The Effect of Various Degrees of Renal or Hepatic Impairment on the Pharmacokinetic Properties of Once-Weekly Insulin Icodec.

BACKGROUND AND OBJECTIVE: Icodec is a once-weekly insulin being developed to provide basal insulin coverage in diabetes mellitus. This study evaluated the effects of renal or hepatic impairment on icodec pharmacokinetics. METHODS: Two open-label, parallel-group, single-dose (1.5 U/kg subcutaneously) trials were conducted. In a renal impairment trial, 58 individuals were allocated to normal renal function (measured glomerular filtration rate ≥ 90 mL/min), mild (60 to < 90 mL/min), moderate (30 to < 60 mL/min) or severe (< 30 mL/min) renal impairment or end-stage renal disease. In a hepatic impairment trial, 25 individuals were allocated to normal hepatic function or mild (Child-Pugh Classification grade A), moderate (grade B) or severe (grade C) hepatic impairment. Blood was sampled frequently for a pharmacokinetic analysis until 35 days post-dose. RESULTS: The shape of the icodec pharmacokinetic profile was not affected by renal or hepatic impairment. Total icodec exposure was greater for mild (estimated ratio [95% confidence interval]: 1.12 [1.01; 1.24]), moderate (1.24 [1.12; 1.37]) and severe (1.28 [1.16; 1.42]) renal impairment, and for end-stage renal disease (1.14 [1.03; 1.28]), compared with normal renal function. It was also greater for mild (1.13 [1.00; 1.28]) and moderate (1.15 [1.02; 1.29]) hepatic impairment versus normal hepatic function. There was no statistically significant difference between severe hepatic impairment and normal hepatic function. Serum albumin levels (range 2.7-5.1 g/dL) did not statistically significantly influence icodec exposure. CONCLUSIONS: The clinical relevance of the slightly higher icodec exposure with renal or hepatic impairment is limited as icodec should be dosed according to individual need. No specific icodec dose adjustment is required in renal or hepatic impairment. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT03723785 and NCT04597697.

A flow equilibrium of zinc in cells of Cupriavidus metallidurans.

The hypothesis was tested that a kinetical flow equilibrium of uptake and efflux reactions is responsible for balancing the cellular zinc content. The experiments were done with the metal-resistant bacterium Cupriavidus metallidurans. In pulse-chase experiments, the cells were loaded with radioactive 65Zn and chased with the 100-fold concentration of non-radioactive zinc chloride. In parallel, the cells were loaded with isotope-enriched stable 67Zn and chased with non-enriched zinc to differentiate between zinc pools in the cell. The experiments demonstrated the existence of a kinetical flow equilibrium, resulting in a constant turnover of cell-bound zinc ions. The absence of the metal-binding cytoplasmic components, polyphosphate and glutathione, metal uptake, and metal efflux systems influenced the flow equilibrium. The experiments also revealed that not all zinc uptake and efflux systems are known in C. metallidurans. Cultivation of the cells under zinc-replete, zinc-, and zinc-magnesium-starvation conditions influenced zinc import and export rates. Here, magnesium starvation had a stronger influence compared to zinc starvation. Other metal cations, especially cobalt, affected the cellular zinc pools and zinc export during the chase reaction. In summary, the experiments with 65Zn and 67Zn demonstrated a constant turnover of cell-bound zinc. This indicated that simultaneously occurring import and export reactions in combination with cytoplasmic metal-binding components resulted in a kinetical flow equilibrium that was responsible for the adjustment of the cellular zinc content. IMPORTANCE: Understanding the biochemical action of a single enzyme or transport protein is the pre-requisite to obtain insight into its cellular function but this is only one half of the coin. The other side concerns the question of how central metabolic functions of a cell emerge from the interplay of different proteins and other macromolecules. This paper demonstrates that a flow equilibrium of zinc uptake and efflux reactions is at the core of cellular zinc homeostasis and identifies the most important contributors to this flow equilibrium: the uptake and efflux systems and metal-binding components of the cytoplasm.

A gold speciation that adds a second layer to synergistic gold-copper toxicity in Cupriavidus metallidurans.

The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.

Protecting the Achilles heel: three FolE_I-type GTP-cyclohydrolases needed for full growth of metal-resistant Cupriavidus metallidurans under a variety of conditions.

