Ruthenium 4d-to-2p X-ray Emission Spectroscopy: A Simultaneous Probe of the Metal and the Bound Ligands.

Ruthenium 4d-to-2p X-ray emission spectroscopy (XES) was systematically explored for a series of Ru2+ and Ru3+ species. Complementary density functional theory calculations were utilized to allow for a detailed assignment of the experimental spectra. The studied complexes have a range of different coordination spheres, which allows the influence of the ligand donor/acceptor properties on the spectra to be assessed. Similarly, the contributions of the site symmetry and the oxidation state of the metal were analyzed. Because the 4d-to-2p emission lines are dipole-allowed, the spectral features are intense. Furthermore, in contrast with K- or L-edge X-ray absorption of 4d transition metals, which probe the unoccupied levels, the observed 4p-to-2p XES arises from electrons in filled-ligand- and filled-metal-based orbitals, thus providing simultaneous access to the ligand and metal contributions to bonding. As such, 4d-to-2p XES should be a promising tool for the study of a wide range of 4d transition-metal compounds.

Clinical and microbiological features of bacteraemia with Gram-positive anaerobic cocci: a population-based retrospective study.

OBJECTIVES: Gram-positive, anaerobic cocci (GPAC) can cause infections in humans. Only a few cases of bacteraemia with GPAC have been reported. We describe the clinical and microbiological characteristics of GPAC bacteraemia. METHODS: A retrospective population-based study of GPAC bacteraemia 2012-2016 in southern Sweden was performed. GPAC were identified using matrix-associated laser desorption ionization time-of-flight mass spectrometry or 16S rRNA gene sequencing. Etests were used to determine antibiotic susceptibilities. Data on patient and infection characteristics, treatment, and outcome were collected from the medical records. RESULTS: A total of 226 episodes of GPAC bacteraemia in adults were studied; this corresponds to an annual incidence of 3.4 cases per 100,000 persons per year. The bacteria identified were Anaerococcus spp. (n = 43), Atopobium spp. (n = 7), Blautia spp. (n = 1), Finegoldia spp. (n = 15), Parvimonas spp. (n = 100), Peptoniphilus spp. (n = 52), Peptostreptococcus spp. (n = 2), and Ruminococcus spp. (n = 9) of which 200 isolates were identified to the species level. Resistance to imipenem and piperacillin was not identified, whereas resistance among the 229 isolates to penicillin was detected in four, to metronidazole in six, and clindamycin in 16 isolates. The median age of patients was 73 years (55-83, IQR), 57% were male and comorbidities were common. Fifty-one per cent of infections were polymicrobial. In 60% of cases a focus of infection was identified. Forty per cent of patients had either organ dysfunction or shock. The 30-day mortality was 11%, and nosocomial infections were over-represented among the deceased. CONCLUSIONS: GPAC bacteraemia is much more common than previously reported. GPAC-bacteraemia is a condition with significant mortality mainly affecting elderly persons with comorbidities.

Transport of chaotic trajectories from regions distant from or near to structures of regular motion of the Fermi-Ulam model.

The chaotic portion of phase space of the simplified Fermi-Ulam model is studied under the context of transport of trajectories in two scenarios: (i) the trajectories are originated from a region distant from the islands of regular motion and are transported to a region located at a high portion of phase space and (ii) the trajectories are originated from chaotic regions around the islands of regular motion and are transported to other regions around islands of regular motion. The transport is investigated in terms of the observables histogram of transport and survival probability. We show that the histogram curves are scaling invariant and we organize the survival probability curves in four kinds of behavior, namely (a) transition from exponential decay to power law decay, (b) transition from exponential decay to stretched exponential decay, (c) transition from an initial fast exponential decay to a slower exponential decay, and (d) a single exponential decay. We show that, depending on choice of the regions of origin and destination, the transport process is weakly affected by the stickiness of trajectories around islands of regular motion.

Clinical presentation of infective endocarditis caused by different groups of non-beta haemolytic streptococci.

