Matthew P. Lazio

Low-Level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria gonorrhoeae

ABSTRACT The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

A comparison of analgesic management for emergency department patients with sickle cell disease and renal colic.

ObjectivesTo determine whether there is a difference in time to initial analgesic for patients with acute pain from sickle cell disease (SCD) versus renal colic (RC) and to identify factors contributing to variance in time to analgesic. MethodsA retrospective cohort study of the adult emergency department (ED) patients with acute pain from SCD and RC in an urban ED (final ED discharge ICD-9 diagnosis codes were included). A structured medical record review abstracted the demographics, arrival shift, triage level, initial pain score, triage time, and time of initial analgesic dose. Data were compared with Kaplan-Meier plots of time to initial analgesic for both RC and SCD with the log-rank test to test for differences by disease category. A multivariable Cox regression model estimated differences in time to initial analgesic by disease category while controlling for other possible confounders. ResultsMedian time to initial analgesic was 80 minutes for patients with SCD (interquartile range, 48 to 145) versus 50 minutes for patients with RC (interquartile range: 30 to 96). Patients with SCD reported a higher pain score on arrival when compared with RC patients and were more frequently assigned a higher triage priority level (P=0.05). Covariates that contributed the most delays to the model were afternoon arrival [hazard ratio (HR): 0.35, P<0.01], low acuity triage level (HR: 0.42, P<0.01), SCD diagnosis (HR: 0.61, P<0.01), and inability to obtain intravenous access (HR: 0.71, P=0.01). DiscussionED patients with SCD experienced longer delays in the administration of the initial analgesic compared with RC patients, despite higher arrival pain scores and triage acuity levels.

Analysis of the Piv Recombinase-Related Gene Family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.

Postexercise Peripheral Oxygen Saturation After Completion of the 6-Minute Walk Test Predicts Successfully Reaching the Summit of Aconcagua

OBJECTIVE The 6-minute walk test (6MWT) is a single measurement of functional status in patients with cardiovascular disease. It has not been studied at high altitude. We investigate the screening value of 6-minute walk distance (6MWD) and postexercise vital sign (VS) measurements as predictors of successfully reaching the summit or development of acute mountain sickness (AMS) on Aconcagua (6962 m). METHODS Prospective observational cohort in Aconcagua Provincial Park, Argentina. Adults climbing the normal route who registered with base camp physicians were included. There were no exclusion criteria. VSs were measured before (resting) and after (postexercise) completion of 6MWT while volunteers acclimatized at Plaza de Mulas base camp (4365 m). Volunteers proceeded towards the summit at their own pace and upon descent returned a questionnaire with maximum altitude reached and Lake Louise AMS Self-report Score (LLSelf). RESULTS One hundred twenty-four volunteers completed the 6MWT. Sixty-four volunteers (51.6%) completed questionnaires; 56% summited. Median LLSelf was 4 (IQR: 3.0-6.5). There was no association between any resting or postexercise VS measurements and AMS. However, mean postexercise SpO(2) was 80.8% in summiters and 76.4% in nonsummiters, a difference of -4.4% (95% CI: -6.7 to -2.0, p = 0.0005). Postexercise SpO(2) < 75% had 97.2% sensitivity and negative likelihood ratio of 0.086 in predicting the outcome of successfully reaching the summit: only one climber with SpO(2) < 75% successfully reached the summit. CONCLUSIONS This study provides the first published data on 6MWD recorded in the field at high altitude. Postexercise SpO(2) < 75% may be a useful screening test for predicting the outcome of successfully reaching the summit of Aconcagua.