In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli. FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro, the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions.IMPORTANCETetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This "Achilles heel" of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.

Full Copper Resistance in Cupriavidus metallidurans Requires the Interplay of Many Resistance Systems.

The metal-resistant bacterium Cupriavidus metallidurans uses its copper resistance components to survive the synergistic toxicity of copper ions and gold complexes in auriferous soils. The cup, cop, cus, and gig determinants encode as central component the Cu(I)-exporting PIB1-type ATPase CupA, the periplasmic Cu(I)-oxidase CopA, the transenvelope efflux system CusCBA, and the Gig system with unknown function, respectively. The interplay of these systems with each other and with glutathione (GSH) was analyzed. Copper resistance in single and multiple mutants up to the quintuple mutant was characterized in dose-response curves, Live/Dead-staining, and atomic copper and glutathione content of the cells. The regulation of the cus and gig determinants was studied using reporter gene fusions and in case of gig also RT-PCR studies, which verified the operon structure of gigPABT. All five systems contributed to copper resistance in the order of importance: Cup, Cop, Cus, GSH, and Gig. Only Cup was able to increase copper resistance of the Δcop Δcup Δcus Δgig ΔgshA quintuple mutant but the other systems were required to increase copper resistance of the Δcop Δcus Δgig ΔgshA quadruple mutant to the parent level. Removal of the Cop system resulted in a clear decrease of copper resistance in most strain backgrounds. Cus cooperated with and partially substituted Cop. Gig and GSH cooperated with Cop, Cus, and Cup. Copper resistance is thus the result of an interplay of many systems. IMPORTANCE The ability of bacteria to maintain homeostasis of the essential-but-toxic "Janus"-faced element copper is important for their survival in many natural environments but also in case of pathogenic bacteria in their respective host. The most important contributors to copper homeostasis have been identified in the last decades and comprise PIB1-type ATPases, periplasmic copper- and oxygen-dependent copper oxidases, transenvelope efflux systems, and glutathione; however, it is not known how all these players interact. This publication investigates this interplay and describes copper homeostasis as a trait emerging from a network of interacting resistance systems.

The Sensory Histidine Kinase CusS of Escherichia coli Senses Periplasmic Copper Ions.

Two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR) are often associated with transenvelope efflux systems, which export transition metal cations from the periplasm directly out of the cell. Although much work has been done in this field, more evidence is needed for the hypothesis that the respective two-component regulatory systems are indeed sensing periplasmic ions. If so, a regulatory circuit between the concentration of periplasmic metal cations, sensing of these metals, and control of expression of the genes for transenvelope efflux systems that remove periplasmic cations can be assumed. Escherichia coli possesses only one transenvelope efflux system for metal cations, the Cus system for export of Cu(I) and Ag(I). It is composed of the transenvelope efflux system CusCBA, the periplasmic copper chaperone CusF, and the two-component regulatory system CusS (HK) and CusR (RR). Using phoA- and lacZ-reporter gene fusions, it was verified that an assumed periplasmic part of CusS is located in the periplasm. CusS was more important for copper resistance in E. coli under anaerobic conditions than under aerobic conditions and in complex medium more than in mineral salts medium. Predicted copper-binding sites in the periplasmic part of CusS were identified that, individually, were not essential for copper resistance but were in combination. In summary, evidence was obtained that the two-component regulatory system CusSR that controls expression of cusF and cusCBA does indeed sense periplasmic copper ions. IMPORTANCE Homeostasis of essential-but-toxic transition metal cations such as Zn(II) and Cu(II)/Cu(I) is an important contributor to the fitness of environmental bacteria and pathogenic bacteria during their confrontation with an infected host. Highly efficient removal of threatening concentrations of these metals can be achieved by the combined actions of an inner membrane with a transenvelope efflux system, which removes periplasmic ions after their export from the cytoplasm to this compartment. To understand the resulting metal cation homeostasis in the periplasm, it is important to know if a regulatory circuit exists between periplasmic metal cations, their sensing, and the subsequent control of the expression of the transenvelope efflux system. This publication adds evidence to the hypothesis that two-component regulatory systems in control of the expression of genes for transenvelope efflux systems do indeed sense metal cations in the periplasm.

Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes.

Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR2S2, and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR2 were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR2S2 interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.

Importance of RpoD- and Non-RpoD-Dependent Expression of Horizontally Acquired Genes in Cupriavidus metallidurans.

The genome of the metal-resistant, hydrogen-oxidizing bacterium Cupriavidus metallidurans contains a large number of horizontally acquired plasmids and genomic islands that were integrated into its chromosome or chromid. For the C. metallidurans CH34 wild-type strain growing under nonchallenging conditions, 5,763 transcriptional starting sequences (TSSs) were determined. Using a custom-built motif discovery software based on hidden Markov models, patterns upstream of the TSSs were identified. The pattern TTGACA, -35.6 ± 1.6 bp upstream of the TSSs, in combination with a TATAAT sequence 15.8 ± 1.4 bp upstream occurred frequently, especially upstream of the TSSs for 48 housekeeping genes, and these were assigned to promoters used by RNA polymerase containing the main housekeeping sigma factor RpoD. From patterns upstream of the housekeeping genes, a score for RpoD-dependent promoters in C. metallidurans was derived and applied to all 5,763 TSSs. Among these, 2,572 TSSs could be associated with RpoD with high probability, 373 with low probability, and 2,818 with no probability. In a detailed analysis of horizontally acquired genes involved in metal resistance and not involved in this process, the TSSs responsible for the expression of these genes under nonchallenging conditions were assigned to RpoD- or non-RpoD-dependent promoters. RpoD-dependent promoters occurred frequently in horizontally acquired metal resistance and other determinants, which should allow their initial expression in a new host. However, other sigma factors and sense/antisense effects also contribute-maybe to mold in subsequent adaptation steps the assimilated gene into the regulatory network of the cell. IMPORTANCE In their natural environment, bacteria are constantly acquiring genes by horizontal gene transfer. To be of any benefit, these genes should be expressed. We show here that the main housekeeping sigma factor RpoD plays an important role in the expression of horizontally acquired genes in the metal-resistant hydrogen-oxidizing bacterium C. metallidurans. By conservation of the RpoD recognition consensus sequence, a newly arriving gene has a high probability to be expressed in the new host cell. In addition to integrons and genes travelling together with that for their sigma factor, conservation of the RpoD consensus sequence may be an important contributor to the overall evolutionary success of horizontal gene transfer in bacteria. Using C. metallidurans as an example, this publication sheds some light on the fate and function of horizontally acquired genes in bacteria.

Loss of Mobile Genomic Islands in Metal-Resistant, Hydrogen-Oxidizing Cupriavidus metallidurans.

The genome of the metal-resistant, hydrogen-oxidizing bacterium Cupriavidus metallidurans strain CH34 contains horizontally acquired plasmids and genomic islands. Metal-resistance determinants on the two plasmids may exert genetic dominance over other related determinants. To investigate whether these recessive determinants can be activated in the absence of the dominant ones, the transcriptome of the highly zinc-sensitive deletion mutant Δe4 (ΔcadA ΔzntA ΔdmeF ΔfieF) of the plasmid-free parent AE104 was characterized using gene arrays. As a consequence of some unexpected results, close examination by PCR and genomic resequencing of strains CH34, AE104, Δe4, and others revealed that the genomic islands CMGI2, 3, 4, D, and E, but no other islands or recessive determinants, were deleted in some of these strains. Provided that wild-type CH34 was kept under alternating zinc and nickel selection pressure, no comparable deletions occurred. All current data suggest that genes were actually deleted and were not, as surmised previously, silenced in the respective strain. As a consequence, a cured database was compiled from the newly generated and previously published gene array data. An analysis of data from this database indicated that some genes of recessive, no longer needed determinants were nevertheless expressed and upregulated. Their products may interact with those of the dominant determinants to mediate a mosaic phenotype. The ability to contribute to such a mosaic phenotype may prevent deletion of the recessive determinant. The data suggest that the bacterium actively modifies its genome to deal with metal stress and at the same time ensures metal homeostasis. IMPORTANCE In their natural environment, bacteria continually acquire genes by horizontal gene transfer, and newly acquired determinants may become dominant over related ones already present in the host genome. When a bacterium is taken into laboratory culture, it is isolated from the horizontal gene transfer network. It can no longer gain genes but instead may lose them. This phenomenon was indeed observed in Cupriavidus metallidurans for the loss key metal resistance determinants when no selection pressure was kept continuously. However, some recessive metal resistance determinants were maintained in the genome. It is proposed that they might contribute some accessory genes to related dominant resistance determinants, for instance periplasmic metal-binding proteins or two-component regulatory systems. Alternatively, they may remain in the genome only because their DNA serves as a scaffold for the nucleoid. Using C. metallidurans as an example, this study sheds light on the fate and function of horizontally acquired genes in bacteria.

