Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K_d, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC₅₀ of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and ¹⁵N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including ¹⁵N-¹H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.     

Detecting and mapping vegetation distribution on the Antarctic Peninsula from remote sensing data

We present the first regional map of vegetation of anywhere on the Antarctic continent based on remote sensing (RS) data. We have used a normalized difference vegetation index (NDVI) for the examination of Landsat ETM data on the Antarctic Peninsula. The results show that 44.6 km² (0.086%) of the study area (74,468 km²) is classed with a probability of vegetation of over 50%. The NDVI analysis is ground-truthed against vegetation surveys in Ryder Bay on the Antarctic Peninsula, and the results have been corrected for several factors influencing low NDVI readings in this environment. This methodology has been applied to 13 Landsat scenes covering Graham Land in the Northern part of the Antarctic Peninsula to examine the distribution of vegetation in the region. The Antarctic Peninsula region is important, as it has shown rapid warming of over 3°C during the past 50 years, and predictions indicate accelerated future warming. A baseline survey of the amount and distribution of vegetation is required against which to monitor future change. The results give a comprehensive coverage and allow us to present the first remote sensing-based vegetation map in Antarctica. However, initial results point to the need for further investigation of apparent errors resulting from geology on bare ground.     

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.     

Synthesis and evaluation of novel heterocyclic inhibitors of GSK-3

A set of novel heterocyclic pyrimidyl hydrazones has been synthesized as inhibitors of glycogen synthase kinase-3 (GSK-3) with the most active exhibiting low nanomolar activity. Quantum mechanical calculations indicate that of the conformational factors that could determine binding affinity, the planarity of the phenyl ring in relation to the central core and the conformation of the hydrazone chain may be the most influential.     

Excision margins for nonmelanotic skin cancer

Scientific evidence for advisable excision margins for nonmelanotic skin carcinoma is poorly documented. Recommended excision margins vary from 2 to 15 mm. A prospective study was performed on 150 skin lesions excised over a 9-month period in an outpatient facility at the authors' institution. Primary nonmelanotic skin lesions were clinically diagnosed as either basal cell carcinoma (nodular, superficial, infiltrating, or sclerosing) or squamous cell carcinoma (well, moderately, or poorly differentiated). Macroscopic surgical excision margins were individually assessed, measured, and excised. Histopathologic analysis was then independently performed to determine the correct diagnosis and to measure the actual microscopic lateral and deep excision margins.Sixty-one percent of lesions were basal cell carcinoma, 25 percent were squamous cell carcinoma, and 15 percent were benign or premalignant. Diagnostic accuracy was 81 percent for basal cell and 59 percent for squamous cell carcinoma. The average diameter of the basal cell carcinoma was 12.1 mm; 47 percent of these lesions had a diameter of less than 10 mm. The average diameter of the squamous cell carcinoma was 16.9 mm; 26 percent of these lesions had a diameter of less than 10 mm. The mean surgical margin was 4.2 mm (3.2 mm adjusted for shrinkage), whereas the mean microscopic lateral margin was 3.4 mm. Overall, complete excision was achieved for 98 percent of basal cell carcinoma and 100 percent of squamous cell carcinoma. The raw data were analyzed to assess the suitability of 1-, 2-, 3-, or 4-mm surgical excision margins. A 4-mm surgical margin would give a microscopic lateral margin beyond one microscopic high-power field (0.5 mm) in 96 percent of cases of basal cell carcinoma and in 97 percent of cases of squamous cell carcinoma.The authors recommend a 4-mm surgical margin as the optimal treatment for skin lesions clinically diagnosed as basal cell or squamous cell carcinoma that are suitable for excision in an outpatient facility. Well-demarcated lesions, such as a nodular basal cell carcinoma, may be excised with a 3-mm margin.