Overlapping functionality of the Pht proteins in zinc homeostasis of streptococcus pneumoniae

Streptococcus pneumoniae is a globally significant pathogen that causes a range of diseases, including pneumonia, sepsis, meningitis, and otitis media. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including transition metal ions such as zinc. The pneumococcus employs a number of surface proteins to achieve this, among which are four highly similar polyhistidine triad (Pht) proteins. It has previously been established that these proteins collectively aid in the delivery of zinc to the ABC transporter substrate-binding protein AdcAII. Here we have investigated the contribution of each individual Pht protein to pneumococcal zinc homeostasis by analyzing mutant strains expressing only one of the four pht genes. Under conditions of low zinc availability, each of these mutants showed superior growth and zinc accumulation profiles relative to a mutant strain lacking all four genes, indicating that any of the four Pht proteins are able to facilitate delivery of zinc to AdcAII. However, optimal growth and zinc accumulation in vitro and pneumococcal survival and proliferation in vivo required production of all four Pht proteins, indicating that, despite their overlapping functionality, the proteins are not dispensable without incurring a fitness cost. We also show that surface-attached forms of the Pht proteins are required for zinc recruitment and that they do not contribute to defense against extracellular zinc stress

The first histidine triad motif of phtd is critical for zinc homeostasis in Streptococcus pneumoniae

Streptococcus pneumoniae is the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination of in vitro growth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence

Experimental evaluation of CTD package hydrodynamic behavior and recommendations for improved lowering techniques

This report is the last of a series of three reports on a comprehensive study of CTD instrument lowering mechanics. The first report, WHOI 79-81, "A Study of CTD Cables and Lowering Systems", examines the causes and modes of lowering cable failures, both mechanical and electrical, and makes recommendations to improve existing instrument packages and lowering procedures. The second report, WHOI 81-76, "Hydrodynamics of CTD Instrument Packages", is a theoretical study of instrument package stability when cable lowered or free falling. The model is used to predict the hydrodynamic response of CTD packages in their present or improved configuration. This report, WHOI 83-21, is more factual. It describes the tests performed on scale models and actual CTD packages to actually observe and/or measure their hydrodynamic behavior. Analytical results and experimental data obtained in this study are used to draw recommendations for CTD package improvement and future lowering procedures.Prepared for the Office of Naval Research under Contract N00014-72-C-0019

Involvement of Mhc Loci in immune responses that are not Ir-gene-controlled

Twenty-nine randomly chosen, soluble antigens, many of them highly complex, were used to immunize mice of two strains, C3H and B10.RIII. Lymphnode cells from the immunized mice were restimulated in vitro with the priming antigens and the proliferative response of the cells was determined. Both strains were responders to 28 of 29 antigens. Eight antigens were then used to immunize 11 congenic strains carrying different H-2 haplotypes, and the T-cell proliferative responses of these strains were determined. Again, all the strains responded to seven of the eight antigens. These experiments were then repeated, but this time -antibodies specific for the A (AA) or E (EE) molecules were added to the culture to block the in vitro responsiveness. In all but one of the responses, inhibition with both A-specific and E-specific antibodies was observed. The response to one antigen (Blastoinyces) was exceptional in that some strains were nonresponders to this antigen. Furthermore, the response in the responder strains was blocked with A-specific, but not with E-specific, antibodies. The study demonstrates that responses to antigens not controlled by Irr genes nevertheless require participation of class II Mhc molecules. In contrast to Ir gene-controlled responses involving either the A- or the E-molecule controlling loci (but never both), the responses not Ir-controlled involve participation of both A- and E-controlling loci. The lack of Ir-gene control is probably the result of complexity of the responses to multiple determinants. There is thus no principal difference between responses controlled and those not controlled by Ir genes: both types involve the recognition of the antigen, in the context of Mhc molecules

Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae.

Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes

The Air that we Breathe: Neutral and volatile PFAS in Indoor Air

Sources of exposure to per- and polyfluorinated alkyl substances (PFAS) include food, water, and, given that humans spend typically 90% of their time indoors, air and dust. Quantifying PFAS that are prevalent indoors, such as neutral, volatile PFAS, and estimating their exposure risk to humans are thus important. To accurately measure these compounds indoors, polyethylene (PE) sheets were employed and validated as passive detection tools and analyzed by gas chromatography–mass spectrometry. Air concentrations were compared to dust and carpet concentrations reported elsewhere. Partitioning between PE sheets of different thicknesses suggested that interactions of the PEs with the compounds are occurring by absorption. Volatile PFAS, specifically fluorotelomer alcohols (FTOHs), were ubiquitous in indoor environments. For example, in carpeted Californian kindergarten classrooms, 6:2 FTOH dominated with concentrations ranging from 9 to 600 ng m–3, followed by 8:2 FTOH. Concentrations of volatile PFAS from air, carpet, and dust were closely related to each other, indicating that carpets and dust are major sources of FTOHs in air. Nonetheless, air posed the largest exposure risk of FTOHs and biotransformed perfluorinated alkyl acids (PFAA) in young children. This research highlights inhalation of indoor air as an important exposure pathway and the need for further reduction of precursors to PFAA

Association between hospital private equity acquisition and outcomes of acute medical conditions among Medicare beneficiaries