Behind the shield of Czc: ZntR controls expression of the gene for the zinc-exporting P-type ATPase ZntA in Cupriavidus metallidurans.

In the metallophilic beta-proteobacterium Cupriavidus metallidurans, the plasmid-encoded Czc metal homeostasis system adjusts the periplasmic zinc, cobalt and cadmium concentration, which influences subsequent uptake of these metals into the cytoplasm. Behind this shield, the PIB2-type APTase ZntA is responsible for removal of surplus cytoplasmic zinc ions, thereby providing a second level of defense against toxic zinc concentrations. ZntA is the counterpart to the Zur-regulated zinc uptake system ZupT and other import systems; however, the regulator of zntA expression was unknown. The chromid-encoded zntA gene is adjacent to the genes czcI2C2B2', which are located on the complementary DNA strand and transcribed from a common promoter region. These genes encode homologs of plasmid pMOL30-encoded Czc components. Candidates for possible regulators of zntA were identified and subsequently tested: CzcI, CzcI2, and the MerR-type gene products of the locus tags Rmet_2302, Rmet_0102, Rmet_3456. This led to the identification of Rmet_3456 as ZntR, the main regulator of zntA expression. Moreover, both CzcIs decreased Czc-mediated metal resistance, possibly to avoid "over-excretion" of periplasmic zinc ions, which could result in zinc starvation due to diminished zinc uptake into the cytoplasm. Rmet_2302 was identified as CadR, the regulator of the cadA gene for an important cadmium-exporting PIB2-type ATPase, which provides another system for removal of cytoplasmic zinc and cadmium. Rmet_0102 was not involved in regulation of the metal resistance systems examined here. Thus, ZntR forms a complex regulatory network with CadR, Zur and the CzcIs. Moreover, these discriminating regulatory proteins assign the efflux systems to their particular function.ImportanceZinc is an essential metal for numerous organisms from humans to bacteria. The transportome of zinc uptake and efflux systems controls the overall cellular composition and zinc content in a double feed-back loop. Zinc starvation mediates, via the Zur regulator, an up-regulation of the zinc import capacity via the ZIP-type zinc importer ZupT and an amplification of zinc storage capacity, which together raise the cellular zinc content again. On the other hand, an increasing zinc content leads to ZntR-mediated up-regulation of the zinc efflux system ZntA, which decreases the zinc content. Together, the Zur regulon components and ZntR/ZntA balance the cellular zinc content under both high external zinc concentrations and zinc starvation conditions.

Albumin-based nanoparticles as contrast medium for MRI: vascular imaging, tissue and cell interactions, and pharmacokinetics of second-generation nanoparticles.

This multidisciplinary study examined the pharmacokinetics of nanoparticles based on albumin-DTPA-gadolinium chelates, testing the hypothesis that these nanoparticles create a stronger vessel signal than conventional gadolinium-based contrast agents and exploring if they are safe for clinical use. Nanoparticles based on human serum albumin, bearing gadolinium and designed for use in magnetic resonance imaging, were used to generate magnet resonance images (MRI) of the vascular system in rats ("blood pool imaging"). At the low nanoparticle doses used for radionuclide imaging, nanoparticle-associated metals were cleared from the blood into the liver during the first 4 h after nanoparticle application. At the higher doses required for MRI, the liver became saturated and kidney and spleen acted as additional sinks for the metals, and accounted for most processing of the nanoparticles. The multiple components of the nanoparticles were cleared independently of one another. Albumin was detected in liver, spleen, and kidneys for up to 2 days after intravenous injection. Gadolinium was retained in the liver, kidneys, and spleen in significant concentrations for much longer. Gadolinium was present as significant fractions of initial dose for longer than 2 weeks after application, and gadolinium clearance was only complete after 6 weeks. Our analysis could not account quantitatively for the full dose of gadolinium that was applied, but numerous organs were found to contain gadolinium in the collagen of their connective tissues. Multiple lines of evidence indicated intracellular processing opening the DTPA chelates and leading to gadolinium long-term storage, in particular inside lysosomes. Turnover of the stored gadolinium was found to occur in soluble form in the kidneys, the liver, and the colon for up to 3 weeks after application. Gadolinium overload poses a significant hazard due to the high toxicity of free gadolinium ions. We discuss the relevance of our findings to gadolinium-deposition diseases.