Streptococci are common causes of infective endocarditis (IE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has provided a practical tool for their species determination. We aimed to investigate if particular groups of non-beta heamolytic streptococci were associated with IE or to specific presentations thereof. The Swedish Registry of Infective Endocarditis was used to identify cases of IE caused by streptococci and a local database to identify cases of streptococcal bacteremia. The bacteria were grouped using MALDI-TOF MS and the clinical characteristics of IE caused by different groups were compared. We identified a group of 201 streptococcal IE isolates: 18 isolates belonged to the anginosus, 19 to the bovis, 140 to the mitis, 17 to the mutans, and seven to the salivarius groups. The mitis and mutans groups were significantly more common and the anginosus group less common among IE cases as compared to all cause bacteremia. Patients infected with the bovis group isolates were older, had more cardiac devices, and had more commonly prosthetic valve IE compared to IE caused by streptococci of the other groups. Twenty-one percent of patients needed surgery, and in-hospital mortality was 8% with no significant differences between the groups. Grouping of non-beta haemolytic streptococci using MALDI-TOF MS can provide a basis for decision-making in streptococcal bacteremia. IE caused by bovis group isolates have clinical characteristics distinguishing them from IE caused by other groups of Streptococcus.

Paradigms and public policies on drought in northeast Brazil: a historical perspective.

This paper describes the evolution of drought-related public policies in Northeast Brazil (NEB). Using a historical approach, we show that the evolution of public policy has not been characterized by abrupt shifts, but has instead been shaped through debates between renowned intellectuals. The resulting public policies formed a hydrological infrastructure that delivers clean water needed for robust economic activity. However, outcomes of the 2012-2013 drought show that populations that depend on rain fed agriculture are as vulnerable to drought as they were at the start of the 20th century. Although government, social, and emergency programs have aided drought victims, drought analysts agree that rain fed agriculture has remained vulnerable since drought policies were first formulated. Drought policies formulate integrated water resources management (IWRM) strategies that are geared toward supplying safe drinking water, and debates surrounding the IWRM paradigm have been affected by outcomes of major international events such as the World Water Forum.

Secas e políticas públicas no semiárido: ideias, pensadores e períodos / Drought and public policy in the semiarid: ideas, thinkers and periods

O artigo analisa a evolução das políticas públicas contra as secas praticadas no Nordeste a partir do período Colonial. Uma proposta de periodização com base nas políticas predominantes é apresentada. A periodização é organizada em cinco fases: 1) defrontando-se com as secas; 2) a busca do conhecimento; 3) a hidráulica da solução; 4) o desenvolvimento regional; 5) a gestão das águas e o desenvolvimento sustentável. As lógicas e os pensamentos dos principais intelectuais que deram suporte a essas políticas são objetos de análise e discussão.

The article analyzes the evolution of public policies against drought in the Northeast since the colonial period. A timeline based on the prevailing policies is proposed. The periodization was divided into five phases: 1) facing the droughts, 2) the construction of the knowledge, 3) the hydraulic phase of the solution, 4) the regional development, 5) the water management and sustainable development. The logic and the thoughts of the leading intellectuals who have supported these policies are subject to review and discussion.

Comparison of the levels of quality of inpatient care delivered by pediatrics residents and by private, community pediatricians at one hospital.

PURPOSE: To compare the quality and cost of inpatient care provided by pediatrics residents with the quality and cost of that provided by private, practicing pediatricians. METHOD: Valley Children's Hospital--a 175-bed, private, nonprofit, university-affiliated pediatric hospital located in Fresno, California--has both a resident service and a community physician service. From May 1994 to March 1995 a total of 154 pediatric patients (64 from the resident service, 90 from the community service) were selected for the study using incidental sampling. In order to be included in the study, patients had discharge diagnoses of uncomplicated gastroenteritis or asthma. The parent or guardian of each patient completed a satisfaction questionnaire at discharge and agreed to a home visit one month later, when the same questionnaire and another were completed. Patients' charts were studied for treatment procedures, and follow-up data for a year were compiled. Statistical analyses were conducted with analysis of variance for continuous measures and chi-squares for dichotomous variables. RESULTS: The findings indicated no difference between the two services in terms of the parents' and guardians' satisfaction with patient care, hospital charges, and degrees of adherence to follow-up care after discharge. The community physicians were more likely than were the residents to employ non-standard laboratory and management procedures with both the asthma and gastroenteritis patients. The asthma patients cared for by community physicians had significantly more frequent asthma attacks after discharge, with a higher frequency of patients being subsequently treated in the emergency room and readmitted to the hospital, than did the asthma patients cared for by the residents. CONCLUSION: Common perceptions that physicians-in-training (1) overuse medical services and (2) fail, due to their inexperience, to provide high-quality services were not supported in this study.

Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity.

Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.

Targeting of retroviral vectors through protease-substrate interactions.

Targetable, injectable vectors would greatly facilitate the development of in vivo therapy strategies. Viral and nonviral vectors can be targeted through ligand-receptor interactions, but protease-substrate interactions have not previously been exploited for vector targeting. Epidermal growth factor (EGF) was fused to a retroviral envelope glycoprotein via a cleavable linker comprising a factor Xa protease recognition signal. Vector particles displaying the cleavable EGF domain could bind to EGF receptors on human cells but did not transfer their genes until they were cleaved by factor Xa protease, whereupon gene delivery proceeded normally. Proteolytic activation of receptor-targeted vectors can therefore provide the basis for a novel two-step targeting strategy that may facilitate efficient targeted in vivo delivery of therapeutic genes.

Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU.

We previously reported a strategy to redirect the retroviral host range by expressing single-chain antibodies (S. J. Russell, R. E. Hawkins, and G. Winter, Nucleic Acids Res. 21:1081-1085, 1993) or ligands (F.-L. Cosset, F. J Morling, Y. Takeuchi, R. A. Weiss, M. K. L. Collins, and S. J. Russell, J. Virol. 69:6314-6322, 1995) at the N terminus of Moloney murine leukemia virus (MoMLV) surface proteins (SU). Although such chimeric envelopes were able to bind the new receptors, the transduction efficiency of retargeted viruses was generally low. We hypothesized that conformational rearrangements of envelope glycoproteins were not optimally triggered following binding, and to overcome these postbinding blocks, we have generated here a set of chimeric MoMLV-derived envelopes targeted to the Ram-1 phosphate transporter in which we have varied the spacing between the Ram-1-binding domain and the MoMLV SU. All of the recombinant envelopes were correctly expressed on virions, and all bound efficiently to Ram-1. However, the interdomain spacing greatly affected the efficiency of gene transfer by retroviral vectors that had bound to Ram-1 via their chimeric envelopes. Optimal interdomain spacing allowed a 100-fold-increased viral transduction via Ram-1 compared to our previous results.

Structure and stability of protein H and the M1 protein from Streptococcus pyogenes. Implications for other surface proteins of gram-positive bacteria.

M proteins and other members of the M protein family, expressed on the surface of Streptococcus pyogenes, bind host proteins such as immunoglobulins, albumin, and fibrinogen. Protein H and the M1 protein are expressed by adjacent genes and both belong to the M protein family. In this work, the structure and stability of these two proteins have been investigated. As judged from sequence analysis and circular dichroism spectroscopy, the proteins are almost entirely in an alpha-helix conformation. The amino acids are arranged in a seven-residue (heptad) repeat pattern along the greater part of the proteins. These observations support the previously accepted model of M proteins as coiled-coil dimers. However, it was also found that the structures of both proteins were thermally unstable; i.e., the content of helix conformation was greatly reduced at 37 degrees C as compared to 25 degrees C or below. Together with previous findings that these proteins appear as monomers at 37 degrees C and dimers at low temperatures, the results suggest that the coiled-coil dimers are unfolded at 37 degrees C. The heptad patterns of protein H and the M1 protein showed a nonoptimal distribution of residues expected for a coiled-coil conformation. This is a possible explanation for the low thermal stability of the proteins. It was also demonstrated that the proteins were stabilized in the presence of the ligands IgG and/or albumin. Protein H and M1 protein show a high degree of sequence similarity in their C-terminal regions, and a fragment from this region displayed a high content of helix conformation, whereas fragments from the nonsimilar N-terminal parts did not adopt any stable folded structure. Thus, the C-terminal parts, which are conserved within the M protein family, may constitute a framework for the formation of the parallel helical coiled-coil structure, and we propose that the less stable N-terminal part may also participate in antiparallel interaction with M proteins on adjacent bacteria. The results suggest that temperature fluctuations in the environment could change the properties of bacterial surface proteins, thereby affecting the molecular interactions between the bacterium and its host.

