Impact of Diabetes on 90-Day Episodes of Care After Elective Total Joint Arthroplasty Among Medicare Beneficiaries.

BACKGROUND: In an effort to improve quality and reduce costs, reimbursement for total knee arthroplasty (TKA) and total hip arthroplasty (THA) in the United States is being based on the value of care provided, with adjustments for some qualifying comorbidities, including diabetes in its most severe form and excluding many diabetes codes. The aims of this study were to examine the effects of diabetes on elective TKA or THA complications and readmission risks among Medicare beneficiaries. METHODS: Complication (n = 521,230) and readmission (n = 515,691) data were extracted from Medicare files in 2013 and 2014. Diabetes status (no diabetes, controlled-uncomplicated diabetes, controlled-complicated diabetes, and uncontrolled diabetes) was identified with ICD-9 (International Classification of Diseases, 9th Revision) codes. TKA or THA complications and readmission odds based on diabetes status were estimated using logistic regression and adjusted for sociodemographic and clinical characteristics, including comorbidities. RESULTS: Compared with no diabetes, the odds ratio (OR) of TKA complications was significantly higher for uncontrolled diabetes (1.29, 95% confidence interval [CI] = 1.06 to 1.57). The OR of THA complications was significantly higher for controlled-complicated diabetes (1.45, 95% CI = 1.17 to 1.80). The OR of readmission was significantly higher for all diabetes groups (1.21 to 1.48 for TKA, 1.20 to 1.70 for THA). CONCLUSIONS: Readmission odds were higher in all diabetes categories. The uncontrolled-diabetes group had the greatest TKA readmission and complication odds. The controlled-complicated diabetes group had the greatest THA readmission and complication odds. The findings suggest that including diabetes and associated systemic complications in cost adjustments in alternative payment models for arthroplasty should be considered. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Targeted nanopore sequencing with Cas9-guided adapter ligation.

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1-4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 µg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.

The association of discharge destination with 30-day rehospitalization rates among older adults receiving lumbar spinal fusion surgery.

BACKGROUND: As defined by Medicare (United States), post-acute rehabilitation services include care provided in inpatient rehabilitation units and facilities, skilled nursing facilities, long-term acute hospitals, and by home health services. METHODS: We retrospectively evaluated the use of rehabilitation-based post-acute services among Medicare beneficiaries who were hospitalized for lumbar spinal fusion (ICD-9-CM procedure codes 81.04-81.08) in 2012-2014, examined the case-mix for those discharged to rehabilitation- and non-rehabilitation based services, and determined the association between these categories of discharge disposition and 30-day rehospitalization. The independent effect of rehabilitation-based discharge destination on 30-day readmissions was examined with a generalized linear mixed model, first adjusting for patient characteristics and then stratified by clusters that delineated more homogenous clinical profiles. RESULTS: Among 261,558 Medicare beneficiaries with lumbar spinal fusion surgery, 50.8% were discharged to a rehabilitation-based post-acute services. Patients discharged to rehabilitation-based services were older and had more comorbidities, and had longer hospital lengths of stays. After adjusting for patient and hospital characteristics, patients discharged to rehabilitation-based post-acute care had increased odds of 30-day rehospitalization than those without discharge to other destinations (OR 1.36; 95%CI = 1.31, 1.40). Analysis of patients by clinical profile clusters found similar results. CONCLUSIONS: Clinical profiles of Medicare beneficiaries who had lumbar spinal fusion surgery and were discharged to rehabilitation-based post-acute services included more comorbidities than those discharged to non-rehabilitation based settings. Controlling for these differences did not mediate the negative association between use of rehabilitation-based post-acute services and 30-day readmission.

Ghrelin and Leptin Have a Complex Relationship with Risk of Barrett's Esophagus.