Mutant Strains of Escherichia coli and Methicillin-Resistant Staphylococcus aureus Obtained by Laboratory Selection To Survive on Metallic Copper Surfaces.

Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or "shallow" persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains.IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.

A copper site is required for iron transport by the periplasmic proteins P19 and FetP.

Campylobacter jejuni is a leading cause of food-borne gastrointestinal disease in humans and uropathogenic Escherichia coli is a leading cause of urinary tract infections. Both human pathogens harbour a homologous iron uptake system (termed cjFetM-P19 in C. jejuni and ecFetM-FetP in E. coli). Although these systems are important for growth under iron limitation, the mechanisms by which these systems function during iron transport remain undefined. The copper ions bound to P19 and FetP, the homologous periplasmic proteins, are coordinated in an uncommon penta-dentate manner involving a Met-Glu-His3 motif and exhibit positional plasticity. Here we demonstrate the function of the Met and Glu residues in modulating copper binding and controlling copper positioning through site-directed variants, binding assays, and crystal structures. Growth of C. jejuni strains with these p19 variants is impaired under iron limited conditions as compared to the wild-type strain. Additionally, an acidic residue-rich secondary site is required for binding iron and function in vivo. Finally, western blot analyses demonstrate direct and specific interactions between periplasmic P19 and FetP with the large periplasmic domain of their respective inner membrane transporters cjFetM and ecFetM.

Nanoscale Organolanthanum Clusters: Nuclearity-Directing Role of Cyclopentadienyl and Halogenido Ligands.

Tetramethylaluminato/halogenido(X) ligand exchange reactions in half-sandwich complexes [CpR La(AlMe4 )2 ] are feasible in non-coordinating solvents and provide access to large coordination clusters of the type [CpR LaX2 ]x . Incomplete exchange reactions generate the hexalanthanum clusters [CpR 6 La6 X8 (AlMe4 )4 ] (CpR =Cp*=C5 Me5 , X=I; CpR =Cp'=C5 H4 SiMe3 , X=Br, I). Treatment of [Cp*La(AlMe4 )2 ] with two equivalents Me3 SiI gave the nonalanthanum cluster [Cp*LaI2 ]9 , while the exhaustive reaction of [Cp'La(AlMe4 )2 ] with the halogenido transfer reagents Me3 GeX and Me3 SiX (X=I, Br, Cl) produced a series of monocyclopentadienyl rare-earth-metal clusters with distinct nuclearity. Depending on the halogenido ion size the homometallic clusters [Cp'LaCl2 ]10 and [Cp'LaX2 ]12 (X=Br, I) could be isolated, whereas different crystallization techniques led to the aggregation of clusters of distinct structural motifs, including the desilylated cyclopentadienyl-bridged cluster [(µ-Cp)2 Cp'8 La8 I14 ] and the heteroaluminato derivative [Cp'10 La10 Br18 (AlBr2 Me2 )2 ]. The use of the Cp' ancillary ligand facilitates cluster characterization by means of NMR spectroscopy.

Head motion predictability explains activity-dependent suppression of vestibular balance control.

