Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in C. elegans.

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, C. elegans . The egl-19 locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized egl-19 expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of egl-19 expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

Regional and national review of factors associated with burn wound cellulitis.

Burn wound cellulitis (BWC) is the second leading complication reported in burns. In this study we sought to identify demographic variables, burn factors, and other factors that may predispose patients to BWC. Regional data was obtained through retrospective medical record review of burn patients treated between May 2009 to April 2013 for BWC within 8 days of the injury. The patients were matched 1:2 with contemporaneously treated patients. Similarly, the National Burn Repository was queried to identify burn patients with BWC between the years 2002 to 2011, which were then compared to the remaining entries who did not have BWC reported. The data sets were analyzed separately. Univariate and multiple variable analyses were performed to evaluate risk factors for BWC. The risk factors that were consistent regionally and nationally were older age, male sex, African-American race (protective), lower extremity burns, scald burns, and full thickness burns. The treatment delay was only collected regionally, and was associated with an eight times increased risk. The factors that were inconsistent or significant in one sample only were smoking status, psychiatric conditions, upper extremity burns, and the place of injury. Cellulitis remains a significant problem for the burn community. Future prospective analyses need to clarify the impact of these factors as well as other factors on the development of BWC. Preventing BWC from occurring through earlier intervention or targeted prophylactic antibiotics may help reduce morbidity and decrease associated healthcare costs.

Delay in diphtheria, pertussis, tetanus vaccination is associated with a reduced risk of childhood asthma.

BACKGROUND: Early childhood immunizations have been viewed as promoters of asthma development by stimulating a T(H)2-type immune response or decreasing microbial pressure, which shifts the balance between T(H)1 and T(H)2 immunity. OBJECTIVE: Differing time schedules for childhood immunizations may explain the discrepant findings of an association with asthma reported in observational studies. This research was undertaken to determine whether timing of diphtheria, pertussis, tetanus (DPT) immunization has an effect on the development of childhood asthma by age 7 years. METHODS: This was a retrospective longitudinal study of a cohort of children born in Manitoba in 1995. The complete immunization and health care records of cohort children from birth until age 7 years were available for analysis. The adjusted odds ratio for asthma at age 7 years according to timing of DPT immunization was computed from multivariable logistic regression. RESULTS: Among 11, 531 children who received at least 4 doses of DPT, the risk of asthma was reduced to (1/2) in children whose first dose of DPT was delayed by more than 2 months. The likelihood of asthma in children with delays in all 3 doses was 0.39 (95% CI, 0.18-0.86). CONCLUSION: We found a negative association between delay in administration of the first dose of whole-cell DPT immunization in childhood and the development of asthma; the association was greater with delays in all of the first 3 doses. The mechanism for this phenomenon requires further research.