Mass spectrometry studies of the fragmentation patterns and mechanisms of protonated peptoids

Peptoids belong to a class of sequence-controlled polymers comprising of N-alkylglycine. This study focuses on using tandem mass spectrometry techniques to characterize the fragmentation patterns of a set of singly and doubly protonated peptoids consisting of one basic residue placed at different positions. The singly protonated peptoids fragment by producing predominately high-abundant C-terminal ions called Y-ions and low-abundant N-terminal ions called B-ions. Computational studies suggest that the proton affinity (PA) of the C-terminal fragments is generally higher than that of the N-terminal fragments, and the PA of the former increases as the fragments are elongated. The B-ions are likely formed upon dissociating the proton-activated amide bonds via an oxazolone structure, and the Y-ions are produced subsequently by abstracting a proton from the newly formed B-ions, which is energetically favored. The doubly protonated peptoids prefer to fragment closest to either the N- or the C-terminus and produce corresponding B/Y-ion pairs. The basic residue seems to dictate the preferred fragmentation site, which may be the result of minimizing the repulsion between the two charges. Water and terminal neutral losses are a facile process accompanying the peptoid fragmentation in both charge states. The patterns appear to be highly influenced by the location of the basic residue.     

Markov Model Predicts Changes in STH Prevalence during Control Activities Even with a Reduced Amount of Baseline Information

Background

Estimating the reduction in levels of infection during implementation of soil-transmitted helminth (STH) control programmes is important to measure their performance and to plan interventions. Markov modelling techniques have been used with some success to predict changes in STH prevalence following treatment in Viet Nam. The model is stationary and to date, the prediction has been obtained by calculating the transition probabilities between the different classes of intensity following the first year of drug distribution and assuming that these remain constant in subsequent years. However, to run this model longitudinal parasitological data (including intensity of infection) are required for two consecutive years from at least 200 individuals. Since this amount of data is not often available from STH control programmes, the possible application of the model in control programme is limited. The present study aimed to address this issue by adapting the existing Markov model to allow its application when a more limited amount of data is available and to test the predictive capacities of these simplified models.

Method

We analysed data from field studies conducted with different combination of three parameters: (i) the frequency of drug administration; (ii) the drug distributed; and (iii) the target treatment population (entire population or school-aged children only). This analysis allowed us to define 10 sets of standard transition probabilities to be used to predict prevalence changes when only baseline data are available (simplified model 1). We also formulated three equations (one for each STH parasite) to calculate the predicted prevalence of the different classes of intensity from the total prevalence. These equations allowed us to design a simplified model (SM2) to obtain predictions when the classes of intensity at baseline were not known. To evaluate the performance of the simplified models, we collected data from the scientific literature on changes in STH prevalence during the implementation of 26 control programmes in 16 countries. Using the baseline data observed, we applied the simplified models and predicted the onward prevalence of STH infection at each time-point for which programme data were available. We then compared the output from the model with the observed data from the programme.

Results

The comparison between the model-predicted prevalence and the observed values demonstrated a good accuracy of the predictions. In 77% of cases the original model predicted a prevalence within five absolute percentage points from the observed figure, for the simplified model one in 69% of cases and for the simplified model two in 60% of cases. We consider that the STH Markov model described here could be an important tool for programme managers to monitor the progress of their control programmes and to select the appropriate intervention. We also developed, and made freely available online, a software tool to enable the use of the STH Markov model by personnel with limited knowledge of mathematical models.     

A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem‐like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24‐ cell populations) and the mature luminal cells (CD49f‐EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label‐free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24‐, ALDH+ versus CD49f‐EpCAM+ and CD44+CD24‐ versus CD49f‐EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti‐CSC therapeutics.     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

Effect of streptomycin on lipid composition with particular reference to cyclic depsipeptide biosynthesis in Serratia marcescens and other micro-organisms

The addition of low concentrations of streptomycin (5-10mug/ml of medium) to Serratia marcescens caused significant alterations in the lipid composition of this organism, but neither growth nor pigmentation was affected. The acetone-soluble cyclic depsipeptides, which comprise on average 15% of the total lipid, were decreased almost to zero and the total lipid phosphorus was more than doubled in the presence of streptomycin. Most of the phospholipid increase was due to an increase in phosphatidylethanolamine. Cyclic depsipeptides were not leached from the cell in the presence of streptomycin, indicating a definite inhibition of the biosynthetic pathway. The effect of streptomycin on the reported peptidolipids of Rhodopseudomonas spheroides, Halobacterium halobium, Nocardia asteroides and Pseudomonas tabaci was investigated. In the case of the only strictly comparable cellular cyclic depsipeptide (that of N. asteroides) the biosynthesis was strongly inhibited by streptomycin, but cell weight was maintained or even slightly increased. A possible mode and site of action of low concentrations of streptomycin on bacterial lipids is discussed.     

Tumor-suppressive effects of psoriasin (S100A7) are mediated through the β-catenin/T cell factor 4 protein pathway in estrogen receptor-positive breast cancer cells

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα(-)) invasive breast cancers, its role in ERα-positive (ERα(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα(+) breast cancer cells and observed that S100A7 overexpression in ERα(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα(+) breast cancer cells. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα(+) breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the β-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα(-) cells, our studies suggest that S100A7 may possess differential activities in ERα(+) compared with ERα(-) cells.     

Neonatal androgen levels and avoidance learning in prepubescent and adult male rats

Neonatal male rats were injected with 1.25 mg of testosterone propionate (TP) and compared with oil-injected controls on the acquisition of an active and passive avoidance response at 25 days of age. The TP treated animals acquired the active avoidance response significantly faster than controls, but no differences were found between groups tested on the step-down passive avoidance task. The active-avoidance paradigm was repeated at 70 days of age, with experimental and control animals receiving the same neonatal treatment as the prepubescent subjects. Again the TP group showed facilitated acquisition of the active avoidance response. The TP treatment also produced an increase in activity levels and aversion threshold to footshock in the prepubescent animals. Therefore the active avoidance effect may be interpreted more parsimoniously as a reflection of these latter effects, rather than learning per se.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.