Longitudinal intravital imaging of the bone marrow for analysis of the race for the surface in a murine osteomyelitis model.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/h on the implant from 2 to 12 h post-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Longitudinal Patterns of Alcohol Use Following Traumatic Brain Injury in an Active Duty and Young Veteran Military Sample: A VA TBI Model Systems Study.

OBJECTIVE: To describe alcohol use among younger military active duty service members and veterans (SMVs) in the first 5 years after traumatic brain injury (TBI) and examine whether differential alcohol use patterns emerge as a function of brain injury severity and active duty service at time of injury. SETTING: Veterans Affairs (VA) Polytrauma Rehabilitation Centers (PRCs). PARTICIPANTS: In total, 265 SMVs enrolled in the VA Traumatic Brain Injury Model Systems (TBIMS) PRC national database. Participants sustained a TBI of any severity level; received inpatient care at a PRC within 1 year of injury; were younger than 40 years; and completed survey interviews or questionnaires regarding their pre- and postinjury alcohol use for at least 3 of 4 time points (preinjury, postinjury years 1, 2, and 5). MAIN MEASURES: Self-reported alcohol use, defined as amount of weekly consumption and endorsement of binge drinking. Participant information related to demographics, injury, TBI severity, active duty status, mental health treatment, and FIM (Functional Independence Measure) total scores was also obtained to examine impact of these as covariates in the analyses. RESULTS: Alcohol use generally increased following an initial period of reduced consumption for SVMs with moderate-to-severe TBI. Individuals with mild TBI showed an opposite trend, with an initial period of increased use, followed by a decline and return to baseline levels in the long term. However, alcohol use did not significantly differ over time within this subsample after adjusting for covariates. CONCLUSIONS: The current study identified longitudinal alcohol use among a young, military/veteran cohort with a history of TBI, an at-risk population for problematic alcohol use. Patterns of self-reported alcohol consumption suggest the time frame of 2 to 5 years postinjury may be a critical window of opportunity for further intervention to maintain lowered levels of alcohol use, particularly among SVMs with moderate-to-severe TBI.

Phosphorylation Regulates CAP1 (Cyclase-Associated Protein 1) Functions in the Motility and Invasion of Pancreatic Cancer Cells.

Pancreatic cancer has the worst prognosis among major malignancies, largely due to its highly invasive property and difficulty in early detection. Mechanistic insights into cancerous transformation and especially metastatic progression are imperative for developing novel treatment strategies. The actin-regulating protein CAP1 is implicated in human cancers, while the role still remains elusive. In this study, we investigated roles for CAP1 and its phosphor-regulation in pancreatic cancer cells. No evidence supports remarkable up-regulation of CAP1 in the panel of cancer cell lines examined. However, knockdown of CAP1 in cancer cells led to enhanced stress fibers, reduced cell motility and invasion into Matrigel. Phosphorylation of CAP1 at the S308/S310 tandem regulatory site was elevated in cancer cells, consistent with hyper-activated GSK3 reported in pancreatic cancer. Inhibition of GSK3, a kinase for S310, reduced cell motility and invasion. Moreover, phosphor mutants had defects in alleviating actin stress fibers and rescuing the reduced invasiveness in the CAP1-knockdown PANC-1 cells. These results suggest a required role for transient phosphorylation for CAP1 function in controlling cancer cell invasiveness. Depletion of CAP1 also reduced FAK activity and cell adhesion, but did not cause significant alterations in ERK or cell proliferation. CAP1 likely regulates cancer cell invasiveness through effects on both actin filament turnover and cell adhesion. Finally, the growth factor PDGF induced CAP1 dephosphorylation, suggesting CAP1 may mediate extracellular signals to control cancer cell invasiveness. These findings may ultimately help develop strategies targeting CAP1 or its regulatory signals for controlling the invasive cycle of the disease.

Attitudes and practices of hookah smokers in the San Francisco Bay Area.