BACKGROUND: Abdominal obesity is a risk factor for Barrett's esophagus independent of GERD symptoms, but little is understood about the biological mechanisms between obesity and the carcinogenic pathway of esophageal adenocarcinoma. AIMS: To evaluate whether ghrelin and leptin may partially explain the association between obesity and Barrett's esophagus. METHODS: We conducted a case-control study using patients with a new diagnosis of Barrett's esophagus (cases) and two control groups frequency matched to cases for age, gender, and geographic region: (1) patients with gastroesophageal reflux disease (GERD) and (2) a sample of the general population. We generated odds ratios using logistic regressions to evaluate quartiles of serum ghrelin or serum leptin, adjusting for known risk factors for Barrett's esophagus. We evaluated potential interaction variables using cross products and ran stratified analyses to generate stratum-specific odds ratios. RESULTS: A total of 886 participants were included in the analysis. Higher ghrelin concentrations were associated with an increased risk of Barrett's esophagus, when compared to the population controls, but not the GERD controls. Ghrelin concentrations were not associated with the frequency of GERD symptoms, but ghrelin's relationship with Barrett's esophagus varied significantly with the frequency of GERD symptoms. Leptin concentrations were positively associated with at least weekly GERD symptoms among the population controls and were inversely associated with Barrett's esophagus only among the GERD controls. Adjusting for waist circumference did not change the main associations. CONCLUSION: Higher levels of ghrelin were associated with an increased risk of Barrett's esophagus among the general population. In contrast, leptin was positively associated with frequent GERD symptoms, but inversely associated with the risk of Barrett's esophagus among the GERD controls.

The effect of obesity on incidence of disability and mortality in Mexicans aged 50 years and older.

OBJECTIVE: To examine the effect of obesity on incidence of disability and mortality among non-disabled older Mexicans at baseline. MATERIALS AND METHODS: The sample included 8 415 Mexicans aged ≥ 50 years from the Mexican Health and Aging Study (2001 -2012), who reported no limitations in activities of daily living (ADLs) at baseline and have complete data on all covariates. Sociodemographics, smoking status, comorbidities, ADL activities, and body mass index (BMI) were collected. RESULTS: The lowest hazard ratio (HR) for disability was at BMI of 25 to < 30 (HR = 0.97;95% confidence interval [CI], 0.85-1.12).The lowest HR for mortality were seen among participants with BMIs 25 to < 30 (HR = 0.85; 95%CI, 075-0.97), 30 to < 35 (HR = 0.86; 95 %CI, 0.72-1.02), and > 35 (HR = 0.92; 95 %CI, 0.70-1.22). CONCLUSION: Mexican older adults with a BMI of 25 to < 30 were at less risk for both disability and mortality.

Adiponectin May Modify the Risk of Barrett's Esophagus in Patients With Gastroesophageal Reflux Disease.

BACKGROUND & AIMS: Abdominal obesity and increasing body mass index are risk factors for esophageal adenocarcinoma and its main precursor, Barrett's esophagus; however, there are no known biological mechanisms for these associations or regarding why only some patients with gastroesophageal reflux disease develop Barrett's esophagus. We evaluated the association between Barrett's esophagus and multimers of an adipose-associated hormone, adiponectin. METHODS: We conducted a case-control study evaluating the associations between adiponectin (total, high-molecular-weight, and low-/medium-molecular-weight) and Barrett's esophagus within the Kaiser Permanente Northern California population. Patients with a new diagnosis of Barrett's esophagus (cases) were matched to patients with gastroesophageal reflux disease (GERD) without Barrett's esophagus and to population controls. RESULTS: Complete serologic and epidemiologic data were available for 284 cases, 294 GERD controls, and 285 population controls. Increasing adiponectin levels were a risk factor for Barrett's esophagus among patients with GERD (total adiponectin fourth vs first quartile odds ratio [OR], 1.96; 95% confidence interval [CI], 1.17-3.27; high-molecular-weight adiponectin OR, 1.65; 95% CI, 1.00-2.73; low-/medium-molecular-weight adiponectin OR, 2.18; 95% CI, 1.33-3.56), but not compared with population controls. The associations were significantly stronger among patients reporting frequent GERD symptoms and among smokers (P values interaction < .01). CONCLUSIONS: Adiponectin levels are associated positively with the risk of Barrett's esophagus among patients with GERD and among smokers, but not among population controls without GERD symptoms. Higher adiponectin concentrations either independently may contribute to the aberrant healing of esophageal injury into Barrett's esophagus or be a marker for other factors.

The effect of obesity on incidence of disability and mortality in Mexicans aged 50 years and older / El efecto de la obesidad sobre la incidencia de discapacidad y mortalidad en adultos mexicanos mayores de 50 años

Objective. To examine the effect of obesity on incidence of disability and mortality among non-disabled older Mexicans at baseline. Materials and methods. The sample included 8 415 Mexicans aged ≥ 50 years from the Mexican Health and Aging Study (2001 -2012), who reported no limitations in activities of daily living (ADLs) at baseline and have complete data on all covariates. Sociodemographics, smoking status, comorbidities, ADL activities, and body mass index (BMI) were collected. Results. The lowest hazard ratio (HR) for disability was at BMI of 25 to < 30 (HR = 0.97;95% confidence interval [CI], 0.85-1.12).The lowest HR for mortality were seen among participants with BMIs 25 to < 30 (HR = 0.85; 95%CI, 075-0.97), 30 to < 35 (HR = 0.86; 95 %CI, 0.72-1.02), and > 35 (HR = 0.92; 95 %CI, 0.70-1.22). Conclusion. Mexican older adults with a BMI of 25 to < 30 were at less risk for both disability and mortality.

Objetivo. Examinar el efecto de la obesidad sobre la incidencia de discapacidad y mortalidad en adultos mayores mexicanos sin discapacidad al inicio del estudio. Material y métodos. La muestra incluyó 8 415 Mexicanos ≥ 50 años de edad del Estudio Nacional de Salud y Envejecimiento en México (2001 -2012), quienes no reportaron discapacidad en las actividades de la vida diaria en la encuesta basal y tenían información completa de todas las covariables. Resultados. La razón de riesgo más baja (HR) para discapacidad se observó con un IMC de 25 a < 30 (HR=0.97;95%CI, 0.85-1.12). La razón de riesgo más baja para mortalidad se observó con IMC de 25 a < 30 (HR = 0.85;95%CI,075-0.97),de 30 a < 35 (HR = 0.86; 95%CI, 0.72-1.02), y > 35 (HR = 0.92; 95%CI, 0.70-1.22). Conclusión. Los adultos mayores mexicanos con un IMC de 25 a < 30 tuvieron menor riesgo de discapacidad y mortalidad.

Provider continuity prior to the diagnosis of advanced lung cancer and end-of-life care.

BACKGROUND: Little is known about the effect of provider continuity prior to the diagnosis of advanced lung cancer and end-of-life care. METHODS: Retrospective analysis of 69,247 Medicare beneficiaries aged 67 years or older diagnosed with Stage IIIB or IV lung cancer between January 1, 1993 and December 31, 2005 who died within two years of diagnosis. We examined visit patterns to a primary care physician (PCP) and/or any provider one year prior to the diagnosis of advanced lung cancer as measures of continuity of care. Outcome measures were hospitalization, ICU use and chemotherapy use during the last month of life, and hospice use during the last week of life. RESULTS: Seeing a PCP or any provider in the year prior to the diagnosis of advanced lung cancer increased the likelihood of hospitalization, ICU care, chemotherapy and hospice use during the end of life. Patients with 1-3, 4-7 or >7 visits to their PCP in the year prior to the diagnosis of lung cancer had 1.0 (reference), 1.08 (95% CI; 1.04-1.13), and 1.14 (95% CI; 1.08-1.19) odds of hospitalization during the last month of life, respectively. Odds of hospice use during the last week of life were higher in patients with visits to multiple PCPs (OR 1.10: 95% CI; 1.06-1.15) compared to those whose visits were all to the same PCP. CONCLUSION: Provider continuity in the year prior to the diagnosis of advanced lung cancer was not associated with lower use of aggressive care during end of life. Our study did not have information on patient preferences and result should be interpreted accordingly.

Bacterial volatiles and diagnosis of respiratory infections.

