Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase

Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mo?ssbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mo?ssbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔE(Q)| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 °C.     

Draft genome sequence of Lactobacillus malefermentans KCTC 3548

We announce the draft genome sequence of the type strain Lactobacillus malefermentans KCTC 3548 (2,003,922 bp, with a G+C content of 41.1%), which is one of the most prevalent lactic acid bacteria present during the manufacturing process of beer; the genome consists of 172 large contigs (>100 bp in size). All of the contigs were assembled by using Newbler Assembler 2.3 (454 Life Science).     

Genome sequence of Lactobacillus farciminis KCTC 3681

Lactobacillus farciminis is one of the most prevalent lactic acid bacterial species present during the manufacturing process of kimchi, the best-known traditional Korean dish. Here, we present the draft genome sequence of the type strain Lactobacillus farciminis KCTC 3681 (2,498,309 bp, with a G+C content of 36.4%), which consists of 5 scaffolds.     

Eponymous instruments in plastic surgery

Surgeons use eponymous instruments daily, yet the stories behind these instruments are often lost in history. The authors have selected eponymous instruments commonly used in plastic surgery and provide a brief biography of the surgeons who invented them. This list represents more than two centuries of surgical history, and the physicians come from a number of disciplines, including general surgery, plastic surgery, ophthalmic surgery, and rural medicine. Remembering the life stories of surgeon inventors enriches our understanding of the history of our profession and allows us to appreciate our instruments instead of taking them for granted.     

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.     

Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution

Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native D- and L-sugars (both α- and β-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.     

Synthesis and antifungal activity of benzofuran-5-ols

Benzofuran-5-ol derivatives were synthesized and tested for in vitro antifungal activity against Candida, Aspergillus species, and Cryptococcus neoformans. Among them tested, many benzofuran-5-ols showed good antifungal activity. The results suggest that benzofuran-5-ols would be promising antifungal agents.     

Selective Lentiviral Gene Delivery to CD133-Expressing Human Glioblastoma Stem Cells

Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.     

Presumptive Treatment of Malaria from Formal and Informal Drug Vendors in Nigeria

Background

Despite policies that recommend parasitological testing before treatment for malaria, presumptive treatment remains widespread in Nigeria. The majority of Nigerians obtain antimalarial drugs from two types of for-profit drug vendors—formal and informal medicine shops—but little is known about the quality of malaria care services provided at these shops.

Aims

This study seeks to (1) describe the profile of patients who seek treatment at different types of drug outlets, (2) document the types of drugs purchased for treating malaria, (3) assess which patients are purchasing recommended drugs, and (4) estimate the extent of malaria over-treatment.

Methods

In urban, peri-urban, and rural areas in Oyo State, customers exiting proprietary and patent medicine vendor (PPMV) shops or pharmacies having purchased anti-malarial drugs were surveyed and tested with malaria rapid diagnostic test. A follow-up phone survey was conducted four days after to assess self-reported drug administration. Bivariate and multivariate regression analysis was conducted to determine the correlates of patronizing a PPMV versus pharmacy, and the likelihood of purchasing an artemisinin-combination therapy (ACT) drug.

Results

Of the 457participants who sought malaria treatment in 49 enrolled outlets, nearly 92% had diagnosed their condition by themselves, a family member, or a friend. Nearly 60% pharmacy customers purchased an ACT compared to only 29% of PPMV customers, and pharmacy customers paid significantly more on average. Multivariate regression results show that patrons of PPMVs were younger, less wealthy, waited fewer days before seeking care, and were less likely to be diagnosed at a hospital, clinic, or laboratory. Only 3.9% of participants tested positive with a malaria rapid diagnostic test.

Conclusions

Poorer individuals seeking care at PPMVs are more likely to receive inappropriate malaria treatment when compared to those who go to pharmacies. Increasing accessibility to reliable diagnosis should be explored to reduce malaria over-treatment.     

Proteomic Analysis in Type 2 Diabetes Patients before and after a Very Low Calorie Diet Reveals Potential Disease State and Intervention Specific Biomarkers

Very low calorie diets (VLCD) with and without exercise programs lead to major metabolic improvements in obese type 2 diabetes patients. The mechanisms underlying these improvements have so far not been elucidated fully. To further investigate the mechanisms of a VLCD with or without exercise and to uncover possible biomarkers associated with these interventions, blood samples were collected from 27 obese type 2 diabetes patients before and after a 16-week VLCD (Modifast ∼450 kcal/day). Thirteen of these patients followed an exercise program in addition to the VCLD. Plasma was obtained from 27 lean and 27 obese controls as well. Proteomic analysis was performed using mass spectrometry (MS) and targeted multiple reaction monitoring (MRM) and a large scale isobaric tags for relative and absolute quantitation (iTRAQ) approach. After the 16-week VLCD, there was a significant decrease in body weight and HbA1c in all patients, without differences between the two intervention groups. Targeted MRM analysis revealed differences in several proteins, which could be divided in diabetes-associated (fibrinogen, transthyretin), obesity-associated (complement C3), and diet-associated markers (apolipoproteins, especially apolipoprotein A-IV). To further investigate the effects of exercise, large scale iTRAQ analysis was performed. However, no proteins were found showing an exercise effect. Thus, in this study, specific proteins were found to be differentially expressed in type 2 diabetes patients versus controls and before and after a VLCD. These proteins are potential disease state and intervention specific biomarkers.

Trial Registration

Controlled-Trials.com ISRCTN76920690