As many as 10 million people will die annually by the year 2030 due to tobacco-related causes. While much research has focused on cigarettes, the increasing popularity of smoking hookah (water pipe) has received much less attention. Epidemiological studies have been carried out in India, Pakistan, Syria, Kuwait, and Lebanon, but there are few in the United States. Hookah smoking is typically a social activity and there are many myths and rumors about the relative safety of smoking hookah compared to cigarettes. The aim of this study was to identify the knowledge, attitudes, and practices of hookah smokers in the San Francisco Bay Area. We sampled 50 participants (25 male and 25 female) who were mostly college students at the University of California, Berkeley. Hookah smoking was occasional among those sampled, with only six participants (12%) reporting weekly hookah smoking. The majority of respondents considered hookah smoking to be harmful to their health (88%), yet 52% had no intention of quitting. More definitive studies conveying the possible harm of hookah smoking are necessary to serve as a basis for health education programs and policy changes towards this potentially harmful activity.

Carcinogen exposure during short-term switching from regular to "light" cigarettes.

OBJECTIVES: "Light" cigarettes are extremely popular and are perceived by many smokers as less hazardous than higher-yield cigarettes. The objectives of this study were (a) to assess a battery of biomarkers of tobacco smoke exposure that includes tobacco smoke carcinogens, (b) to examine the behavioral nature of compensation, and (c) to examine the consistency of an individual's tobacco smoke exposure when smoking the same cigarette at different times. METHODS: The study was a 3-week crossover study in which smokers smoked their usual cigarettes during weeks 1 and 3, and a light cigarette, with a machine-determined nicotine yield of about 50% of the usual cigarette, during week 2. Blood and urine biomarkers of exposure and subjective questionnaires were collected weekly. RESULTS: Based on cotinine and carboxyhemoglobin levels, compensation averaged 78% and 83%, respectively. Urinary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-butanol, a metabolite of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-butanone, and a number of polycyclic aromatic hydrocarbon metabolites was similar in all conditions. Compensation was accomplished both by smoking cigarettes more intensively and by smoking more cigarettes per day. Exposures to various tobacco smoke constituents while smoking the usual brand of cigarette in weeks 1 and 3 were highly correlated. CONCLUSION: Our findings support the idea that smokers compensate to a high degree when switched from their usual brand to a light cigarette. Short-term switching resulted in no significant reduction in carcinogen exposure. Our assessment, based on measures of biochemical exposures, supports the idea that switching to light cigarettes is unlikely to reduce the health risks of cigarette smoking.

Nicotine metabolite ratio as an index of cytochrome P450 2A6 metabolic activity.

BACKGROUND: Nicotine and a variety of other drugs and toxins are metabolized by cytochrome P450 (CYP) 2A6. Our objective was to evaluate the use of oral nicotine with measurement of the trans-3'-hydroxycotinine (3HC)/cotinine (COT) metabolite ratio as a noninvasive probe of CYP2A6 activity. METHODS: Sixty-two healthy volunteers received an oral solution of deuterium-labeled nicotine (2 mg) and its metabolite cotinine (10 mg). Plasma nicotine and plasma and saliva cotinine and 3HC concentrations were measured over time. RESULTS: The 3HC/COT ratio derived from deuterium-labeled cotinine, measured in either plasma (2-8 hours after administration) or saliva (at 6 hours), was strongly correlated with the oral clearance of nicotine (r = 0.76-0.83, depending on the time of measurement). The 6-hour 3HC/COT ratio from nicotine derived from tobacco in 14 smokers was highly correlated with the ratio derived from deuterium-labeled nicotine (r = 0.88) and was also highly correlated with the oral clearance of nicotine (r = 0.90). Two subjects homozygous for inactive CYP2A6 alleles produced no 3HC, confirming the specificity of the metabolite ratio. The 3HC/COT ratio was also highly correlated with the clearance and half-life of cotinine, consistent with the fact that cotinine is also primarily metabolized by CYP2A6. CONCLUSIONS: The 3HC/COT ratio derived from nicotine either administered as a probe drug or from tobacco use, measured in either plasma or saliva, is highly correlated with the oral clearance of nicotine. The ratio appears to be a useful noninvasive marker of the rate of nicotine metabolism (which is important in studying nicotine addiction and smoking behavior), as well as a general marker of CYP2A6 activity (which is important in studying drug and toxin metabolism).