Structure based design of type II dehydroquinase inhibitors against Mycobacterium tuberculosis

There is currently an alarming increase in the world-wide incidence of tuberculosis (TB), the disease caused due to infection by Mycobacterium tuberculosis. There is no easy treatment for TB which currently requires a regime of four drugs administered over a six month time course. The rise in strains of M. tuberculosis resistant to one or more of these frontline anti-tubercular drugs, is a strong incentive to develop novel anti-tubercular agents. The shikimate pathway has been identified as a potential target for the development of new antimicrobial drugs and knockout strains of M. tuberculosis lacking shikimate kinase have been shown to be non-viable.In this study the third enzyme in the pathway, type II dehydroquinase is considered an attractive target as many pathogenic bacteria possess the type II enzyme whereas many bacteria that inhabit the human gut posses the type I form. Structure based ligand design was used to identify potential inhibitors of the enzyme type II 3-dehydroquinate deydratase (DHQase) from M. tuberculosis (MTDHQase). This methodology allows the identification of non-substrate like inhibitors that have solubility profiles more amenable to development as potential drugs. The crystal structures of MTDHQase available in house, were analysed to generate Pharmacophore models, which were subsequently used to screen a virtual compound library, to generate a list of potential inhibitors. This process was repeated for the type II DHQases from Streptomyces coelicolor (SCDHQase) and Helicobacter pylori (HPDHQase) and the hit lists for each enzyme compared. The hits unique to MTDHQase were screened in vitro, identifying six compounds that were active inhibitors. In tandem the cloning and structural analysis of the type II 3-dehydroquinase from Helocobacter pylori was undertaken as a series of inhibitors of this enzyme obtained from a traditional high throughput screen (HTS) against a compound library was available from GlaxoSmithKline. The structure of HPDHQase in complex with the transition state analogue 2,3-anhydroquinic acid was determined to 3.1A resolution. While the structure of HPDHQase in complex with inhibitor AH9095, an HPDHQase specific non-substrate-like inhibitor, was determined to 1.5A resolution. Both structures were solved using the molecular replacement method. Comparison of the structures of HPDHQase with those of SCDHQase and MTDHQase provided significant insight into the factors that affect ligand specificity. The complex of HPDHQase and AH9095 provided the first crystal structure of a type II 3-dehydroquinase with a non-substrate like ligand. This structure allowed the identification of new, unexploited areas of the type II 3-dehydroquinase active site which may be utilised to develop specificity and potency of inhibitors. The present assay for 3-dehydroquinase activity is based upon UV absorbance at 234nm from the product 3-dehydroshikimate. Detection of UV absorbance at this wavelength is not possible when the assay is carried out in plastic 96 well plates, therefore not amenable for high throughput methods. Development of a simple colorimetric assay for 3-dehydroquinase activity was investigated to permit high throughput testing of compounds. By coupling the dehydroquinase step to dehydroshikimate dehydratase, the third enzyme in the quinate degradation pathway, the product protoctaechuate can be detected by a strong colour change in the presence of ferric chloride or sodium molybdate. The amount of protocatechuate detectable by this method, in 96 well plate format, was investigated and expression studies were carried out upon the coupling enzyme dehydroshikimate dehydratase from Acinetobacter coalceticus

Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS. Results: We found that in the heart, t(½) of complex I deactivation during ischemia was 10 min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H(2)O(2) or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis. Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting. Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R. Antioxid. Redox Signal. 19, 1459–1468

Functional morphology and efficiency of the antenna cleaner in Camponotus rufifemur ants.

Contamination of body surfaces can negatively affect many physiological functions. Insects have evolved different adaptations for removing contamination, including surfaces that allow passive self-cleaning and structures for active cleaning. Here, we study the function of the antenna cleaner in Camponotus rufifemur ants, a clamp-like structure consisting of a notch on the basitarsus facing a spur on the tibia, both bearing cuticular 'combs' and 'brushes'. The ants clamp one antenna tightly between notch and spur, pull it through, and subsequently clean the antenna cleaner itself with the mouthparts. We simulated cleaning strokes by moving notch or spur over antennae contaminated with fluorescent particles. The notch removed particles more efficiently than the spur, but both components eliminated more than 60% of the particles with the first stroke. Ablation of bristles, brush and comb strongly reduced the efficiency, indicating that they are essential for cleaning. To study how comb and brush remove particles of different sizes, we contaminated antennae of living ants, and anaesthetized them immediately after they had performed the first cleaning stroke. Different-sized beads were trapped in distinct zones of the notch, consistent with the gap widths between cuticular outgrowths. This suggests that the antenna cleaner operates like a series of sieves that remove the largest objects first, followed by smaller ones, down to the smallest particles that get caught by adhesion