Vestibular balance control is dynamically weighted during locomotion. This might result from a selective suppression of vestibular inputs in favor of a feed-forward balance regulation based on locomotor efference copies. The feasibility of such a feed-forward mechanism should however critically depend on the predictability of head movements (HMP) during locomotion. To test this, we studied in 10 healthy subjects the differential impact of a stochastic vestibular stimulation (SVS) on body sway (center-of-pressure, COP) during standing and walking at different speeds and compared it to activity-dependent changes in HMP. SVS-COP coupling was determined by correlation analysis in frequency and time domains. HMP was quantified as the proportion of head motion variance that can be explained by the average head trajectory across the locomotor cycle. SVS-COP coupling decreased from standing to walking and further dropped with faster locomotion. Correspondingly, HMP increased with faster locomotion. Furthermore, SVS-COP coupling depended on the gait-cycle-phase with peaks corresponding to periods of least HMP. These findings support the assumption that during stereotyped human self-motion, locomotor efference copies selectively replace vestibular cues, similar to what was previously observed in animal models.

The ancient alarmone ZTP and zinc homeostasis in Bacillus subtilis.

In Bacillus subtilis a sophisticated regulatory circuit that involves Z nucleoside triphosphate (ZTP) is recruited to optimize cellular zinc distribution when cytoplasmic zinc is scarce. This process uses enzymatic reactions to measure the pool of available zinc ions and amplifies this signal to control the activity of zinc chaperones. The ZTP-dependent regulatory circuit that is exploited for zinc homeostasis controls purine and folate biosynthesis, which starts with GTP as initial substrate. Low concentrations of formyl-tetrahydrofolate (fTHF) lead to accumulation of the intermediate 5'-phosphoribosyl-4-carboxyamide-5-aminoimidazole (AICAR or ZMP), which is pyrophosphorylated by another intermediate to ZTP. This alarmone activates expression of genes using a ZTP-dependent riboswitch in many bacterial strains. In this way, the cellular folate concentration controls folate biosynthesis via the enzymatic activity of the fTHF-dependent AICAR-transforming reaction. Zinc distribution control is layered onto this circuit. The 'sensor' is the activity of the initial reaction of folate synthesis from GTP, which is catalyzed by a zinc-dependent enzyme FolEIA or its metal-cambialistic paralog FolEIB . Consequently, low zinc lowers folate levels, causing AICAR accumulation and ZTP formation. In addition to the riboswitch, ZTP activates the zinc chaperone ZagA of the COG0523 protein family, which efficiently allocate zinc to zinc-dependent enzymes such as FolEIA .

Interplay between the Zur Regulon Components and Metal Resistance in Cupriavidus metallidurans.

The Zur regulon is central to zinc homeostasis in the zinc-resistant bacterium Cupriavidus metallidurans It comprises the transcription regulator Zur, the zinc importer ZupT, and three members of the COG0523 family of metal-chaperoning G3E-type GTPases, annotated as CobW1, CobW2, and CobW3. The operon structures of the zur and cobW1 loci were determined. To analyze the interplay between the Zur regulon components and metal resistance, deletion mutants were constructed from the wild-type strain CH34 and various other strains. The Zur regulon components interacted with the plasmid-encoded and chromosomally encoded metal resistance factors to acquire metals from complexes of EDTA and for homeostasis of and resistance to zinc, nickel, cobalt, and cadmium. The three G3E-type GTPases were characterized in more detail. CobW1 bound only 1 Zn atom per mol of protein with a stability constant slightly above that of 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (Zincon) and an additional 0.5 Zn with low affinity. The CobW1 system was necessary to obtain metals from EDTA complexes. The GTPase CobW2 is a zinc storage compound and bound 0.5 to 1.5 Zn atoms tightly and up to 6 more with lower affinity. The presence of MgGTP unfolded the protein partially. CobW3 had no GTPase activity and equilibrated metal import by ZupT with that of the other metal transport systems. It sequestered 8 Zn atoms per mol with decreasing affinity. The three CobWs bound to the metal-dependent protein FolEIB2, which is encoded directly downstream of cobW1 This demonstrated an important contribution of the Zur regulon components to metal homeostasis in C. metalliduransIMPORTANCE Zinc is an important transition metal cation and is present as an essential component in many enzymes, such as RNA polymerase. As with other transition metals, zinc is also toxic at higher concentrations so that living cells have to maintain strict control of their zinc homeostasis. Members of the COG0523 family of metal-chaperoning GE3-type GTPases exist in archaea, bacteria, and eucaryotes, including humans, and they may be involved in delivery of zinc to thousands of different proteins. We used a combination of molecular, physiological, and biochemical methods to demonstrate the important but diverse functions of COG0523 proteins in C. metallidurans, which are produced as part of the Zur-controlled zinc starvation response in this bacterium.

