MBLinhibitors.com, a Website Resource Offering Information and Expertise for the Continued Development of Metallo--Lactamase Inhibitors.

In an effort to facilitate the discovery of new, improved inhibitors of the metallo--lactamases (MBLs), a new, interactive website called MBLinhibitors.com was developed. Despite considerable efforts from the science community, there are no clinical inhibitors of the MBLs, which are now produced by human pathogens. The website, MBLinhibitors.com, contains a searchable database of known MBL inhibitors, and inhibitors can be searched by chemical name, chemical formula, chemical structure, Simplified Molecular-Input Line-Entry System (SMILES) format, and by the MBL on which studies were conducted. The site will also highlight a "MBL Inhibitor of the Month", and researchers are invited to submit compounds for this feature. Importantly, MBLinhibitors.com was designed to encourage collaboration, and researchers are invited to submit their new compounds, using the "Submit" function on the site, as well as their expertise using the "Collaboration" function. The intention is for this site to be interactive, and the site will be improved in the future as researchers use the site and suggest improvements. It is hoped that MBLinhibitors.com will serve as the one-stop site for any important information on MBL inhibitors and will aid in the discovery of a clinically useful MBL inhibitor.

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions.

To understand the evolution of Verona integron-encoded metallo-ß-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-ß-lactamase families, although compensating mutations use different mechanisms to enhance resistance.IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-ß-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-ß-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.

A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-ß-lactamase SPS-1.

In an effort to evaluate whether a recently reported putative metallo-ß-lactamase (MßL) contains a novel MßL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not ß-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar to that in NDM-1 and other subclass B1 MßLs; however, the Zn1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MßLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of ß-lactam antibiotics. This study reveals a novel metal binding site in MßLs and suggests that the targeting of metal binding sites in MßLs with inhibitors is now more challenging with the identification of this new MßL.

Species-specific viromes in the ancestral holobiont Hydra.

Recent evidence showing host specificity of colonizing bacteria supports the view that multicellular organisms are holobionts comprised of the macroscopic host in synergistic interdependence with a heterogeneous and host-specific microbial community. Whereas host-bacteria interactions have been extensively investigated, comparatively little is known about host-virus interactions and viral contribution to the holobiont. We sought to determine the viral communities associating with different Hydra species, whether these viral communities were altered with environmental stress, and whether these viruses affect the Hydra-associated holobiont. Here we show that each species of Hydra harbors a diverse host-associated virome. Primary viral families associated with Hydra are Myoviridae, Siphoviridae, Inoviridae, and Herpesviridae. Most Hydra-associated viruses are bacteriophages, a reflection of their involvement in the holobiont. Changes in environmental conditions alter the associated virome, increase viral diversity, and affect the metabolism of the holobiont. The specificity and dynamics of the virome point to potential viral involvement in regulating microbial associations in the Hydra holobiont. While viruses are generally regarded as pathogenic agents, our study suggests an evolutionary conserved ability of viruses to function as holobiont regulators and, therefore, constitutes an emerging paradigm shift in host-microbe interactions.

An experimental model for the measurement of inspired gas temperatures in ventilated neonates.

OBJECTIVE: To determine the inspired gas temperature at points from the endo-tracheal tube (ETT) circuit manifold to the tip of the ETT in a model neonatal lung. DESIGN: A model lung attached to standard ventilator circuit, autofeed chamber and humidifier was ventilated using typical pressure-limited, time cycled settings. Temperatures were measured at various distances along the ETT using a K-type thermocouple temperature probe. RESULTS: The inspired gas temperature dropped from the circuit temperature probe site (40 degrees C) to the proximal end of the ETT (37 degrees C). The temperature dropped further as it passed through the exposed part of the ETT (34 degrees C) but then warmed again on entering the lung model so that the inspired gas at the distal end of the ETT was 37 degrees C. Statistically significant differences were found with a one-way ANOVA P-value of <0.0001. The differences between each pair of mean temperatures were statistically significant (all P<0.001) except when comparing the proximal end of the ETT with midway down the ETT (Bonferroni's Multiple Comparison Test, P>0.05). CONCLUSIONS: Inspired gas temperature drops as it passes through the circuit temperature probe site, the proximal end of the ETT and the exposed part of the ETT. The inspired gas rewarms on entering the model lung and exits the ETT at the desired temperature. The effect of measuring temperature closer to the patient, setting the circuit temperature higher and/or increasing the ambient temperature through which the circuit passes, need to be evaluated.

Establishing normal values of central venous pressure in very low birth weight infants.

The objective of this paper is to establish a reference range of central venous pressure (CVP) values during the first 4 days of life in very low birth weight (VLBW) infants. A prospective observational study with continuous monitoring of CVP in VLBW newborns who had an umbilical venous catheter (UVC) positioned in or near the right atrium is conducted. All UVCs were inserted as part of normal care of the infants. The mean CVP (mCVP) was monitored for 72 h from recruitment, or until the UVC was removed. The mean mCVP was calculated for each infant. The median of the mean mCVPs was then calculated. Data were analysed in 17 infants. The median gestational age was 27 weeks and median birth weight was 940 g. Sixteen were mechanically ventilated and of these, six also received continuous positive airway pressure (CPAP) during the study period. One infant received no respiratory support. One infant died during the study period. The lowest mean mCVP was 2.8 mmHg and the highest was 13.9 mmHg. The median mean mCVP was 4.9 mmHg (interquartile range 4.4-6.1). The normal range of CVP in VLBW infants during the first 4 days of life is wider than previously suggested.

Inspired gas temperature in ventilated neonates.

The warming and humidification of inspired gases for ventilated neonates are routine. There are no data on the temperature of the gas at the airway opening in ventilated neonates. Is the inspired gas temperature at the airway opening, as expected and set on the humidifier, around 37 degrees C? We aimed to measure temperature at the airway opening and compare this with the circuit temperature. This was an observational study in a neonatal intensive care unit. Twenty-five mechanically ventilated infants were studied. All had humidifiers with chamber temperature set at 36 degrees C and the circuit temperature set at 37 degrees C. Two temperature probes were inserted and rested at the circuit-exit and at the airway opening, and temperatures were measured for 2 min in each infant. At this time, the circuit temperature was also noted. The mean (SD) temperature at the airway opening in infants nursed in incubators was 34.9 (1.2) degrees C, compared with radiant warmers where the mean (SD) was 33.1 (0.5) degrees C. The mean (SD) difference in temperature from the circuit temperature probe to the airway opening was greater under radiant warmers, with a mean (SD) drop of 3.9 (0.6) degrees C compared with a mean (SD) drop of 2.0 (1.3) degrees C in the incubators. In conclusion, the temperature at the circuit temperature probe does not reflect the temperature at the airway opening. Inspired gas temperatures are lower than the expected 37 degrees C with the normal circuits and usual humidifier settings.

A novel expiratory circuit for recovery of perfluorocarbon liquid during partial liquid ventilation.

OBJECTIVE: To determine whether perfluorocarbon liquid can be condensed from gases containing perfluorocarbon vapour and whether the amount recovered varies with background flow rate. DESIGN AND SETTING: Bench-top experimental study in a neonatal laboratory. INTERVENTIONS: The expiratory limb of a standard ventilator circuit set-up was mimicked, with the addition of a chilled water jacket (Liebig) condenser. Perfluorocarbon vapour was passed through the circuit at a number of flow rates. MEASUREMENTS AND RESULTS: Perfluorocarbon vapour was passed through the circuit and the percentage recovery of liquid measured. More than 60% of the perfluorocarbon vapour was recovered at all flow rates (1, 2, 5 and 10 l/min), with significantly higher recovery obtained (up to 74%) at low flow rates (1 l/min). CONCLUSIONS: Using a simple condenser, more than 60% of perfluorocarbon liquid can be recovered without altering the function of an expiratory limb of a ventilator circuit.

In vivo somatic cell gene transfer of an engineered Noggin mutein prevents BMP4-induced heterotopic ossification.

BACKGROUND: The formation of the skeleton requires inductive signals that are balanced with their antagonists in a highly regulated negative feedback system. Inappropriate or excessive expression of BMPs (bone morphogenetic proteins) or their antagonists results in genetic disorders affecting the skeleton, such as fibrodysplasia ossificans progressiva. BMP signaling mediated through binding to its receptors is a critical step in the induction of abnormal ossification. Therefore, we hypothesized that engineering more effective inhibitors of this BMP-signaling process may lead to the development of therapies for such conditions. METHODS: BMP4-induced heterotopic ossification was used as a model for testing the ability of the BMP antagonist Noggin to block de novo bone formation, either by local or systemic delivery. Since Noggin naturally acts locally, a Noggin mutein, hNOGDeltaB2, was engineered and was shown to circulate systemically, and its ability to block heterotopic ossification was tested in a mouse model with use of adenovirus-mediated somatic cell gene transfer. RESULTS: A mouse model of BMP4-induced heterotopic ossification was developed. Local delivery of wild-type NOG inhibited heterotopic ossification, but systemic administration was ineffective. In contrast, systemic delivery of the adenovirus encoding hNOGDeltaB2 resulted in systemic levels that persisted for more than two weeks and were sufficient to block BMP4-induced heterotopic ossification. CONCLUSIONS: BMP4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild-type Noggin or after somatic cell gene transfer of a Noggin mutein, hNOGDeltaB2. Furthermore, the data in the present study provide proof of concept that a naturally occurring factor can be engineered for systemic delivery toward a desirable pharmacological outcome. CLINICAL RELEVANCE: Blocking bone formation is clinically relevant to disorders of heterotopic ossification in humans, such as fibrodysplasia ossificans progressiva. Furthermore, development of BMP antagonists as therapeutic agents may provide modalities for the treatment of other pathologic conditions that arise from aberrant expression of BMPs and/or from a lack of their antagonists.