On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins.

Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.

Increased risk of Pneumocystis carinii pneumonia in patients with Wegener's granulomatosis.

Combining cyclophosphamide (Cy) and corticosteroids has dramatically improved the prognosis of Wegener's granulomatosis (WG). But this treatment carries the risks of severe infectious complications and drug toxicity. During a 10-month period, we observed 6 cases of Pneumocystis carinii pneumonia (PCP) in 23 patients with biopsy-proven WG and renal involvement. These 23 patients were enrolled in a multicenter controlled clinical trial designed to evaluate the efficacy and safety of either intermittent high-dose pulse Cy or daily oral low-dose Cy in combination with oral prednisone. Mean delay of onset of PCP was 2.5 months after the beginning of the immunosuppressive therapy. In all cases, the diagnosis of PCP was established by cytological examination of bronchoalveolar lavage fluid. None of the patients experienced severe leukopenia at the time of diagnosis, but the mean lymphocyte count decreased to 495/mm3 (range 100 to 830/mm3) and 2 patients had inverted CD4/CD8 T-cell ratios. Renal function was significantly impaired (creatininemia = 493.5 vs 195.4 micromol/l; p = 0.03) in the 6 patients presenting PCP vs those without. High-dose co-trimoxazole therapy was successful in 3 patients, but 3 others who required mechanical ventilation died. Treatment of WG with daily prednisone and either pulse or oral Cy may have contributed to higher rates of PCP in the past than previously thought and, therefore, patients currently receiving such a regimen may be at greater risk for PCP. For these patients, this opportunistic infection must remain highly suspect in order to reach a diagnosis earlier and rapidly initiate treatment. In addition, recommendations for prophylactic therapy are needed.

Purification of antibodies using protein L-binding framework structures in the light chain variable domain.

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

On the interaction between protein L and immunoglobulins of various mammalian species.

Protein L, a cell wall molecule of certain strains of the anaerobic bacterial species Peptostreptococcus magnus, shows high affinity for human immunoglobulin (Ig) light chains. In the present study protein L was tested against a panel of human myeloma proteins of the IgG, IgM, IgA and IgE classes, and strong binding was seen with antibodies carrying kappa light chains. A high degree of specificity for Ig was demonstrated in binding experiments with human plasma proteins. Apart from human Ig, strong protein L-binding activity was also detected in the serum of 12 out of 23 tested additional mammalian species, including other primates and rodents. Subsequent analysis with purified Ig samples demonstrated the binding of protein L to Ig of important laboratory animal species such as the mouse, the rat and the rabbit. The affinity constants for the interactions between protein L and polyclonal IgG of these species were 2.6 x 10(9), 3.9 x 10(8) and 7.4 x 10(7), respectively. In non-human species, the binding of protein L was also found to be mediated through Ig light chains, and the results demonstrate the potential value of protein L as an immunochemical tool.

Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain.

Protein L is an immunoglobulin light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The major variable region subgroups of human kappa and lambda light chains were tested for protein L binding; V kappa I, V kappa III, and V kappa IV bound protein L, whereas no binding occurred with proteins of the V kappa II subgroup or with any lambda light chain subgroups. Studies of the protein L binding capacity of naturally occurring VL fragments, and VL- and CL-related trypsin- and pepsin-derived peptides prepared from a kappa I light chain, localized the site of interaction to the VL domain. The affinity constant for the binding to an isolated V kappa I fragment was comparable to that for the native protein (Ka 0.9 x 10(9) M-1 and Ka 1.5 x 10(9) M-1, respectively). No binding occurred with CL-related fragments. Extensive reduction and alkylation of the V kappa fragment or the native kappa chain resulted in complete loss of protein L binding. Although it is possible, from comparative amino acid sequence data, to identify certain VL-framework region residues that account for the selective binding of protein L by kappa I, kappa III, and kappa IV proteins, our studies indicate that this interaction is essentially dependent upon the tertiary structural integrity of the kappa chain VL domain.

Characterization of monoclonal anti-alpha 1-microglobulin antibodies: binding strength, binding sites, and inhibition of lymphocyte stimulation.

