The results were best demonstrated by sigmoidal curves (pFe 18.8–21.7, Fe3+ = 10−18.8–10−21.7 M) with the linear range extending from pFe 19.6–21.5 (Fe3+ = 10−19.6–10−21.5 M) after a 12-h incubation time. Optimal conditions for the use of this bioreporter to sense the iron bioavailability were determined to be: a 12-h exposure time, initial cell density of OD730 nm = 0.06, high nitrate (100 μM), high phosphate (10 μM), moderate Co2+ (0.1–22.5 nM), Zn2+ (0.16–12 nM), Cu2+ (0.04–50 nM),

and wide range of Mn2+ concentration (0.92–2300 nM). The applicability of using this iron bioreporter to assess iron availability in the natural environment http://www.selleckchem.com/products/MK-1775.html has been tested using water samples from eutrophic Taihu, Donghu, and Chaohu lakes. It is indicated that the bioreporter is a useful tool to assess bioavailable iron in various water quality samples, especially in eutrophic lakes with high bioavailable iron. Iron is an essential nutrient for organisms. As the fourth most abundant element in the crust of the earth, it generally exists in two forms, Fe2+ and Fe3+, in aquatic environments. In oxic environments, Fe2+ can be quickly oxidized into Fe3+ and then

transformed into insoluble and inaccessible ferric hydroxide. In addition, iron also exists in the form of colloids and can be complexed http://www.selleckchem.com/products/Fulvestrant.html by organic ligands. Although various iron chelates, including siderophores and grazing byproducts, and iron-organic compounds have been shown to act as sources of iron to phytoplankton (Hutchins et al., 1999; Poorvin et al., 2004), iron bioavailability is still low in many aquatic environments and constrains phytoplankton growth in areas of the open ocean characterized as ‘high-nutrient, low-chlorophyll’ regions (Martin et al., 1991; Coale et al., 1996), coastal waters (Hutchins et al., 1998), and some freshwater systems (Twiss et al., 2000). Although rapid and reliable chemical protocols are available to measure absolute

levels of iron in water samples, whole-cell bioreporters provide data on the capacity of the biota to acquire and assimilate iron. Recombinant bioluminescent bacterial ROS1 strains have been successfully applied in monitoring iron (Durham et al., 2002; Mioni et al., 2003) and the availability of other metal ions (Peca et al., 2008) in environmental samples. The bicistronic isiAB operon is in part regulated by the iron-dependent repressor Fur (ferric uptake regulator) in cyanobacteria (Ghassemian & Straus, 1996). The first gene isiA codes for a protein that is very similar to CP43, a chlorophyll-binding core protein of photosystem II. Flavodoxin coded by gene isiB has been revealed to have the ability to replace ferredoxin as carrier in the electron transfer chain.

The results were best demonstrated by sigmoidal curves (pFe 18.8–21.7, Fe3+ = 10−18.8–10−21.7 M) with the linear range extending from pFe 19.6–21.5 (Fe3+ = 10−19.6–10−21.5 M) after a 12-h incubation time. Optimal conditions for the use of this bioreporter to sense the iron bioavailability were determined to be: a 12-h exposure time, initial cell density of OD730 nm = 0.06, high nitrate (100 μM), high phosphate (10 μM), moderate Co2+ (0.1–22.5 nM), Zn2+ (0.16–12 nM), Cu2+ (0.04–50 nM),

and wide range of Mn2+ concentration (0.92–2300 nM). The applicability of using this iron bioreporter to assess iron availability in the natural environment this website has been tested using water samples from eutrophic Taihu, Donghu, and Chaohu lakes. It is indicated that the bioreporter is a useful tool to assess bioavailable iron in various water quality samples, especially in eutrophic lakes with high bioavailable iron. Iron is an essential nutrient for organisms. As the fourth most abundant element in the crust of the earth, it generally exists in two forms, Fe2+ and Fe3+, in aquatic environments. In oxic environments, Fe2+ can be quickly oxidized into Fe3+ and then