Breath testing has enormous potential in the medical diagnostic field. The underlying complexity and perceived availability of adequate specimens, combined with a lack of knowledge of the metabolic pathways that give rise to compounds that are sources of analytes detectable in breath, has greatly slowed development. These real obstacles have recently been largely overcome in the use of breath testing to identify patients with cystic fibrosis associated Pseudomonas aeruginosa infection and tuberculosis. This review summarizes progress made in the characterization of microbial volatiles produced by major lower respiratory tract bacterial pathogens, and their potential use as diagnostic markers in patient breath testing.

Effects of opsonization of Rhodococcus equi on bacterial viability and phagocyte activation.

OBJECTIVE: To investigate the effect of opsonization of Rhodococcus equi with R. equi-specific antibodies in plasma on bacterial viability and phagocyte activation in a cell culture model of infection. SAMPLE: Neutrophils and monocyte-derived macrophages from 6 healthy 1-week-old foals and 1 adult horse. PROCEDURES: Foal and adult horse phagocytes were incubated with either opsonized or nonopsonized bacteria. Opsonization was achieved by use of plasma containing high or low concentrations of R. equi-specific antibodies. Phagocyte oxidative burst activity was measured by use of flow cytometry, and macrophage tumor necrosis factor (TNF)-α production was measured via an ELISA. Extracellular and intracellular bacterial viability was measured with a novel R. equi-luciferase construct that used a luminometer. RESULTS: Opsonized bacteria increased oxidative burst activity in adult horse phagocytes, and neutrophil activity was dependent on the concentration of specific antibody. Secretion of TNF-α was higher in macrophages infected with opsonized bacteria. Opsonization had no significant effect on bacterial viability in macrophages; however, extracellular bacterial viability was decreased in broth containing plasma with R. equi-specific antibodies, compared with viability in broth alone. CONCLUSIONS AND CLINICAL RELEVANCE: The use of plasma enriched with specific antibodies for the opsonization of R. equi increased the activation of phagocytes and decreased bacterial viability in the extracellular space. Although opsonized R. equi increased TNF-α secretion and oxidative burst in macrophages, additional factors may be necessary for effective intracellular bacterial killing. These data have suggested a possible role of plasma antibody in protection of foals from R. equi pneumonia.

Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA.

FtsK is a homohexameric, RecA-like dsDNA translocase that plays a key role in bacterial chromosome segregation. The FtsK regulatory γ-subdomain determines directionality of translocation through its interaction with specific 8 base pair chromosomal sequences [(KOPS); FtsK Orienting/Polarizing Sequence(s)] that are cooriented with the direction of replication in the chromosome. We use millisecond-resolution ensemble translocation and ATPase assays to analyze the assembly, initiation, and translocation of FtsK. We show that KOPS are used to initiate new translocation events rather than reorient existing ones. By determining kinetic parameters, we show sigmoidal dependences of translocation and ATPase rates on ATP concentration that indicate sequential cooperative coupling of ATP hydrolysis to DNA motion. We also estimate the ATP coupling efficiency of translocation to be 1.63-2.11 bp of dsDNA translocated/ATP hydrolyzed. The data were used to derive a model for the assembly, initiation, and translocation of FtsK hexamers.

Obesity and disability: relation among older adults living in Latin America and the Caribbean.

The prevalence and incidence of both obesity and disability are projected to increase in the coming decades. The authors examined the relation between obesity and disability in older adults from 6 Latin American cities participating in the Health, Well-Being and Aging in Latin America and the Caribbean (SABE) Study (1999-2000). The sample included 6,166 participants aged 65 years or more. Data on sociodemographic factors, smoking status, medical conditions, body mass index (BMI; weight (kg)/height (m)(2)), and self-reported activities of daily living (ADL) were obtained. The prevalence of obesity (BMI > or = 30) ranged from 13.3% in Havana, Cuba, to 37.6% in Montevideo, Uruguay. Using a BMI of 18.5-<25 as the reference category and controlling for all covariates, the lowest odds ratio for ADL limitation was for a BMI of 25-<30 (odds ratio = 1.10, 95% confidence interval: 0.93, 1.30), and the highest odds ratio for ADL limitation was for a BMI of 35 or higher (odds ratio = 1.63, 95% confidence interval: 1.26, 2.11). The results indicated that obesity is an independent factor contributing to ADL disability in these populations and should be included in future planning to reduce the impact of disability on global health.