Visual Analog Scale (VAS) for Assessment of Acute Mountain Sickness (AMS) on Aconcagua

OBJECTIVE The Lake Louise AMS Self-Report Score (LLSelf) is a commonly used, validated assessment of acute mountain sickness (AMS). We compared LLSelf and visual analog scales (VAS) to quantify AMS on Aconcagua (6962 m). METHODS Prospective observational cohort study at Plaza de Mulas base camp (4365 m), Aconcagua Provincial Park, Argentina. Volunteers climbing in January 2009 were enrolled at base camp and ascended at their own pace. They completed the LLSelf, an overall VAS [VAS(o)], and 5 individual VAS [VAS(i)] corresponding to the items of the LLSelf when symptoms were maximal. Composite VAS [VAS(c)] was calculated as the sum of the 5 VAS(i). RESULTS A total of 127 volunteers consented to the study. Response rate was 52.0%. AMS occurred in 77.3% of volunteers, while 48.5% developed severe AMS. Median (interquartile range, IQR) LLSelf was 4 (3-7). Median (IQR) VAS(o) was 36 mm (23-59). VAS(o) was linear and correlated with LLSelf: slope = 6.7 (95% CI: 4.4-9.0), intercept = 3.0 (95% CI: -10.0-16.1), ρ = 0.71, τ = 0.55, R(2) = 0.45, p < 0.001. Median (IQR) VAS(c) was 29 (13-44). VAS(c) was also linear and correlated with LLSelf: slope = 5.9 (95% CI: 4.9-6.9), intercept = -0.6 (95% CI: -6.3-5.1), ρ = 0.83, τ = 0.68, R(2) = 0.73, p < 0.001. The relationship between the 5 VAS(i) and LLSelf(i) was less significant and less linear than that between VAS(o), VAS(c), and LLSelf. CONCLUSIONS While both VAS(o) and VAS(c) for assessment of AMS appear to be linear with respect to LLSelf, the amount of scatter within the VAS is considerable. The LLSelf remains the gold standard for the diagnosis of AMS.

Mountaineering Fatalities on Aconcagua: 2001–2012

High altitude mountaineering is a dangerous endeavor due to the hypoxic hypobaric environment, extreme weather, and technical skills required. One of the seven summits, Aconcagua (6962 m) is the highest mountain outside of Asia. Its most popular route is nontechnical, attracting >3000 mountaineers annually. Utilizing data from the Servicio Médico Aconcagua (park medical service), we performed a retrospective descriptive analysis with the primary objective of deriving a fatality rate on Aconcagua from 2001 to 2012. The fatality rate on Aconcagua was then compared to other popular mountains. For climbers who died, we report all available demographic data, mechanisms of death, and circumstances surrounding the death. Between 2001 and 2012, 42,731 mountaineers attempted to summit Aconcagua. There were 33 fatalities. The fatality rate was 0.77 per 1000, or 0.077%. The fatality rate on Aconcagua is lower than that on Everest or Denali but higher than that on Rainier.

Prior Altitude Experience of Climbers Attempting to Summit Aconcagua

Aconcagua (6962 m) is one of the seven summits and the highest mountain outside of Asia. Climbers of varying experience are drawn to its nontechnical route. Our objective was to detail the prior altitude experience of climbers attempting to summit Aconcagua. We asked all climbers on the normal route of Aconcagua to complete questionnaires with demographics and prior high altitude experience while acclimatizing at Plaza de Mulas base camp during 9 nonconsecutive days in January 2009. 127 volunteers from 22 countries were enrolled. Mean age was 39.8 years and 88.2% were male. Median altitude at place of residence was 200 m (IQR: 30, 700). Median previous maximum altitude reached was 5895 m (IQR: 5365, 6150). 7.1% of climbers had never been above 4000 m. Median previous maximum sleeping altitude was 4800 m (IQR: 4300, 5486). 12.6% of climbers had never slept above 4000 m. Climbers who performed acclimatization treks spent a mean of 3.6 (2.5, 4.7) days at>3000 m in the previous 2 months. However, 50.4% of climbers performed no acclimatization treks. Although the majority of mountaineers who attempt Aconcagua have prior high altitude experience, a substantial minority has never been above 4000 m.