transformed into insoluble and inaccessible ferric hydroxide. In addition, iron also exists in the form of colloids and can be complexed learn more by organic ligands. Although various iron chelates, including siderophores and grazing byproducts, and iron-organic compounds have been shown to act as sources of iron to phytoplankton (Hutchins et al., 1999; Poorvin et al., 2004), iron bioavailability is still low in many aquatic environments and constrains phytoplankton growth in areas of the open ocean characterized as ‘high-nutrient, low-chlorophyll’ regions (Martin et al., 1991; Coale et al., 1996), coastal waters (Hutchins et al., 1998), and some freshwater systems (Twiss et al., 2000). Although rapid and reliable chemical protocols are available to measure absolute

levels of iron in water samples, whole-cell bioreporters provide data on the capacity of the biota to acquire and assimilate iron. Recombinant bioluminescent bacterial Thymidylate synthase strains have been successfully applied in monitoring iron (Durham et al., 2002; Mioni et al., 2003) and the availability of other metal ions (Peca et al., 2008) in environmental samples. The bicistronic isiAB operon is in part regulated by the iron-dependent repressor Fur (ferric uptake regulator) in cyanobacteria (Ghassemian & Straus, 1996). The first gene isiA codes for a protein that is very similar to CP43, a chlorophyll-binding core protein of photosystem II. Flavodoxin coded by gene isiB has been revealed to have the ability to replace ferredoxin as carrier in the electron transfer chain.

Regarding oral health behaviour there were no differences, except that PT children more often used dental floss and extra fluoride supplements. PT children reported more medical health problems than C children. Conclusions.  Preterm (PT) children 12- to 14-years-old, as well as

C of same age group, seem to be satisfied find more with their dental care and display low prevalence of DFA. Still, a higher frequency of medical health problems in the PT children suggests that these children should be regarded as potential risk patients for oral health problems. “
“This study aimed to compare the clinical and radiographic effectiveness of Low Level Laser Therapy in vital pulp of human primary teeth. Sixty mandibular primary molars of children aged between 5–9 years were assigned into four groups: Diluted Formocresol (FC), Calcium Hydroxide (CH), Low Level Laser Therapy (LLLT) and Calcium Hydroxide preceded by Low Level Laser Therapy (LLLT + CH). The clinical and radiographic evaluations were performed at 6, 12 and

18 post-operative months. All the groups studied were successful in the clinical evaluation over the follow-up period. At 6 months, the radiographic success rate for FC group was 100%, 60% for CH group, 80% for LLLT group and 85.7% for LLLT + CH APO866 manufacturer group. After 12 months, the radiographic success rate was 100% for FC group, 50% for CH group, 80% for LLLT group and 78.6% for LLLT + CH group. At the 18 months follow-up, 100% of the FC group, 66.7% of CH group, 73.3% of the LLLT group and 75% of the LLLT + CH group. These findings suggest

that Low Level Laser Therapy may be considered as an adjuvant alternative for vital pulp therapy on human primary teeth. Low Level Laser Therapy preceding the use of calcium hydroxide showed satisfactory results. “
“International Journal of Paediatric Tideglusib Dentistry 2013; 23: 166–172 Objective.  Our in vitro study evaluated calcium fluoride formation in enamel and the anticaries effect of seven resin-based varnishes under cariogenic challenge. Methods.  Enamel blocks were subjected to pH cycling. The experimental groups received fluoride varnish application, the positive control received topical fluoride gel treatment, and the negative control did not receive any treatment. The pH cycling surface hardness (SH1) and integrated loss of subsurface hardness (ΔKHN) were then determined. We measured the amount of fluoride released into the demineralizing and remineralizing (DE–RE) solutions used in pH cycling. The fluoride concentration in the enamel was determined 24 h after application of the products as loosely bound fluoride and firmly bound fluoride. Results.  Higher deposits of loosely bound fluoride were observed for Duofluorid, followed by Biophat. For Duraphat, Bifluorid, Duraflur, and Duofluorid, no difference was observed in the SH1 and ΔKHN values, with the lowest mineral loss compared to the other groups.