Evaluation of a biosurfactant extract obtained from corn for dermal application.

At present, there is an increasing demand to improve the sustainability of surface-active compounds in dermal formulations. Biosurfactants, which are derived from living cells, are considered to be more environmentally friendly than synthetic surfactants. Thus, the use of biosurfactants is a promising strategy for the formulation of more environmentally friendly and sustainable dermal products. In this work, a biosurfactant extract (BS) obtained from corn wet-milling industry was studied for its potential use in dermal formulations. The corn derived BS possesses good surface-active properties and was found to be a suitable co-stabilizer for nanoemulsions and nanocrystals for dermal application. It also possesses antioxidative and skin protective properties and was also able to increase the dermal penetration efficacy for lipophilic actives. In dermal formulations the BS can therefore be used as co-stabilizer with antioxidative and penetration enhancing properties at the same time.

Strategies for Gaze Stabilization Critically Depend on Locomotor Speed.

Locomotion involves complex combinations of translational and rotational head movements. For gaze stability, this necessitates the interplay of angular and linear vestibulo-ocular reflexes (VOR) as well as the integration of visual feedback about the desired viewing distance. Furthermore, gaze stabilizing systems must be able to cope with vast differences in head motion brought about by changing locomotor speeds and patterns (walking vs. running). The present study investigated horizontal and vertical angular VOR (aVOR) and linear gaze stabilization (lGS) as well as compensation for linear head movements by angular counter rotation of the head during treadmill walking and running at different velocities (0.4 to 2.4 m/s) while fixating either a close (0.5 m) or distant (2.0 m) target. In the horizontal plane, the aVOR predominated throughout all locomotor speeds, whereas the compensation of linear translations was highly variable and generally insufficient. In contrast, in the vertical plane, eye and angular head motion steadily became more in phase with increasing locomotor speed, which served to optimize linear motion compensation. Furthermore, the timing of the vertical aVOR became more automated and independent of visual feedback during faster locomotion. Thus, horizontal and vertical gaze stabilization strategies appear to be considerably different. Whereas horizontal gaze control is likely governed by passive sensorimotor reflexes throughout all locomotor speeds, vertical gaze stabilization switches to an automated feed-forward control at faster locomotion. This switch is presumably driven by efference copies from spinal locomotor commands that were previously shown to govern gaze stabilization in animal models during stereotypic locomotion.

The third pillar of metal homeostasis in Cupriavidus metallidurans CH34: preferences are controlled by extracytoplasmic function sigma factors.

The role of extracytoplasmic function (ECF) sigma factors in multiple metal homeostasis of the metallophilic bacterium Cupriavidus metallidurans was studied. RNA sequencing was used to predict 3084 operons in the genome of this bacterium, including 11 for ECF sigma factors, and to measure transcript abundances. Mutants carrying multiple deletions in genes for ECF sigma factors were constructed and characterized. Mutants and parent were challenged with a metal mix, changes in the global gene expression profile and the overall metal content determined. All 11 ECF sigma factors were involved in metal homeostasis. The three ECF sigma factors RpoI, RpoJ and RpoK synchronized iron homeostasis with that of other divalent metal cations, RpoO, RpoL and RpoM magnesium and phosphorous homeostasis with that of zinc and with cadmium resistance. Factors RpoE, CnrH and RpoP controlled the response to nickel and cobalt, RpoQ and RpoR may be assigned to the thiol and sulfide metabolism. All 11 ECF sigma factors overlap in their function and control gene expression involved in metal homeostasis, however, except CnrH, no other ECF sigma factor was needed for up-regulation of 63 predicted operons responding to metal shock, 48 of these encoding metal efflux pumps. Moreover, disturbance of the cellular metal content resulting from missing sigma factors also affected silencing and un-silencing of genomic islands. Together, these data demonstrate on a global and systemic level how a robust network of ECF sigma factors and other regulators allow C. metallidurans to handle a mixture of toxic transition metal cations, which are conditions the bacterium faces in its natural environment. Iron homeostasis is to be maintained at any cost, followed by the necessity for magnesium, phosphorous and zinc homeostasis on the second level, and cobalt plus nickel coming last.