FtsK translocation on DNA stops at XerCD-dif.

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKgamma regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.

Mexican Americans and frailty: findings from the Hispanic established populations epidemiologic studies of the elderly.

OBJECTIVES: We examined the prevalence of frailty among Mexican American older adults and explored the correlates associated with becoming frail to determine their affect on disability and morbidity in this population. METHODS: We studied the trajectory of frailty over 10 years in 2049 Mexican Americans participating in the Hispanic Established Populations Epidemiologic Studies of the Elderly. We constructed a frailty index based on weight loss, exhaustion, grip strength, walking speed, and physical activity and collected data on sociodemographic and health status, comorbidities, and functional measures of performance. RESULTS: The sample was 58% female, with a mean age of 74.43 years (SD = 6.04) at baseline. Fifty-five percent of participants at baseline and 75% of the surviving sample at follow-up (n = 777) were classified as prefrail or frail. Of persons identified as frail at baseline, 84% died by the end of follow-up. Baseline age, diabetes, arthritis, smoking status, body mass index, cognition, negative affect, and number of comorbid conditions were predictors of frailty at follow-up (R(2) = 0.29; P < .05). CONCLUSIONS: Further research into ways to reduce the number of Mexican American older adults who become frail and disabled and therefore lose their independence is needed. Future studies should continue to examine the trajectory of frailty as a dynamic process that includes psychosocial and cognitive components.

Gender and ethnic differences in rehabilitation outcomes after hip-replacement surgery.

OBJECTIVE: To examine gender and ethnic differences in functional status and living setting for patients after hip arthroplasty. DESIGN: Retrospective cohort study of 69,793 patients receiving inpatient medical rehabilitation after hip arthroplasty included in the Uniform Data System for Medical Rehabilitation database for the period of 2002-2003. Primary measures included functional status as assessed by the FIM instrument and discharge living setting (home vs. not home). The sample included non-Hispanic white, non-Hispanic black, Hispanic, and Asian patients. RESULTS: Multivariate regression models showed the greatest FIM instrument change scores from admission to discharge among non-Hispanic whites (mean [SE]: 23.42 [0.18]) and among women (mean [SE]: 22.79 [0.23]). Asians had the lowest mean change scores (mean [SE]: 22.00 [0.53]). Estimates from multivariate logistic models showed that being of nonwhite ethnicity was associated with higher odds of home discharge (black: OR [CI]: 1.23, CI 95% = 1.07, 1.41; Hispanic: OR [CI]: 1.51, CI 95% = 1.15-1.99). Compared with women, men had higher odds of home discharge (OR [CI]: 1.08, CI 95% = 1.01, 1.17). CONCLUSIONS: The findings suggest that ethnic and gender disparities exist in postacute care outcomes for persons with hip arthroplasty.

Glycine betaine uptake by the ProXVWZ ABC transporter contributes to the ability of Mycobacterium tuberculosis to initiate growth in human macrophages.

Mycobacterium tuberculosis maintains a large genetic capacity necessary for growth in different environments during infection and survival upon aerosol transmission to new hosts. Screening for bacterial RNAs produced in response to host interactions produced candidate lists where we noted proXVWZ, annotated as encoding a putative glycine betaine or proline transporter. As high surface-to-volume ratios make bacterial cells particularly vulnerable to changes in water availability, we investigated the contributions of this transporter to the ability of M. tuberculosis to colonize macrophages. An H37Rv proXVWZ mutant was impaired for initial survival and intracellular growth and exhibited reduced growth at elevated medium osmolarity. This defect could be complemented by restoring proXVWZ and was attributable to a failure to accumulate the compatible solute glycine betaine. We then demonstrated that ProXVWZ allows M. tuberculosis to obtain betaine from host macrophages and thereby contributes to early steps in colonizing this niche.