Importance: As private equity (PE) acquisitions of short-term acute care hospitals (ACHs) continue, their impact on the care of medically vulnerable older adults remains largely unexplored. Objective: To investigate the association between PE acquisition of ACHs and access to care, patient outcomes, and spending among Medicare beneficiaries hospitalized with acute medical conditions. Design, Setting, and Participants: This cross-sectional study used a generalized difference-in-differences approach to compare 21 091 222 patients admitted to PE-acquired vs non-PE-acquired short-term ACHs between January 1, 2001, and December 31, 2018, at least 3 years before to 3 years after PE acquisition. The analysis was conducted between December 28, 2020, and February 1, 2022. Differences were estimated using both facility and hospital service area fixed effects. To assess the robustness of findings, regressions were reestimated after including fixed effects of patient county of origin to account for geographic differences in underlying health risks. Two subset analyses were also conducted: (1) an analysis including only hospitals in hospital referral regions with at least 1 PE acquisition and (2) an analysis stratified by participation in the Hospital Corporation of America 2006 acquisition. The study included Medicare beneficiaries 66 years and older who were hospitalized with 1 of 5 acute medical conditions: acute myocardial infarction (AMI), acute stroke, chronic obstructive pulmonary disease exacerbation, congestive heart failure exacerbation, and pneumonia. Exposures: Acquisition of hospitals by PE firms. Main Outcomes and Measures: Comorbidity burden (measured by Elixhauser comorbidity score), hospital length of stay, in-hospital mortality, 30-day mortality, 30-day readmission, and 30-day episode payments. Results: Among 21 091 222 total Medicare beneficiaries admitted to ACHs between 2001 and 2018, 20 431 486 patients received care at non-PE-acquired hospitals, and 659 736 received care at PE-acquired hospitals. Across all admissions, the mean (SD) age was 79.45 (7.95) years; 11 727 439 patients (55.6%) were male, and 4 550 012 patients (21.6%) had dual insurance; 2 996 560 (14.2%) patients were members of racial or ethnic minority groups, including 2 085 128 [9.9%] Black and 371 648 [1.8%] Hispanic; 18 094 662 patients (85.8%) were White. Overall, 3 083 760 patients (14.6%) were hospitalized with AMI, 2 835 777 (13.4%) with acute stroke, 3 674 477 (17.4%) with chronic obstructive pulmonary disease exacerbation, 5 868 034 (27.8%) with congestive heart failure exacerbation, and 5 629 174 (26.7%) with pneumonia. Comorbidity burden decreased slightly among patients admitted with acute stroke (difference, -0.04 SDs; 95% CI, -0.004 to -0.07 SDs) at acquired hospitals compared with nonacquired hospitals but was unchanged across the other 4 conditions. Among patients with AMI, a greater decrease in in-hospital mortality was observed in PE-acquired hospitals compared with non-PE-acquired hospitals (difference, -1.14 percentage points, 95% CI, -1.86 to -0.42 percentage points). In addition, a greater decrease in 30-day mortality (difference, -1.41 percentage points; 95% CI, -2.26 to -0.56 percentage points) was found at acquired vs nonacquired hospitals. However, 30-day spending and readmission rates remained unchanged across all conditions. The extent and directionality of estimates were preserved across all robustness assessments and subset analyses. Conclusions and Relevance: In this cross-sectional study using a difference-in-differences approach, PE acquisition had no substantial association with the patient-level outcomes examined, although it was associated with a moderate improvement in mortality among Medicare beneficiaries hospitalized with AMI

The Influence of Quadrature Errors on Isogeometric Mortar Methods

Mortar methods have recently been shown to be well suited for isogeometric analysis. We review the recent mathematical analysis and then investigate the variational crime introduced by quadrature formulas for the coupling integrals. Motivated by finite element observations, we consider a quadrature rule purely based on the slave mesh as well as a method using quadrature rules based on the slave mesh and on the master mesh, resulting in a non-symmetric saddle point problem. While in the first case reduced convergence rates can be observed, in the second case the influence of the variational crime is less significant

Progressive osseous heteroplasia: A case report with an unexpected trigger

Progressive osseous heteroplasia (POH) is a rare genetic disorder characterised by progressive heterotopic ossification (HO) within the skin and subcutaneous tissues. The condition is caused by heterozygous inactivating mutations of the GNAS gene and usually presents in infancy. We describe the case of a white male ex-preterm who was first referred because of subcutaneous calcium deposits along the right arm after extravasation of parenteral nutrition. As these lesions progressed, a skin biopsy was undertaken which revealed intramembranous ossification. Genetic testing revealed a constitutional, de novo, heterozygous, nonsense variant in the GNAS gene that has not previously been described, but which is consistent with patient's clinical diagnosis of POH. No endocrine abnormalities or other signs congruent with overlapping conditions were detected. To the best of our knowledge, this is the first case describing an inflammatory trigger in POH. Trials with intravenous bisphosphonate and glucocorticoid as well as with topical sodium thiosulphate were attempted without clinical improvement. Excision of the calcifications and physiotherapy seem to have provided a partial improvement on mobility of the elbow. This case widens the spectrum of phenotypes seen in GNAS mutation disorders and suggests that alternative anti-inflammatory treatments may be effective. Mutations in GNAS should be considered in cases of significant progressive calcium deposition after extravasation injury