Do naps benefit novel word learning? Developmental differences and white matter correlates.

Memory representations of newly learned words undergo changes during nocturnal sleep, as evidenced by improvements in explicit recall and lexical integration (i.e., after sleep, novel words compete with existing words during online word recognition). Some studies have revealed larger sleep-benefits in children relative to adults. However, whether daytime naps play a similar facilitatory role is unclear. We investigated the effect of a daytime nap (relative to wake) on explicit memory (recall/recognition) and lexical integration (lexical competition) of newly learned novel words in young adults and children aged 10-12 years, also exploring white matter correlates of the pre- and post-nap effects of word learning in the child group with diffusion weighted MRI. In both age groups, a nap maintained explicit memory of novel words and wake led to forgetting. However, there was an age group interaction when comparing change in recall over the nap: children showed a slight improvement whereas adults showed a slight decline. There was no evidence of lexical integration at any point. Although children spent proportionally more time in slow-wave sleep (SWS) than adults, neither SWS nor spindle parameters correlated with over-nap changes in word learning. For children, increased fractional anisotropy (FA) in the uncinate fasciculus and arcuate fasciculus were associated with the recognition of novel words immediately after learning, and FA in the right arcuate fasciculus was further associated with changes in recall of novel words over a nap, supporting the importance of these tracts in the word learning and consolidation process. These findings point to a protective role of naps in word learning (at least under the present conditions), and emphasize the need to better understand both the active and passive roles that sleep plays in supporting vocabulary consolidation over development.

Period-dependent Associations between Hypotension during and for Four Days after Noncardiac Surgery and a Composite of Myocardial Infarction and Death: A Substudy of the POISE-2 Trial.

BACKGROUND: The relative contributions of intraoperative and postoperative hypotension to perioperative morbidity remain unclear. We determined the association between hypotension and a composite of 30-day myocardial infarction and death over three periods: (1) intraoperative, (2) remaining day of surgery, and (3) during the initial four postoperative days. METHODS: This was a substudy of POISE-2, a 10,010-patient factorial-randomized trial of aspirin and clonidine for prevention of myocardial infarction. Clinically important hypotension was defined as systolic blood pressure less than 90 mmHg requiring treatment. Minutes of hypotension was the exposure variable intraoperatively and for the remaining day of surgery, whereas hypotension status was treated as binary variable for postoperative days 1 to 4. We estimated the average relative effect of hypotension across components of the composite using a distinct effect generalized estimating model, adjusting for hypotension during earlier periods. RESULTS: Among 9,765 patients, 42% experienced hypotension, 590 (6.0%) had an infarction, and 116 (1.2%) died within 30 days of surgery. Intraoperatively, the estimated average relative effect across myocardial infarction and mortality was 1.08 (98.3% CI, 1.03, 1.12; P < 0.001) per 10-min increase in hypotension duration. For the remaining day of surgery, the odds ratio was 1.03 (98.3% CI, 1.01, 1.05; P < 0.001) per 10-min increase in hypotension duration. The average relative effect odds ratio was 2.83 (98.3% CI, 1.26, 6.35; P = 0.002) in patients with hypotension during the subsequent four days of hospitalization. CONCLUSIONS: Clinically important hypotension-a potentially modifiable exposure-was significantly associated with a composite of myocardial infarction and death during each of three perioperative periods, even after adjustment for previous hypotension.

Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation.

Autophagy is a highly dynamic intracellular process involving interactions between protein complexes and membranes. Direct observation of these components in living cells provides information on how they interact and when and where they are involved in the autophagy pathway. This chapter provides an overview of methods used to acquire images of fluorescently labeled components of the autophagy pathway in living cells using wide-field microscopy. Due to the diffraction-limited nature of this technique further details are provided on how to acquire postfixation correlative super resolution images from the same cells that have previously been imaged live. Combining these techniques offers an opportunity to follow the processes of autophagy in living cells with unprecedented detail.

The Clinical Utility of Next Generation Sequencing Results in a Community-Based Hereditary Cancer Risk Program.