The expected 806-bp hxk1 fragments were obtained after PCR using reverse-transcribed RNAs from five different transformants as templates, indicating transgenic hxk1 expression in these transformants (Fig. 2c). To visualize expression of the EGFP reporter, positive transformants were investigated by microscopy. Most of the investigated transformants showed the typical green fluorescence (Fig. 3). The fluorescence could be detected easily by fluorescence microscopy, suggesting the EGFP expression levels were high in transformants. About 20% of a total of 50 randomly selected colonies from the transformation plates were regarded as abortive Selleck PI3K inhibitor transformants without

further growth after transfer to fresh selective medium, which was in accordance with a previous report using H. jecorina pyr4 as a homologous marker (Gruber et al., 1990). Individual colonies from a single spore isolated from nonabortive transformants were tested for phenotypic stability. Subcultivation of the transformants RAD001 nmr on MM without selective pressure followed by a growth test for d-mannitol utilization demonstrated that the

HXK+ phenotype remained stable for at least five successive generations. Complementation of an auxotrophic mutation in a recipient strain to prototrophy is probably the most successful strategy for genetic transformation and selection of transformants. Auxotrophic markers are often preferred over dominant markers due to the high cost of the antibiotics used as selective agents and the detrimental effects of such PRKACG agents on the cell during the transformation and selection procedures. In this study, we investigated whether an hxk1-negative strain can be used as a recipient for the development of a carbon source-dependent genetic transformation system. An analysis of phenotypic differences in carbon utilization between the parental strain TU-6 and the hxk1 deletion strain TU-6H showed that the latter strain was not able to metabolize d-mannitol or d-sorbitol, which are both commonly used as effective osmotic stabilizers for fungal transformation (Ruiz-Díez 2002; Li et al., 2006). These physiological characteristics

were the prerequisites for the development of the transformation system in which these polyols could be used as both selective agent and osmotic stabilizer. Transformation of TU-6H with the selectable marker hxk1 showed that this gene is responsible for d-mannitol and d-sorbitol utilization via d-fructose. A comparison of the effect of the two polyols on transformation efficiencies showed that the efficiency was around five times higher for d-mannitol than for d-sorbitol. As d-sorbitol is usually applied as osmotic stabilizer in conventional H. jecorina transformation (Penttiläet al., 1987; Gruber et al., 1990; Mach et al., 1994), the replacement of it by d-mannitol might provide a useful alternative for transformation assays where high transformation efficiencies are required.

The expected 806-bp hxk1 fragments were obtained after PCR using reverse-transcribed RNAs from five different transformants as templates, indicating transgenic hxk1 expression in these transformants (Fig. 2c). To visualize expression of the EGFP reporter, positive transformants were investigated by microscopy. Most of the investigated transformants showed the typical green fluorescence (Fig. 3). The fluorescence could be detected easily by fluorescence microscopy, suggesting the EGFP expression levels were high in transformants. About 20% of a total of 50 randomly selected colonies from the transformation plates were regarded as abortive Antiinfection Compound Library screening transformants without

further growth after transfer to fresh selective medium, which was in accordance with a previous report using H. jecorina pyr4 as a homologous marker (Gruber et al., 1990). Individual colonies from a single spore isolated from nonabortive transformants were tested for phenotypic stability. Subcultivation of the transformants BIBW2992 on MM without selective pressure followed by a growth test for d-mannitol utilization demonstrated that the

HXK+ phenotype remained stable for at least five successive generations. Complementation of an auxotrophic mutation in a recipient strain to prototrophy is probably the most successful strategy for genetic transformation and selection of transformants. Auxotrophic markers are often preferred over dominant markers due to the high cost of the antibiotics used as selective agents and the detrimental effects of such Leukocyte receptor tyrosine kinase agents on the cell during the transformation and selection procedures. In this study, we investigated whether an hxk1-negative strain can be used as a recipient for the development of a carbon source-dependent genetic transformation system. An analysis of phenotypic differences in carbon utilization between the parental strain TU-6 and the hxk1 deletion strain TU-6H showed that the latter strain was not able to metabolize d-mannitol or d-sorbitol, which are both commonly used as effective osmotic stabilizers for fungal transformation (Ruiz-Díez 2002; Li et al., 2006). These physiological characteristics

were the prerequisites for the development of the transformation system in which these polyols could be used as both selective agent and osmotic stabilizer. Transformation of TU-6H with the selectable marker hxk1 showed that this gene is responsible for d-mannitol and d-sorbitol utilization via d-fructose. A comparison of the effect of the two polyols on transformation efficiencies showed that the efficiency was around five times higher for d-mannitol than for d-sorbitol. As d-sorbitol is usually applied as osmotic stabilizer in conventional H. jecorina transformation (Penttiläet al., 1987; Gruber et al., 1990; Mach et al., 1994), the replacement of it by d-mannitol might provide a useful alternative for transformation assays where high transformation efficiencies are required.