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages.

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.

Genetic susceptibility and caspase activation in mouse and human macrophages are distinct for Legionella longbeachae and L. pneumophila.

Legionella pneumophila is the predominant cause of Legionnaires' disease in the United States and Europe, while Legionella longbeachae is the common cause of the disease in Western Australia. Although clinical manifestations by both intracellular pathogens are very similar, recent studies have shown that phagosome biogeneses of both species within human macrophages are distinct (R. Asare and Y. Abu Kwaik, Cell. Microbiol., in press). Most inbred mouse strains are resistant to infection by L. pneumophila, with the exception of the A/J mouse strain, and this genetic susceptibility is associated with polymorphism in the naip5 allele and flagellin-mediated early activation of caspase 1 and pyropoptosis in nonpermissive mouse macrophages. Here, we show that genetic susceptibility of mice to infection by L. longbeachae is independent of allelic polymorphism of naip5. L. longbeachae replicates within bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice, while L. pneumophila replicates in macrophages in vitro and in the lungs of the A/J mouse strain only. Quantitative real-time PCR studies on infected A/J and C57BL/6 mouse bone marrow-derived macrophages show that both L. longbeachae and L. pneumophila trigger similar levels of naip5 expression, but the levels are higher in infected C57BL/6 mouse macrophages. In contrast to L. pneumophila, L. longbeachae has no detectable pore-forming activity and does not activate caspase 1 in A/J and C57BL/6 mouse or human macrophages, despite flagellation. Unlike L. pneumophila, L. longbeachae triggers only a modest activation of caspase 3 and low levels of apoptosis in human and murine macrophages in vitro and in the lungs of infected mice at late stages of infection. We conclude that despite flagellation, infection by L. longbeachae is independent of polymorphism in the naip5 allele and L. longbeachae does not trigger the activation of caspase 1, caspase 3, or late-stage apoptosis in mouse and human macrophages. Neither species triggers caspase 1 activation in human macrophages.

Role for RpoS but not RelA of Legionella pneumophila in modulation of phagosome biogenesis and adaptation to the phagosomal microenvironment.

The induction of virulence traits by Legionella pneumophila at the post-exponential phase has been proposed to be triggered by the stringent response mediated by RelA, which triggers RpoS. We show that L. pneumophila rpoS but not relA is required for early intracellular survival and replication within human monocyte-derived macrophages and Acanthamoeba polyphaga. In addition, L. pneumophila rpoS but not relA is required for expression of the pore-forming activity. We provide evidence that RpoS plays a role in the modulation of phagosome biogenesis and in adaptation to the phagosomal microenvironment. Thus, there is no functional link between the stringent response and RpoS in the pathogenesis of L. pneumophila.

Sequence-specific Rho-RNA interactions in transcription termination.

The bacteriophage lambda tR1 terminator encodes a region of the nascent cro transcript containing RNA residues recognized by termination factor Rho. To identify ribonucleotide-protein interactions contributing to termination, a library of reporter gene plasmids was constructed containing predominantly single-nucleotide substitutions in a 24 nt region previously shown to be critical for efficient termination. Screening 16 822 bacterial transformants identified 110 terminator mutants, most of which contained two or more nucleotide substitutions. Although the vast majority of single base changes did not reduce tR1 function, 11 specific single-nucleotide substitutions at eight positions interspersed in the upstream part of the target region (5'-ATAACCCCGCTCTT ACACATTCCA-3') did reduce termination. About half of these substitutions also reduced Rho-dependent termination on cro gene templates transcribed by purified RNA polymerase, indicating specific residues critical for optimal terminator function. Other termination defects were not reproduced in these in vitro assays, and likely resulted from indirect effects of altering interactions between tR1 and additional cellular factors capable of attenuating Rho function. Our results indicate that while Rho is able to recognize a wide variety of similar rut site sequences by interacting with alternate nucleotides at critical positions, interactions with specific individual ribonucleotides of the tR1 transcript provide highly efficient Rho-dependent termination.