A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual,SPR and NMR screening

NDM-1 can hydrolyze nearly all available β-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of ¹⁵N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.     

Functional lung imaging with synchrotron radiation: Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors'' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants'' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.     

Infrared thermography detects febrile and behavioural responses to vaccination of weaned piglets

An automated, non-invasive system for monitoring of thermoregulation has the potential to mitigate swine diseases through earlier detection. Measurement of radiated temperature of groups of animals by infrared thermography (IRT) is an essential component of such a system. This study reports on the feasibility of monitoring the radiated temperature of groups of animals as a biomarker of immune response using vaccination as a model for febrile disease. In Study A, weaned pigs were either treated with an intramuscular vaccine (FarrowSure Gold), a sham injection of 0.9% saline or left as untreated controls. An infrared thermal camera (FLIR A320) was fixed to the ceiling directly above the pen of animals, and recorded infrared images of the treatment groups at 5 min intervals. The effect on temperature of the spatial distribution of pigs within the pen was significant, with higher temperatures recorded when pigs were grouped together into a single cluster. A higher frequency of clustering behaviour was observed in vaccinated animals compared with controls during a period of the afternoon ~4 to 7 h post-vaccination. The daily mean of the maximum image temperature was significantly higher in vaccinated animals compared with control and sham-treated animals. In the vaccination treated group, the 24 h mean of the maximum temperature was significantly higher during the post-vaccination period compared with the 24 h period before vaccination. Increased temperature in the vaccinated animals occurred from ~3 h, peaked at ~10 h, and remained elevated for up to 20 h post-vaccination. In Study B, the effect of prevalence was tested in terms of the difference in maximum temperature between control and vaccination days. A thermal response to vaccination was detected in a pen of 24 to 26 animals when <10% of the animals were vaccinated. The results support the concept of radiated temperature measurements of groups of animals by IRT as a screening tool for febrile diseases in pig barns.     

Response of instream animal communities to a short-term extreme event and to longer-term cumulative impacts in a strategic water resource area,South Africa

Disturbance plays an integral part in generating heterogeneity required for ecosystem persistence, but the increased amplitude and duration of disturbances linked to drivers of global change could result in ecosystem shifts or collapse. Biomonitoring over time provides insights into trajectories of ecosystem change. The responses of two instream animal taxa to two contrasting disturbance events, a major flood event and the long-term cumulative effects of land-use changes, were assessed in 1999–2012 by quantifying variation and change in abundance of functional groups based on flow rate sensitivity, water quality and metrics of ecological condition. All metrics recovered to pre-flood conditions within seven months after the flood event. Similarly, cumulative impacts of land use effected significant decreases in some but not all metrics. Indices that did not change, including SASS total score and ASPT, were the result of insufficient consideration of the decrease in the abundance of sensitive taxa specifically, and the abundance of all taxa in general. The decrease in abundance of sensitive taxa could signal imminent collapse in certain metrics. Evidence is also provided for a shift in the structure of fish assemblages linked to the decrease and loss of taxa sensitive to ecosystem degradation caused by the longer-term impacts of land-use change.     

Interaction between cystatin-derived peptides and papain

The interaction between papain and synthetic peptides which tentatively mimic cystatin surfaces was investigated both enzymatically and structurally. Measurements of dissociation equilibrium constants for the interaction of papain with these peptides modified by successive deletions or substitutions demonstrated that the QVVAG segment, which is highly conserved throughout members of the cystatin superfamily, is essential for the interaction. The glycylcontaining (N-terminal) fragments and PW-containing (C-terminal) fragments were found to be of lesser importance, since each could be deleted without significantly modifying the interaction. These fragments improved the stability of the interacting QVVAG region, which appeared to be substrate-like in all peptides tested, as it was cleaved at the A-G bond upon peptide-papain interaction. Replacement of the A residue at the scissile bond of the QVVAG by a blocked cysteinyl residue reduced the rate of cleavage of the susceptible bond and therefore shifted the resulting peptide from a substrate to an inhibitor. Derivatization of this substituted peptide at its N- and C-terminal ends by fluoresceinyl groups resulted in a dramatic decrease in theK _i to 0.5 µM. This improvement in the inhibitory properties of the substituted and derivatized peptides was correlated with structural changes as analyzed by molecular dynamic calculations. The results were compared to those proposed for the mechanism of inhibition by natural inhibitors of the cystatin superfamily.