A63-S208 KGF-2 protein. Moreover, the OmpT-mediated cleavage occurred at a novel site (Arg-Ser). From this work, we show that production of the A63-S208 form of KGF-2 in an ompT-deleted E. coli host nearly abolished the N-terminal cleavage issue.     

A mixture model for longitudinal data with application to assessment of noncompliance

In clinical trials of a self-administered drug, repeated measures of a laboratory marker, which is affected by study medication and collected in all treatment arms, can provide valuable information on population and individual summaries of compliance. In this paper, we introduce a general finite mixture of nonlinear hierarchical models that allows estimates of component membership probabilities and random effect distributions for longitudinal data arising from multiple subpopulations, such as from noncomplying and complying subgroups in clinical trials. We outline a sampling strategy for fitting these models, which consists of a sequence of Gibbs, Metropolis-Hastings, and reversible jump steps, where the latter is required for switching between component models of different dimensions. Our model is applied to identify noncomplying subjects in the placebo arm of a clinical trial assessing the effectiveness of zidovudine (AZT) in the treatment of patients with HIV, where noncompliance was defined as initiation of AZT during the trial without the investigators' knowledge. We fit a hierarchical nonlinear change-point model for increases in the marker MCV (mean corpuscular volume of erythrocytes) for subjects who noncomply and a constant mean random effects model for those who comply. As part of our fully Bayesian analysis, we assess the sensitivity of conclusions to prior and modeling assumptions and demonstrate how external information and covariates can be incorporated to distinguish subgroups.     

The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine,halotolerant<Emphasis Type="Bold">,</Emphasis> and halophilic microalgae

Open ponds are the preferred cultivation system for large-scale microalgal biomass production. To be more sustainable, commercial scale biomass production should rely on seawater, as freshwater is a limiting resource, especially in places with high irradiance. If seawater is used for both pond fill and evaporative volume makeup, salinity of the growth media will rise over time. It is not possible for any species to achieve optimum growth over the whole saline spectrum (from seawater salinity level up to salt saturation state). In this study, we investigated the effects of gradual salinity increase (between 35 and 233 ppt) on biomass productivity and biochemical composition (lipid and carbohydrate) of six marine, two halotolerant, and a halophilic microalgae. A gradual and slow stepped salinity increase was found to expand the salinity tolerance range of tested species. A gradual reduction in biomass productivity and maximum photochemical efficiency was observed as a consequence of increased salinity in all tested species. Among the marine microalgae, Tetraselmis showed highest biomass productivity (32 mg L^?1 day^?1) with widest salinity tolerance range (35 to 109 ppt). Halotolerant Amphora and Navicula were able to grow from 35 ppt to 129 ppt salinity. Halophilic Dunaliella was the only species capable of growing between 35 and 233 ppt and showed highest lipid content (56.2%) among all tested species. This study showed that it should be possible to maintain high biomass in open outdoor cultivation utilizing seawater by growing Tetraselmis, Amphora, and Dunaliella one after another as salinity increases in the cultivation system.     

Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of cornelia de Lange syndrome with predominant mental retardation

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.     

Arbuscular mycorrhizal fungi reduce the construction of extrafloral nectaries in <Emphasis Type="Italic">Vicia faba</Emphasis>

Arbuscular mycorrhizal fungi (AMF) can alter the physiology and morphology of their host plant, and therefore may have indirect effects on insect herbivores and pollinators. We conducted this study to test the hypothesis that AMF can also affect insects involved in protection-for-food mutualisms. We examined the constitutive and inducible production of food rewards [extrafloral (EF) nectaries] in Vicia faba plants by manipulating the presence/absence of AMF and by simulating various levels of herbivory. Plants inoculated with AMF produced significantly fewer EF nectaries than uninoculated plants, even after accounting for differences in plant growth. In contrast to earlier studies, EF nectaries were not inducible: damaged plants produced significantly fewer EF nectaries than undamaged plants. Moreover, the effects of mycorrhizal and damage status on EF nectary production were additive. The reduction in EF nectaries in mycorrhizal plants potentially represents a mechanism for indirect effects of AMF on the protective insects that exploit EF nectaries as a food source (e.g., ants). Reduced reward size should result in reduced protection by ants, and could therefore be a previously unappreciated cost of the mycorrhizal symbiosis to host plants. However, the overall effect of AMF will depend upon the extent to which the reduction of EF nectaries affects the number and activity of ants and the extent to which AMF alter other aspects of host plant physiology. Our results emphasize the complexity of multitrophic interactions, particularly those that span belowground and aboveground ecology.     

Acidic pH Strongly Enhances In Vitro Biofilm Formation by a Subset of Hypervirulent ST-17 Streptococcus agalactiae Strains

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability.