EssC:domain structures inform on the elusive translocation channels in the Type VII secretion system.

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein

2- and 3-substituted imidazo [1,2-a] pyrazines as inhibitors of bacterial type IV secretion

A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a]pyrazines has been used to probe the structure–activity relationships of these inhibitors, which show potential as antibacterial agents

Preclinical electrogastrography in experimental pigs

Surface electrogastrography (EGG) is a non-invasive means of recording gastric myoelectric activity or slow waves from cutaneous leads placed over the stomach. This paper provides a comprehensive review of preclinical EGG. Our group recently set up and worked out the methods for EGG in experimental pigs. We gained our initial experience in the use of EGG in assessment of porcine gastric myoelectric activity after volume challenge and after intragastric administration of itopride and erythromycin. The mean dominant frequency in pigs is comparable with that found in humans. EGG in experimental pigs is feasible. Experimental EGG is an important basis for further preclinical projects in pharmacology and toxicology

Heparin-binding protein as a biomarker of acute kidney injury in critical illness

Background: There is no biomarker with high sensitivity and specificity for the development of acute kidney injury (AKI) in a mixed intensive care unit (ICU) population. Heparin-binding protein (HBP) is released from granulocytes and causes increased vascular permeability which plays a role in the development of AKI in sepsis and ischemia. The aim of this study was to investigate whether plasma levels of HBP on admission can predict the development of AKI in a mixed ICU population and in the subgroup with sepsis. Methods: Longitudinal observational study with plasma HBP levels from 245 patients taken on admission to ICU. Presence and severity of AKI was scored daily for 1 week. Results: Mean (95% CI) plasma concentrations of log HBP (ng/ml) in the groups developing different stages of AKI were: stage 0 (n = 175), 3.5 (3.4–3.7); stage 1 (n = 33), 3.7 (3.5–4.0), stage 2 (n = 20), 4.4 (3.5–4.8); and stage 3 (n = 17), 4.6 (3.8–5.2). HBP levels were significantly higher in patients developing AKI stage 3 (P < 0.01) compared to AKI stage 0 and 1. The area under the curve (AUC) for HBP to discriminate the group developing AKI stage 2–3 was 0.70 (CI: 0.58–0.82) and in the subgroup with severe sepsis 0.88 (CI: 0.77–0.99). Conclusion: Heparin-binding protein levels on admission to ICU are associated with the development of severe kidney injury. The relationship between HBP and AKI needs to be further validated in larger studies

Heparin-binding protein in ventilator-induced lung injury

Background: Although mechanical ventilation is often lifesaving, it can also cause injury to the lungs. The lung injury is caused by not only high pressure and mechanical forces but also by inflammatory processes that are not fully understood. Heparin-binding protein (HBP), released by activated granulocytes, has been indicated as a possible mediator of increased vascular permeability in the lung injury associated with trauma and sepsis. We investigated if HBP levels were increased in the bronchoalveolar lavage fluid (BALF) or plasma in a pig model of ventilator-induced lung injury (VILI). We also investigated if HBP was present in BALF from healthy volunteers and in intubated patients in the intensive care unit (ICU). Methods: Anaesthetized pigs were randomized to receive ventilation with either tidal volumes of 8 ml/kg (controls, n = 6) or 20 ml/kg (VILI group, n = 6). Plasma and BALF samples were taken at 0, 1, 2, 4, and 6 h. In humans, HBP levels in BALF were sampled from 16 healthy volunteers and from 10 intubated patients being cared for in the ICU. Results: Plasma levels of HBP did not differ between pigs in the control and VILI groups. The median HBP levels in BALF were higher in the VILI group after 6 h of ventilation compared to those in the controls (1144 ng/ml (IQR 359–1636 ng/ml) versus 89 ng/ml (IQR 33–191 ng/ml) ng/ml, respectively, p = 0.02). The median HBP level in BALF from healthy volunteers was 0.90 ng/ml (IQR 0.79–1.01 ng/ml) as compared to 1959 ng/ml (IQR 612–3306 ng/ml) from intubated ICU patients (p < 0.001). Conclusions: In a model of VILI in pigs, levels of HBP in BALF increased over time compared to controls, while plasma levels did not differ between the two groups. HBP in BALF was high in intubated ICU patients in spite of the seemingly non-harmful ventilation, suggesting that inflammation from other causes might increase HBP levels