, 2005). Indeed, biochemical evidence was obtained that KdpE undergoes a monomer-to-dimer transition upon phosphorylation (Lucassen, 1998). Histidine kinase/response regulator systems are often referred to as ‘two-component systems’ based on the assumption that they consist of only two components. Meanwhile, many systems are known that include accessory proteins responsible for stimulus perception, fine-tuning, cross-talk, or signal integration (Island & Kadner, 1993; Kato & Groisman, 2004; Eguchi et al., 2007; Fleischer et al., 2007; Paul et al., 2008). Accessory

proteins were also identified for CYC202 manufacturer the KdpD/KdpE system. The universal stress protein UspC was identified as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with the Usp domain in KdpD under salt stress (Fig. 2b) (Heermann et al., 2009b). Usp proteins are small soluble proteins that accumulate under diverse stress conditions. They are widespread in living organisms, but their physiological role is poorly understood (Kvint et al., 2003). Scaffolding

proteins are usually known from eukaryotes. These proteins connect proteins and enhance the binding properties in a signaling pathway and thus influence signal transduction (Pawson & Scott, 1997; Garrington & Johnson, 1999; Burack & Shaw, 2000). Under K+-limiting conditions, the Kdp system restores the intracellular K+ concentration, while in response to salt stress, K+ is accumulated far above the normal content VEGFR inhibitor by rapid uptake via Trk. Nevertheless, the Kdp system is induced under salt stress. Because the kinase activity of KdpD is inhibited at high concentrations of K+ (Jung et al., 2000), it has been puzzling how the sensor can be activated in response to salt stress. KdpD has a Usp domain within the N-terminal input domain belonging to the UspA subfamily, and it was hypothesized that KdpD might interact with one or more UspA-subfamily proteins (Heermann et al., 2009b). Escherichia coli encodes Etofibrate three single domain proteins of this subfamily, UspA, UspC, and UspD, and the expression of the corresponding

genes is upregulated under various stress conditions including salt stress (Gustavsson et al., 2002). Among these proteins, only UspC stimulated the in vitro reconstructed signaling cascade (KdpDKdpEDNA), resulting in phosphorylation of KdpE at a K+ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant, KdpFABC production was significantly downregulated when cells were exposed to salt stress, but unaffected under K+ limitation. Biochemical studies revealed that UspC specifically interacts with the Usp domain in the stimulus-perceiving N-terminal domain of KdpD. UspC does not influence the enzymatic activities of KdpD, but stabilizes the KdpD/phospho-KdpE/DNA complex. Therefore, UspC can be regarded as one of the rare examples of bacterial scaffolding proteins (Heermann et al., 2009b).

, 2005). Indeed, biochemical evidence was obtained that KdpE undergoes a monomer-to-dimer transition upon phosphorylation (Lucassen, 1998). Histidine kinase/response regulator systems are often referred to as ‘two-component systems’ based on the assumption that they consist of only two components. Meanwhile, many systems are known that include accessory proteins responsible for stimulus perception, fine-tuning, cross-talk, or signal integration (Island & Kadner, 1993; Kato & Groisman, 2004; Eguchi et al., 2007; Fleischer et al., 2007; Paul et al., 2008). Accessory

proteins were also identified for BAY 73-4506 concentration the KdpD/KdpE system. The universal stress protein UspC was identified as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with the Usp domain in KdpD under salt stress (Fig. 2b) (Heermann et al., 2009b). Usp proteins are small soluble proteins that accumulate under diverse stress conditions. They are widespread in living organisms, but their physiological role is poorly understood (Kvint et al., 2003). Scaffolding