Since the 2013 Supreme Court ruling on BRCA1/BRCA2 patenting, hereditary cancer gene panels now include BRCA1 and BRCA2, making these panels an option for first-tier testing. However, questions remain about the clinical utility and implications of these panels for medical management with inclusion of genes of unknown to moderate penetrance. To better understand how use of these panels affected our practice, we reviewed patients who underwent testing in our clinic from July 1, 2013 through May 23, 2014. Indications for testing included personal and/or family history of breast and/or ovarian cancer. A total of 136 patients underwent panel testing via a single commercial laboratory; 12 (8.8 %) patients were positive for a pathogenic or likely pathogenic mutation (four BRCA2 mutations, two TP53 mutations, one CDH1 mutation, two ATM mutations, and one patient each with a CHEK2, NBN, or PALB2 mutation). Of these positive patients, 100 % met the National Comprehensive Cancer Network (NCCN) guidelines for Hereditary Breast and Ovarian Cancer genetic testing (2.2014). Mutations in seven of twelve (58 %) patients led to changes in medical management; three of seven (43 %) had a non-BRCA1 or BRCA2 gene mutation. Our findings suggest that there is clinical utility of panels that include genes of unknown to moderate penetrance.

A critical role for IRF5 in regulating allergic airway inflammation.

Interferon regulatory factor 5 (IRF5) is a key transcription factor involved in the control of the expression of proinflammatory cytokine and responses to infection, but its role in regulating pulmonary immune responses to allergen is unknown. We used genetic ablation, adenoviral vector-driven overexpression, and adoptive transfer approaches to interrogate the role of IRF5 in pulmonary immunity and during challenge with the aeroallergen, house dust mite. Global IRF5 deficiency resulted in impaired lung function and extracellular matrix (ECM) deposition. IRF5 was also essential for effective responses to inhaled allergen, controlling airway hyperresponsiveness, mucus secretion, and eosinophilic inflammation. Adoptive transfer of IRF5-deficient alveolar macrophages into the wild-type pulmonary milieu was sufficient to drive airway hyperreactivity, at baseline or following antigen challenge. These data identify IRF5-expressing macrophages as a key component of the immune defense of the airways. Manipulation of IRF5 activity in the lung could therefore be a viable strategy for the redirection of pulmonary immune responses and, thus, the treatment of lung disorders.

Impact of infection with extended-spectrum ß-lactamase-producing Escherichia coli or Klebsiella species on outcome and hospitalization costs.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing bacteria are important sources of infection; however, Canadian data evaluating the impact of ESBL-associated infection are lacking. AIM: To determine whether patients infected with ESBL-producing Escherichia coli or Klebsiella species (ESBL-EcKs) exhibit differences in clinical outcome, microbiological outcome, mortality, and/or hospital resource use compared to patients infected with non-ESBL-producing strains. METHODS: A retrospective case-control study of 75 case patients with ESBL-EcKs matched to controls infected with non-ESBL-EcKs who were hospitalized from June 2010 to April 2013 was conducted. Patient-level cost data were provided by the institution's business office. Clinical data were collected using the electronic databases and paper charts. FINDINGS: Median infection-related hospitalization costs per patient were greater for cases than controls (C$10,507 vs C$7,882; median difference: C$3,416; P = 0.04). The primary driver of increased costs was prolonged infection-related hospital length of stay (8 vs 6 days; P = 0.02) with patient location (ward, ICU) and indirect care costs (including costs associated with infection prevention and control) as the leading cost categories. Cases were more likely to experience clinical failure (25% vs 11%; P = 0.03), with a higher all-cause mortality (17% vs 5%; P = 0.04). Less than half of case patients were prescribed appropriate empiric antimicrobial therapy, whereas controls received adequate initial treatment in nearly all circumstances (48% vs 96%; P < 0.01). CONCLUSION: Patients with infection caused by ESBL-EcKs are at increased risk for clinical failure and mortality, with additional cost to the Canadian healthcare system of C$3,416 per patient.

Alveolar macrophages are sentinels of murine pulmonary homeostasis following inhaled antigen challenge.

