Biapenem Inactivation by B2 Metallo β-Lactamases: Energy Landscape of the Post-Hydrolysis Reactions

<div><h3>Background</h3><p>The first line of defense by bacteria against <em>β</em>-lactam antibiotics is the expression of β-lactamases, which cleave the amide bond of the β-lactam ring. In the reaction of biapenem inactivation by B2 metallo β-lactamases (MβLs), after the β-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5–C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo β-lactamase CphA in complex with product.</p> <h3>Methodology/Principal Findings</h3><p>QM/MM and metadynamics simulations of the post-hydrolysis steps in solution and in the enzyme reveal that while the rotation of the hydroxyethyl group can occur in solution or in the enzyme active site, formation of the bicyclic compound occurs primarily in solution, after which the final product binds back to the enzyme. The calculations also suggest that the rotation and cyclization steps can occur at a rate comparable to that observed experimentally for the enzymatic inactivation of biapenem only if the hydrolysis reaction leaves the N4 nitrogen of the β-lactam ring unprotonated.</p> <h3>Conclusions/Significance</h3><p>The calculations support the existence of a common mechanism (in which ionized N4 is the leaving group) for carbapenems hydrolysis in all MβLs, and suggest a possible revision of mechanisms for B2 MβLs in which the cleavage of the β-lactam ring is associated with or immediately followed by protonation of N4. The study also indicates that the bicyclic derivative of biapenem has significant affinity for B2 MβLs, and that it may be possible to obtain clinically effective inhibitors of these enzymes by modification of this lead compound.</p> </div

In Vivo Assessment of Cold Adaptation in Insect Larvae by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Background Temperatures below the freezing point of water and the ensuing ice crystal formation pose serious challenges to cell structure and function. Consequently, species living in seasonally cold environments have evolved a multitude of strategies to reorganize their cellular architecture and metabolism, and the underlying mechanisms are crucial to our understanding of life. In multicellular organisms, and poikilotherm animals in particular, our knowledge about these processes is almost exclusively due to invasive studies, thereby limiting the range of conclusions that can be drawn about intact living systems. Methodology Given that non-destructive techniques like 1H Magnetic Resonance (MR) imaging and spectroscopy have proven useful for in vivo investigations of a wide range of biological systems, we aimed at evaluating their potential to observe cold adaptations in living insect larvae. Specifically, we chose two cold-hardy insect species that frequently serve as cryobiological model systems–the freeze-avoiding gall moth Epiblema scudderiana and the freeze-tolerant gall fly Eurosta solidaginis. Results In vivo MR images were acquired from autumn-collected larvae at temperatures between 0°C and about -70°C and at spatial resolutions down to 27 µm. These images revealed three-dimensional (3D) larval anatomy at a level of detail currently not in reach of other in vivo techniques. Furthermore, they allowed visualization of the 3D distribution of the remaining liquid water and of the endogenous cryoprotectants at subzero temperatures, and temperature-weighted images of these distributions could be derived. Finally, individual fat body cells and their nuclei could be identified in intact frozen Eurosta larvae. Conclusions These findings suggest that high resolution MR techniques provide for interesting methodological options in comparative cryobiological investigations, especially in vivo

High Climate Model Dependency of Pliocene Antarctic Ice-Sheet Predictions

The mid-Pliocene warm period provides a natural laboratory to investigate the long-term response of the Earth’s ice-sheets and sea level in a warmer-than-present-day world. Proxy data suggest that during the warm Pliocene, portions of the Antarctic ice-sheets, including West Antarctica could have been lost. Ice-sheet modelling forced by Pliocene climate model outputs is an essential way to improve our understanding of ice-sheets during the Pliocene. However, uncertainty exists regarding the degree to which results are model-dependent. Using climatological forcing from an international climate modelling intercomparison project, we demonstrate the high dependency of Antarctic ice-sheet volume predictions on the climate model-based forcing used. In addition, the collapse of the vulnerable marine basins of Antarctica is dependent on the ice-sheet model used. These results demonstrate that great caution is required in order to avoid making unsound statements about the nature of the Pliocene Antarctic ice-sheet based on model results that do not account for structural uncertainty in both the climate and ice sheet models

Staggers in horses grazing paspalum infected with Claviceps paspali

Invasion of the flowering heads of grasses by Claviceps spp. can produce sclerotia (ergots) containing several toxins. Ingestion of these toxins, through the consumption of paspalum (Paspalum dilatatum), can induce a range of clinical symptoms, including staggers. Cattle are the most commonly affected species, but although sheep and horses have been reported affected there are no published descriptions of paspalum staggers in horses. We describe two occurrences of paspalum staggers, the first in three Australian Stockhorse foals and the second in mature Standardbred horses. All three foals presented with ataxia in all limbs after consuming infected paspalum. One foal died from misadventure and the other two recovered within 1 week of removal from the infected paddock. In the second case, two of eight mares and geldings grazing in an irrigation channel developed hindquarter paresis. After removal of all horses from the area, one of the affected horses continued to deteriorate. Both horses were treated with antibiotics. The more severely affected horse was also treated with fluids and electrolytes, but had to be euthanased. The second affected horse recovered after 2 days. Paspalum pastures should inspected for Claviceps paspali infection before the introduction of horses

Neuropathy after herniorrhaphy: indication for surgical treatment and outcome.

BACKGROUND: Chronic neuropathy after hernia repair is a neglected problem as very few patients are referred for surgical treatment. The aim of the present study was to assess the outcome of standardized surgical revision for neuropathic pain after hernia repair. METHODS: In a prospective cohort study we evaluated all patients admitted to our tertiary referral center for surgical treatment of persistent neuropathic pain after primary herniorrhaphy between 2001 and 2006. Diagnosis of neuropathic pain was based on clinical findings and a positive Tinel's sign. Postoperative pain was evaluated by a visual analogue scale (VAS) and a pain questionnaire up to 12 months after revision surgery. RESULTS: Forty-three consecutive patients (39 male, median age 35 years) underwent surgical revision, mesh removal, and radical neurectomy. The median operative time was 58 min (range: 45-95 min). Histological examination revealed nerve entrapment, complete transection, or traumatic neuroma in all patients. The ilioinguinal nerve was affected in 35 patients (81%); the iliohypogastric nerve, in 10 patients (23%). Overall pain (median VAS) decreased permanently after surgery within a follow-up period of 12 months (preoperative 74 [range: 53-87] months versus 0 [range: 0-34] months; p&lt;0.0001). CONCLUSIONS: The results of this cohort study suggest that surgical mesh removal with ilioinguinal and iliohypogastric neurectomy is a successful treatment in patients with neuropathic pain after hernia repair