proteins are usually known from eukaryotes. These proteins connect proteins and enhance the binding properties in a signaling pathway and thus influence signal transduction (Pawson & Scott, 1997; Garrington & Johnson, 1999; Burack & Shaw, 2000). Under K+-limiting conditions, the Kdp system restores the intracellular K+ concentration, while in response to salt stress, K+ is accumulated far above the normal content TSA HDAC concentration by rapid uptake via Trk. Nevertheless, the Kdp system is induced under salt stress. Because the kinase activity of KdpD is inhibited at high concentrations of K+ (Jung et al., 2000), it has been puzzling how the sensor can be activated in response to salt stress. KdpD has a Usp domain within the N-terminal input domain belonging to the UspA subfamily, and it was hypothesized that KdpD might interact with one or more UspA-subfamily proteins (Heermann et al., 2009b). Escherichia coli encodes Diflunisal three single domain proteins of this subfamily, UspA, UspC, and UspD, and the expression of the corresponding

genes is upregulated under various stress conditions including salt stress (Gustavsson et al., 2002). Among these proteins, only UspC stimulated the in vitro reconstructed signaling cascade (KdpDKdpEDNA), resulting in phosphorylation of KdpE at a K+ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant, KdpFABC production was significantly downregulated when cells were exposed to salt stress, but unaffected under K+ limitation. Biochemical studies revealed that UspC specifically interacts with the Usp domain in the stimulus-perceiving N-terminal domain of KdpD. UspC does not influence the enzymatic activities of KdpD, but stabilizes the KdpD/phospho-KdpE/DNA complex. Therefore, UspC can be regarded as one of the rare examples of bacterial scaffolding proteins (Heermann et al., 2009b).

The three proteins with amino acid substitutions of this study were tested for their abilities to protect membranes from thermal damage. Interestingly, Y107A was associated with the membrane, but appeared to have an impaired capacity to stabilize membranes, in contrast to the other proteins. It has been described previously that dissociation of the oligomer is a prerequisite for the Hsp16.3 membrane-association process (Zhang et al., 2005). It has also been suggested that Hsp16.3 dissociates into

find more small oligomers to expose certain interfaces that are necessary for the membrane-association process that follows (Zhang et al., 2005). Although the Y107A did not prevent interaction with the membrane, the membrane stabilization activity was abolished. Consequently, we suggest that the amino acid in position 107 may be necessary for this activity or/and for correct insertion at the membrane level. Our PI3K inhibitor drugs data presented here strongly suggest that the amino acids involved in chaperone activity on denaturated proteins

and membrane fluidity regulation are different and are localized in the α-crystallin domain. However, we cannot exclude the existence of amino acids necessary for both activities. The construction and characterization of other proteins with amino acid substitutions should help to understand how Lo18 is able to function on both substrates. This study was supported by the Ministère de l’Education Nationale de la Recherche et de la Technologie and the Université de Bourgogne. We thank M. Guillemin and D. Carrel for their technical assistance and L. Gal for his help in point substitutions of Lo18. We thank Alex Edelman and associates for their reading of the English text. “
“Staphylococcus aureus is a common human pathogenic bacteria that can cause serious infections, including lethal staphylococcal pneumonia. The development of antimicrobial

resistance has limited treatment options for this pathogen; consequently, novel antibiotics and strategies Carteolol HCl are urgently desired to combat these infections. In recent years, virulence factors secreted by pathogenic microorganisms have been developed as targets for drug discovery. Alpha-hemolysin, a pore-forming cytotoxin that is secreted by most S. aureus strains, is essential for the pathogenesis of S. aureus pneumonia. In this study, we report that apigenin, a compound extracted from parsley that has no antimicrobial activity vs. S. aureus in vitro, can remarkably decrease the production of α-hemolysin at low concentrations. When added to the A549 cells and S. aureus co-culture system, apigenin protected A549 cells from α-hemolysin-mediated injury. Furthermore, in vivo tests indicated that apigenin alleviated injury of the lung tissue and decreased cytokine levels in the bronchoalveolar lavage fluid in the mouse model of S. aureus pneumonia.