BACKGROUND: Alveolar macrophages are sentinels of the pulmonary mucosa and central to maintaining immunological homeostasis. However, their role in governing the response to allergen is not fully understood. Inappropriate responses to the inhaled environment manifest as asthma. METHODS: We utilized a mechanistic IL-13-driven model and a house dust mite allergen mucosal sensitization model of allergic airway disease to investigate the role of alveolar macrophages in regulating pulmonary inflammation. RESULTS: IL-13-dependent eosinophilic and Th2 inflammation was enhanced in mice depleted of alveolar macrophages using clodronate liposomes. Similarly, depletion of alveolar macrophages during house dust mite sensitization or established disease resulted in augmented Th2 immunity and increased allergen-specific IgG1 and IgE. Clodronate treatment also delayed the resolution of tissue inflammation following cessation of allergen challenge. Strikingly, tissue interstitial macrophages were elevated in alveolar macrophage-deficient mice identifying a new homeostatic relationship between different macrophage subtypes. A novel role for the macrophage-derived immunoregulatory cytokine IL-27 was identified in modulating Th2 inflammation following mucosal allergen exposure. CONCLUSIONS: In summary, alveolar macrophages are critical regulators of Th2 immunity and their dysregulation promotes an inflammatory environment with exacerbation of allergen-induced airway pathology. Manipulating IL-27 may provide a novel therapeutic strategy for the treatment of asthma.

Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease.

BACKGROUND: Associations between vitamin D status and childhood asthma are increasingly reported, but direct causation and mechanisms underlying an effect remain unknown. We investigated the effect of early-life vitamin D deficiency on the development of murine neonatal allergic airways disease (AAD). METHODS: In utero and early-life vitamin D deficiency was achieved using a vitamin D-deficient diet for female mice during the third trimester of pregnancy and lactation. Offspring were weaned onto a vitamin D-deficient or vitamin D-replete diet, and exposure to intranasal house dust mite (HDM) or saline was commenced from day 3 of life for up to 6 weeks, when airway hyper-responsiveness (AHR), airway inflammation and remodelling were assessed. RESULTS: Neonatal mice that had in utero and early-life vitamin D deficiency had significantly increased pulmonary CD3(+) CD4(+) T1ST2(+) cells and reduced CD4(+) IL-10(+) cells. This effect was enhanced following HDM exposure. AHR in HDM-exposed mice was unaffected by vitamin D status. Introduction of vitamin D into the diet at weaning resulted in a significant reduction in serum IgE levels, reduced pulmonary eosinophilia and peri-bronchiolar collagen deposition. CONCLUSION: Peri-natal vitamin D deficiency alone has immunomodulatory effects including Th2 skewing and reduced IL-10-secreting T regulatory cells, exaggerated with additional allergen exposure. Vitamin D deficiency in early life does not affect AHR, but contributes to disease severity with worse eosinophilic inflammation and airway remodelling. Importantly, supplementation with vitamin D improves both of these pathological abnormalities.

Linear structures in the core of the Coma cluster of galaxies.

The hot x-ray-emitting plasma in galaxy clusters is predicted to have turbulent motion, which can contribute around 10% of the cluster's central energy density. We report deep Chandra X-ray Observatory observations of the Coma cluster core, showing the presence of quasi-linear high-density arms spanning 150 kiloparsecs, consisting of low-entropy material that was probably stripped from merging subclusters. Two appear to be connected with a subgroup of galaxies at a 650-kiloparsec radius that is merging into the cluster, implying coherence over several hundred million years. Such a long lifetime implies that strong isotropic turbulence and conduction are suppressed in the core, despite the unrelaxed state of the cluster. Magnetic fields are presumably responsible. The structures seen in Coma present insight into the past billion years of subcluster merger activity.

Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons.

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

Duration of hospital admission and the need for empirical antipseudomonal therapy.

