Sequential binding and sensing of Zn(II) by Bacillus subtilis Zur

Bacillus subtilis Zur (BsZur) represses high-affinity zinc-uptake systems and alternative ribosomal proteins in response to zinc replete conditions. Sequence alignments and structural studies of related Fur family proteins suggest that BsZur may contain three zinc-binding sites (sites 1–3). Mutational analyses confirm the essential structural role of site 1, while mutants affected in sites 2 and 3 retain partial repressor function. Purified BsZur binds a maximum of two Zn(II) per monomer at site 1 and site 2. Site 3 residues are important for dimerization, but do not directly bind Zn(II). Analyses of metal-binding affinities reveals negative cooperativity between the two site 2 binding events in each dimer. DNA-binding studies indicate that BsZur is sequentially activated from an inactive dimer (Zur2:Zn2) to a partially active asymmetric dimer (Zur2:Zn3), and finally to the fully zinc-loaded active form (Zur2:Zn4). BsZur with a C84S mutation in site 2 forms a Zur2:Zn3 form with normal metal- and DNA-binding affinities but is impaired in formation of the Zur2:Zn4 high affinity DNA-binding state. This mutant retains partial repressor activity in vivo, thereby supporting a model in which stepwise activation by zinc serves to broaden the physiological response to a wider range of metal concentrations

Mass spectrometry of B. subtilis CopZ: Cu(I)-binding and interactions with bacillithiol

CopZ from Bacillus subtilis is a well-studied member of the highly conserved family of Atx1-like copper chaperones. It was previously shown via solution and crystallographic studies to undergo Cu(I)-mediated dimerisation, where the CopZ dimer can bind between one and four Cu(I) ions. However, these studies could not provide information about the changing distribution of species at increasing Cu(I) levels. To address this, electrospray ionisation mass spectrometry using soft ionisation was applied to CopZ under native conditions. Data revealed folded, monomeric CopZ in apo- and Cu(I)-bound forms, along with Cu(I)-bound dimeric forms of CopZ at higher Cu(I) loading. Cu4(CopZ)2 was the major dimeric species at loadings >1 Cu(I)/CopZ, indicating the cooperative formation of the tetranuclear Cu(I)-bound species. As the principal low molecular weight thiol in B. subtilis, bacillithiol (BSH) may play a role in copper homeostasis. Mass spectrometry showed that increasing BSH led to a reduction in Cu(I)-bound dimeric forms, and the formation of S-bacillithiolated apo-CopZ and BSH adducts of Cu(I)-bound forms of CopZ, where BSH likely acts as a Cu(I) ligand. These data, along with the high affinity of BSH for Cu(I), determined here to be β2(BSH) = ∼4 × 1017 M−2, are consistent with a role for BSH alongside CopZ in buffering cellular Cu(I) levels. Here, mass spectrometry provides a high resolution overview of CopZ–Cu(I) speciation that cannot be obtained from less discriminating solution-phase methods, thus illustrating the potential for the wider application of this technique to studies of metal–protein interactions

Cardiovascular changes after administration of aerosolized salbutamol in horses: five cases

Prevention and treatment of intraoperative hypoxemia in horses is difficult and both efficacy and safety of therapeutic maneuvers have to be taken into account. Inhaled salbutamol has been suggested as treatment of hypoxia in horses during general anesthesia, due to safety and ease of the technique. The present report describes the occurrence of clinically relevant unwanted cardiovascular effects (i.e. tachycardia and blood pressure modifications) in 5 horses undergoing general anesthesia in dorsal recumbency after salbutamol inhalation. Balanced anesthesia based on inhalation of isoflurane in oxygen or oxygen and air and continuous rate infusion (CRI) of lidocaine, romifidine, or combination of lidocaine and guaifenesine and ketamine was provided. Supportive measures were necessary to restore normal cardiovascular function in all horses but no long-term adverse effects were noticed in any of the cases

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability

Prochlorococcus contributes significantly to ocean primary productivity. The link between primary productivity and iron in specific ocean regions is well established and iron-limitation of Prochlorococcus cell division rates in these regions has been demonstrated. However, the extent of ecotypic variation in iron metabolism among Prochlorococcus and the molecular basis for differences is not understood. Here, we examine the growth and transcriptional response of Prochlorococcus strains, MED4 and MIT9313, to changing iron concentrations. During steady-state, MIT9313 sustains growth at an order-of-magnitude lower iron concentration than MED4. To explore this difference, we measured the whole-genome transcriptional response of each strain to abrupt iron starvation and rescue. Only four of the 1159 orthologs of MED4 and MIT9313 were differentially-expressed in response to iron in both strains. However, in each strain, the expression of over a hundred additional genes changed, many of which are in labile genomic regions, suggesting a role for lateral gene transfer in establishing diversity of iron metabolism among Prochlorococcus. Furthermore, we found that MED4 lacks three genes near the iron-deficiency induced gene (idiA) that are present and induced by iron stress in MIT9313. These genes are interesting targets for studying the adaptation of natural Prochlorococcus assemblages to local iron conditions as they show more diversity than other genomic regions in environmental metagenomic databases.Gordon and Betty Moore FoundationNational Science Foundation (U.S.) (Biological Oceanography)United States. Office of Naval Research (ONR Young Investigator Award)National Science Foundation (U.S.) (Chemical Oceanography)National Science Foundation (U.S.) (Environmental Genomics grants