The three proteins with amino acid substitutions of this study were tested for their abilities to protect membranes from thermal damage. Interestingly, Y107A was associated with the membrane, but appeared to have an impaired capacity to stabilize membranes, in contrast to the other proteins. It has been described previously that dissociation of the oligomer is a prerequisite for the Hsp16.3 membrane-association process (Zhang et al., 2005). It has also been suggested that Hsp16.3 dissociates into

Tofacitinib clinical trial small oligomers to expose certain interfaces that are necessary for the membrane-association process that follows (Zhang et al., 2005). Although the Y107A did not prevent interaction with the membrane, the membrane stabilization activity was abolished. Consequently, we suggest that the amino acid in position 107 may be necessary for this activity or/and for correct insertion at the membrane level. Our click here data presented here strongly suggest that the amino acids involved in chaperone activity on denaturated proteins

and membrane fluidity regulation are different and are localized in the α-crystallin domain. However, we cannot exclude the existence of amino acids necessary for both activities. The construction and characterization of other proteins with amino acid substitutions should help to understand how Lo18 is able to function on both substrates. This study was supported by the Ministère de l’Education Nationale de la Recherche et de la Technologie and the Université de Bourgogne. We thank M. Guillemin and D. Carrel for their technical assistance and L. Gal for his help in point substitutions of Lo18. We thank Alex Edelman and associates for their reading of the English text. “
“Staphylococcus aureus is a common human pathogenic bacteria that can cause serious infections, including lethal staphylococcal pneumonia. The development of antimicrobial

resistance has limited treatment options for this pathogen; consequently, novel antibiotics and strategies Phospholipase D1 are urgently desired to combat these infections. In recent years, virulence factors secreted by pathogenic microorganisms have been developed as targets for drug discovery. Alpha-hemolysin, a pore-forming cytotoxin that is secreted by most S. aureus strains, is essential for the pathogenesis of S. aureus pneumonia. In this study, we report that apigenin, a compound extracted from parsley that has no antimicrobial activity vs. S. aureus in vitro, can remarkably decrease the production of α-hemolysin at low concentrations. When added to the A549 cells and S. aureus co-culture system, apigenin protected A549 cells from α-hemolysin-mediated injury. Furthermore, in vivo tests indicated that apigenin alleviated injury of the lung tissue and decreased cytokine levels in the bronchoalveolar lavage fluid in the mouse model of S. aureus pneumonia.

The three proteins with amino acid substitutions of this study were tested for their abilities to protect membranes from thermal damage. Interestingly, Y107A was associated with the membrane, but appeared to have an impaired capacity to stabilize membranes, in contrast to the other proteins. It has been described previously that dissociation of the oligomer is a prerequisite for the Hsp16.3 membrane-association process (Zhang et al., 2005). It has also been suggested that Hsp16.3 dissociates into

ABT-888 nmr small oligomers to expose certain interfaces that are necessary for the membrane-association process that follows (Zhang et al., 2005). Although the Y107A did not prevent interaction with the membrane, the membrane stabilization activity was abolished. Consequently, we suggest that the amino acid in position 107 may be necessary for this activity or/and for correct insertion at the membrane level. Our EGFR inhibitor data presented here strongly suggest that the amino acids involved in chaperone activity on denaturated proteins

and membrane fluidity regulation are different and are localized in the α-crystallin domain. However, we cannot exclude the existence of amino acids necessary for both activities. The construction and characterization of other proteins with amino acid substitutions should help to understand how Lo18 is able to function on both substrates. This study was supported by the Ministère de l’Education Nationale de la Recherche et de la Technologie and the Université de Bourgogne. We thank M. Guillemin and D. Carrel for their technical assistance and L. Gal for his help in point substitutions of Lo18. We thank Alex Edelman and associates for their reading of the English text. “
“Staphylococcus aureus is a common human pathogenic bacteria that can cause serious infections, including lethal staphylococcal pneumonia. The development of antimicrobial

resistance has limited treatment options for this pathogen; consequently, novel antibiotics and strategies Florfenicol are urgently desired to combat these infections. In recent years, virulence factors secreted by pathogenic microorganisms have been developed as targets for drug discovery. Alpha-hemolysin, a pore-forming cytotoxin that is secreted by most S. aureus strains, is essential for the pathogenesis of S. aureus pneumonia. In this study, we report that apigenin, a compound extracted from parsley that has no antimicrobial activity vs. S. aureus in vitro, can remarkably decrease the production of α-hemolysin at low concentrations. When added to the A549 cells and S. aureus co-culture system, apigenin protected A549 cells from α-hemolysin-mediated injury. Furthermore, in vivo tests indicated that apigenin alleviated injury of the lung tissue and decreased cytokine levels in the bronchoalveolar lavage fluid in the mouse model of S. aureus pneumonia.