Eleven monoclonal antibodies (MoAb) directed against the immunoregulatory plasma glycoprotein alpha 1-microglobulin were characterized. The MoAb were produced in mice immunized with a mixture of alpha 1-microglobulin homologues from man, guinea pig, rat and rabbit. Using radioimmunoassay, western blotting, affinity chromatography, and Scatchard analysis, the affinities and binding sites of the MoAb were analysed. All antibodies were more or less cross-reactive, but most showed a major specificity for one or two of the alpha 1-microglobulin homologues. None of the antibodies was directed against the carbohydrate moiety of alpha 1-microglobulin. Six of the MoAb had high affinity for the antigen and four of these were directed towards the same part of the molecule though differing in their species specificity. Five showed lower affinity for the antigen and were mainly directed towards epitopes on other parts of the molecule. Only some of the antibodies could block the proliferation of lymphocytes induced by human alpha 1-microglobulin. The blocking efficiency of the different antibodies was similar when tested on the stimulation of human or mouse lymphocytes, suggesting that the same part of the alpha 1-microglobulin molecule is responsible in both species. The magnitude of blocking by the different MoAb was not related to their affinities, emphasizing the importance of where on the alpha 1-microglobulin molecule, rather than how strongly, they bind. The binding of the strongest blocking antibody was shown to be directed to a C-terminal peptide of rat alpha 1-microglobulin, indicating that this part of alpha 1-microglobulin is important for the mitogenic effects. Thus the panel of anti-alpha 1-microglobulin MoAb should be a valuable tool for structural and functional studies of alpha 1-microglobulin.

Antibody response in immunized rabbits measured with bacterial immunoglobulin-binding proteins.

Protein G, an immunoglobulin (Ig)-binding protein isolated from group C or G streptococci, binds to the Fc portion of IgG. Protein L, from the anaerobic bacterium Peptostreptococcus magnus, specifically binds light chains of Ig. In this study, protein G and L were used to measure the production of antibodies in immunized rabbits. Two rabbits were immunized with a mixture of human urinary proteins from a patient with tubular proteinuria, and blood samples were collected regularly from the animals for 6 weeks after the immunization. The antibody levels of the blood samples against six of the proteins in the antigen mixture were then measured by ELISA. Microtiter plates were coated with each of the antigens, incubated with the rabbit serum samples, and the specific antibodies of the IgG class measured by incubation with biotinylated protein G, and antibodies of all Ig classes with biotinylated protein L. Alternatively, Western blotting was employed, where the antibodies which bound to each antigen after separation by SDS-PAGE and transfer to nitrocellulose membranes, were detected by protein G or L. The results showed that antibody production against five of the antigens, albumin, alpha 1 gamma-acid glycoprotein, alpha 1 gamma-microglobulin, Ig light chains, and retinol-binding protein, showed a similar pattern, although the magnitude of the initial IgM response differed somewhat. After 6 weeks, the levels of the protein G-binding antibodies had reached a plateau, while those of protein L-binding antibodies were still increasing. The response to the sixth antigen, beta 2 microglobulin, was considerably different. A dramatic increase of anti-beta 2 gamma-microglobulin antibodies was seen during the 4th week after immunization when protein L was used.

Enzyme linked immunosorbent assay using alkaline phosphatase conjugated with streptococcal protein G.

Protein G, an IgG-binding protein, purified from the surface of group G streptococci, was coupled to alkaline phosphatase. The conjugate was used for detection of polyclonal goat and rabbit antibodies and monoclonal mouse IgG1, IgG2a and IgG2b in an enzyme-linked immunosorbent assay. A two-step coupling procedure was used, in which glutaraldehyde was allowed to react with the enzyme, excess glutaraldehyde was then removed by dialysis, and finally protein G added to the glutaraldehyde-activated and polymerized alkaline phosphatase. The activity and yield of the conjugates were then tested in an enzyme-linked immunosorbent assay. Coupling of 25 micrograms protein G to 5 mg alkaline phosphatase gave a conjugate which could be used for more than 10,000 determinations with maximal antibody binding giving an absorbance of 2.0. Under these conditions, there was no need for separation of the reactants before using the protein G-alkaline phosphatase complex.