Profiling UK injectable aesthetic practitioners: a national cohort analysis

Introduction: The United Kingdom (UK) injectables market has been growing rapidly with a lack of robust regulation and to date, no information regarding the profile of practitioners has been published. Aim: We aim to provide a descriptive and qualitative analysis of the advertised practitioners in the United Kingdom. Methods: We performed a systematic search using the internet search engine Google to perform a qualitative descriptive analysis of aesthetic practitioners in the UK. For each contiguous country in the UK: England, Scotland, and Wales, five searches were performed. The list of practitioners was then cross-referenced with professional regulatory bodies, with extraction of registration number, date of registration and presence or absence from the Specialist Register or General Practitioner register. Results: 3,000 websites were visited and evaluated. 1,224 independent clinics with 4,405 practitioners were identified. 738 were identified as those in business support functions and the remaining 3,667 practitioners were undertaking injectable practice. The profile of professions were doctors 32%, nurses 13%, dentists 24% and dental nurses 8%. Of the 1,163 doctors identified 481 were on the specialist register (41%) and 219 were on the GP register (19%). 27 specialties were represented in this cohort analysis. Plastic Surgery formed the majority of those who were on the specialist register at 37%, followed by Dermatology at 18%. Conclusion: This paper is the first to describe the range of practitioners, their professional backgrounds and experience who perform non-surgical aesthetic interventions. The range of backgrounds may have an impact on the potential risks to patients and will be an important consideration in proposed legislation to introduce licensing to the industry

A 610-MHz survey of the ELAIS-N1 field with the Giant Metrewave Radio Telescope - Observations, data analysis and source catalogue

Observations of the ELAIS-N1 field taken at 610 MHz with the Giant Metrewave Radio Telescope are presented. Nineteen pointings were observed, covering a total area of 9 square degrees with a resolution of 6" x 5", PA +45 deg. Four of the pointings were deep observations with an rms of 40 microJy before primary beam correction, with the remaining fifteen pointings having an rms of 70 microJy. The techniques used for data reduction and production of a mosaicked image of the region are described, and the final mosaic is presented, along with a catalogue of 2500 sources detected above 6 sigma. This work complements the large amount of optical and infrared data already available on the region. We calculate 610-MHz source counts down to 270 microJy, and find further evidence for the turnover in differential number counts below 1 mJy, previously seen at both 610 MHz and 1.4 GHz.Comment: 12 pages, 18 figures, two tables. Table 1 can be found in full via http://www.mrao.cam.ac.uk/surveys/ . Accepted for publication in MNRA

An Information-theoretic Approach to Prompt Engineering Without Ground Truth Labels

Pre-trained language models derive substantial linguistic and factual knowledge from the massive corpora on which they are trained, and prompt engineering seeks to align these models to specific tasks. Unfortunately, existing prompt engineering methods require significant amounts of labeled data, access to model parameters, or both. We introduce a new method for selecting prompt templates \textit{without labeled examples} and \textit{without direct access to the model}. Specifically, over a set of candidate templates, we choose the template that maximizes the mutual information between the input and the corresponding model output. Across 8 datasets representing 7 distinct NLP tasks, we show that when a template has high mutual information, it also has high accuracy on the task. On the largest model, selecting prompts with our method gets 90\% of the way from the average prompt accuracy to the best prompt accuracy and requires no ground truth labels

The European Large Area ISO Survey IX: the 90 micron luminosity function from the Final Analysis sample

We present the 90 micron luminosity function of the Final Analysis of the European Large Area ISO Survey (ELAIS), extending the sample size of our previous analysis (paper IV) by about a factor of 4. Our sample extends to z=1.1, around 50 times the comoving volume of paper IV, and 10^{7.7} < h^{-2}L/Lsun < 10^{12.5}. From our optical spectroscopy campaigns of the northern ELAIS 90 mircon survey (7.4 deg^2 in total, to S(90um)>70mJy), we obtained redshifts for 61% of the sample (151 redshifts) to B<21 identified at 7 microns, 15 microns, 20cm or with bright (B<18.5) optical identifications. The selection function is well-defined, permitting the construction of the 90 micron luminosity function of the Final Analysis catalogue in the ELAIS northern fields, which is in excellent agreement with our Preliminary Analysis luminosity function in the ELAIS S1 field from paper IV. The luminosity function is also in good agreement with the IRAS-based prediction of Serjeant & Harrison (2004), which if correct requires luminosity evolution of (1+z)^{3.4 +/- 1.0} for consistency with the source counts. This implies an evolution in comoving volume averaged star formation rate at z<~1 consistent with that derived from rest-frame optical and ultraviolet surveys.Comment: MNRAS accepted. 7 pages, 5 figures. Uses BoxedEPS (included