To reduce selective pressure for antimicrobial resistance, empirical use of antipseudomonal antibiotics is often reserved for patients with late-onset hospital-acquired infections. We examined the likelihood of isolating Pseudomonas aeruginosa as a function of time from hospital admission. We conducted a retrospective cohort study of all positive bacterial cultures in a tertiary-care hospital between March 2010 and November 2011. The primary outcome was the proportion of positive cultures yielding P. aeruginosa. Multivariable logistic regression was employed to assess the impact of time from admission on the likelihood of isolating P. aeruginosa, after adjusting for other important risk factors. A total of 7,668 positive cultures were obtained from 4,108 unique patients during the study interval, including 633 (8.3%) yielding P. aeruginosa. The probability of isolating P. aeruginosa increased linearly from 79/2,044 (3.9%) positive cultures obtained on admission to 153/664 (23%) in the 10th week of admission or beyond. The unadjusted odds ratio was 1.002/day (95% confidence interval [CI], 1.0016 to 1.0028; P < 0.0001); the adjusted odds ratio (aOR) was 1.0007/day (95% CI, 1.0001 to 1.0013; P = 0.02). Other important predictors of P. aeruginosa isolation included respiratory specimen type (aOR, 13.8; 95% CI, 9.1 to 21.1), recent hospital admission (aOR,1.8; 95% CI, 1.4 to 2.3), prior P. aeruginosa isolation during current admission (aOR, 4.9; 95% CI, 3.7 to 6.4), and prior antipseudomonal (aOR, 1.9; 95% CI, 1.4 to 2.5) or nonantipseudomonal (aOR, 1.8; 95% CI, 1.4 to 2.4) antibiotic exposure. It was determined that as time from admission increases, there is a linear increase in the likelihood of P. aeruginosa isolation. Any guidelines which distinguish early from late hospital-acquired infection must consider the implications of time point selection on the likelihood of inadequate P. aeruginosa empirical coverage.

Audit of antibiotic duration of therapy, appropriateness and outcome in patients with nosocomial pneumonia following the removal of an automatic stop-date policy.

Automatic stop-orders (ASOs) have been utilized to discourage inappropriately prolonged antibiotic therapy. An ASO policy, which required reordering of antibiotics after 7 days of therapy, had been in place at our institution prior to 2002, but was revoked after instances of compromised patient care due to inadvertent and inappropriate interruption of antimicrobial treatment. The objective of this study was to evaluate the impact of revoking the ASO policy on the duration of antibiotic therapy, infection-related outcome (cure vs failure), relapsing infection, occurrence of resistant bacteria and superinfection in patients with nosocomial pneumonia. A retrospective chart review of adult patients (≥ 18 years old) admitted to Sunnybrook Health Sciences Centre with nosocomial pneumonia requiring antibiotic therapy was conducted. Duration of antibiotic therapy, infection-related outcome (cure vs failure), rate of relapsing infection, resistant organisms and superinfection were determined for each cohort. Forty-six eligible adults with nosocomial pneumonia per cohort were included [corrected]. Duration of antibiotic therapy was not significantly different in the pre- (11.4 ± 3.8 days) compared with the post-ASO revocation cohort (10.8 ± 4.1 days; p=0.43). There were also no significant differences between the cohorts with regard to infection-related outcome (cure vs failure), relapsing infection, or the occurrence of resistant bacteria or superinfection (p>0.5). Revocation of the ASO policy for antibiotics at our institution was not associated with a longer duration of antibiotic therapy, or increased incidence of infection-related mortality, relapsing infection, resistant bacteria or superinfection for patients with nosocomial pneumonia.

Comparison of mortality between nosocomial and community-acquired febrile neutropenia patients treated initially with cefazolin plus tobramycin: retrospective chart review.

Cefazolin plus tobramycin have been determined to be effective for community-acquired FN, but have not been evaluated in the treatment of nosocomial FN. This study compared the incidence of mortality from 2002 to 2004 with 2008 to 2009 in patients with nosocomial FN treated with cefazolin plus tobramycin and compared characteristics of patients with nosocomially acquired FN to community acquired FN. A retrospective chart review of 45 nosocomial FN episodes from 2008 to 2009, and 54 episodes from 2002 to 2004 treated with cefazolin plus tobramycin was conducted. Data on the community acquired FN episodes was obtained from our previous research. Nosocomial FN mortality increased from 4% in 2002-2004 to 13% in 2008-2009 (p = 0.08). The nosocomial cohort was at higher risk of medical complications and mortality than the community-acquired cohort based on several variables (neutrophil nadir, duration of neutropenia and fever, hematological malignancy, MASCC and Talcott score; p < 0.05). As a result, the nosocomial cohort was treated with longer courses of antibiotic therapy (14 days vs 7 days; p < 0.0001) and were more likely to require broader spectrum antibiotics (64 out of 99 vs 34 out of 96; p < 0.0001). There was an observed increased risk of mortality from 2002 to 2004 compared with 2008 to 2009 in patients treated with cefazolin plus tobramycin for nosocomial FN, this was notable despite not attaining statistical significance. Therefore, this regimen is not appropriate for nosocomial FN.

Exposures recorded for participants in the UK Chemical Warfare Agent Human Research Programme, 1941-1989.

OBJECTIVES: This study describes exposures to military veterans who participated between 1941 and 1989 in British research at Porton Down on the effects of exposure to chemical warfare agents and to defences against those agents. The study is part of a programme of epidemiological research initiated in response to service veterans' concerns about possible long-term health effects of their participation. METHODS: All entries in 97 books held in the Porton Down historical experimental archive covering the years 1939-1989 were reviewed. For tests between April 1941 and December 1989, data were abstracted on chemicals used, with additional detail abstracted for tests involving vesicants and nerve agents. For tests recorded during 1939-1941, similar data were abstracted for a representative sample of tests. RESULTS: Historical data were abstracted for 17 303 veterans included in the cohort study of 18,276 servicemen who took part in tests at Porton Down between 1941 and 1989. The median number of days per veteran on which tests were carried out was 2 days. The median difference between the last and first day of testing was 4 days. A large number of chemicals were tested over this period (n = 492). The type of chemical tested varied over time. Exposures were often modified by respirator use or use of protective clothing or protective equipment. It was possible to assign a quantitative measure of cumulative exposure to 73% of veterans exposed to the vesicant sulphur mustard--3491 (34%) of exposed veterans had cumulative exposures > or =10.63 mg and for 70% of veterans exposed to the nerve agent sarin--658 (29%) of exposed veterans had cumulative exposures > or =15.0 mg min m(-3). Ninety-three per cent of veterans exposed to sulphur mustard were classified to a semi-quantitative scale of dermal effect--3771 (37%) had a vesicle or necrosed area, and 69% of veterans exposed to sarin could be categorized by change in blood cholinesterase activity--1033 (31%) had a depression in cholinesterase activity of > or =30%. CONCLUSIONS: The experimental archive at Porton Down has proved to be a rich source of data on tests conducted between 1941 and 1989. It has been possible to categorize most veterans according to date of test, chemical group, chemical, type of protection and, for certain chemicals, level of exposure and/or degree of acute toxicity. These categorizations have been used to assign veterans to exposure groups for epidemiological analysis.

Computational methods for the HZETRN code.

Asymptotic expansion has been used to simplify the transport of high charge and energy ions for broad beam applications in the laboratory and space. The solution of the lowest order asymptotic term is then related to a Green's function for energy loss and straggling coupled to nuclear attenuation providing the lowest order term in a rapidly converging Neumann series for which higher order collisions terms are related to the fragmentation events including energy dispersion and downshift. The first and second Neumann corrections were evaluated numerically as a standard for further analytic approximation. The first Neumann correction is accurately evaluated over the saddle point whose width is determined by the energy dispersion and located at the downshifted ion collision energy. Introduction of the first Neumann correction leads to significant simplification of the second correction term allowing application of the mean value theorem and a second saddle point approximation. The regular dependence of the second correction spectral dependence lends hope to simple approximation to higher corrections. At sufficiently high energy nuclear cross-section variations are small allowing non-perturbative methods to all orders and renormalization of the second corrections allow accurate evaluation of the full Neumann series.

Validation of the HZETRN code for laboratory exposures with 1A GeV iron ions in several targets.

A new version of the HZETRN code capable of validation with HZE ions in either the laboratory or the space environment is under development. The computational model consists of the lowest order asymptotic approximation followed by a Neumann series expansion with non-perturbative corrections. The physical description includes energy loss with straggling, nuclear attenuation, nuclear fragmentation with energy dispersion and downshift. Measurements to test the model were performed at the Alternating Gradient Synchrotron and the NASA Space Radiation Laboratory at Brookhaven National Laboratory with iron ions. Surviving beam particles and produced fragments were measured with solid-state detectors. Beam analysis software has been written to relate the computational results to the measured energy loss spectra of the incident ions for rapid validation of modeled target transmission functions.

The Ras binary switch: an ideal processor for decoding complex Ca2+ signals?

Activation of cell-surface receptors often leads to changes in intracellular calcium concentration ([Ca(2+)](i)). Receptor-generated calcium transients are often seen as repetitive spikes of elevated intracellular calcium concentration ([Ca(2+)](i)), whose frequency varies according to the amplitude of the receptor stimuli. This suggests a requirement for molecular decoders, capable of interpreting such complex calcium signals into the correct physiological response. Ras proteins are binary molecular switches controlling a plethora of cellular responses. Whether Ras is in its active GTP-bound, or inactive GDP-bound, form is determined by the activity of guanine nucleotide exchange factors (GEFs) and GTPase-activating protein (GAPs). Calcium-regulated GEFs and GAPs have been identified, some with an exquisite sensitivity to [Ca(2+)](i), implicating a potential role of complex calcium signals in regulating Ras.

Femoral condylar lift-off in vivo in total knee arthroplasty.

We carried out weight-bearing video radiological studies on 40 patients with a total knee arthroplasty (TKA), to determine the presence and magnitude of femoral condylar lift-off. Half (20) had posterior-cruciate-retaining (PCR) and half (20) posterior-cruciate-substituting (PS) prostheses. The selected patients had successful arthroplasties with no pain or instability. Each carried out successive weight-bearing knee bends to maximum flexion, and the radiological video tapes were analysed using an interactive model-fitting technique. Femoral lift-off was seen at some increment of knee flexion in 75% of patients (PCR TKA 70%; PS TKA 80%). The mean values for lift-off were 1.2 mm with a PCR TKA and 1.4 mm with a PS TKA. Lift-off occurred mostly laterally with the PCR TKA, and both medially and laterally with the PS TKA. Separation between the femoral condyles and the articular surface of the tibia was recorded at 0 degrees, 30 degrees, 60 degrees and 90 degrees of flexion. Femoral condylar lift-off may contribute to eccentric polyethylene wear, particularly in designs of TKA which have flatter condyles. Coronal conformity is an important consideration in the design of a TKA.

Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways.

A novel system that leaks beta-galactosidase (beta-gal) without a requirement for secretion or export signals was developed in Lactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage phi31 is an 888-bp fragment (P(15A10)) encoding the transcriptional activator. When a high-copy-number P(15A10)::lacZ.st fusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant beta-gal activity were detected in the supernatant (approximately 85% of the total beta-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P(15A10) fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of beta-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage phi31 late promoter, into the host genome of MG1363 yielded a similar "leaky" phenotype, indicating that holin and lysin might play a critical role in the release of beta-gal into the medium. In addition to beta-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.

Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by nod factors and chitin oligomers.

Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum bv. viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Green). Within 1-2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occurring at a rate of about one spike per minute. These spikes, corresponding to an increase in calcium of approximately 200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to those induced by Nod factors in alfalfa. Calcium responses were analyzed in nonnodulating pea mutants, representing seven loci that affect early stages of the symbiosis. Mutations affecting three loci (sym8, sym10, and sym19) abolished Nod-factor-induced calcium spiking, whereas a normal response was seen in peas carrying alleles of sym2(A), sym7, sym9, and sym30. Chitin oligomers of four or five N-acetylglucosamine residues could also induce calcium spiking, although the response was qualitatively different from that induced by Nod factors; a rapid increase in intracellular calcium was not observed, the period between spikes was lower, and the response was not as sustained. The chitin-oligomer-induced calcium spiking did not occur in nodulation mutants (sym8, sym10, and sym19) that were defective for Nod-factor-induced spiking, suggesting that this response is related to nodulation signaling. From our data and previous observations on the lack of mycorrhizal infection in some of the sym mutants, we propose a model for the potential order of pea nodulation genes in nodulation and mycorrhizal signaling.