13 research outputs found
An insertional trap for conditional gene expression in Toxoplasma gondii: Identification of TAF250 as an essential gene
Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma
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Sentinel lymph node biopsy at the time of mastectomy does not increase the risk of lymphedema: implications for prophylactic surgery
Women diagnosed with or at high risk for breast cancer increasingly choose prophylactic mastectomy. It is unknown if adding sentinel lymph node biopsy (SLNB) to prophylactic mastectomy increases the risk of lymphedema. We sought to determine the risk of lymphedema after mastectomy with and without nodal evaluation. 117 patients who underwent bilateral mastectomy were prospectively screened for lymphedema. Perometer arm measurements were used to calculate weight-adjusted arm volume change at each follow-up. Of 234 mastectomies performed, 15.8 % (37/234) had no axillary surgery, 63.7 % (149/234) had SLNB, and 20.5 % (48/234) had axillary lymph node dissection (ALND). 88.0 % (103/117) of patients completed the LEFT-BC questionnaire evaluating symptoms associated with lymphedema. Multivariate analysis was used to assess clinical characteristics associated with increased weight-adjusted arm volume and patient-reported lymphedema symptoms. SLNB at the time of mastectomy did not result in an increased mean weight-adjusted arm volume compared to mastectomy without axillary surgery (p = 0.76). Mastectomy with ALND was associated with a significantly greater mean weight-adjusted arm volume change compared to mastectomy with SLNB (p < 0.0001) and without axillary surgery (p = 0.0028). Patients who underwent mastectomy with ALND more commonly reported symptoms associated with lymphedema compared to those with SLNB or no axillary surgery (p < 0.0001). Patients who underwent mastectomy with SLNB or no axillary surgery reported similar lymphedema symptoms. Addition of SLNB to mastectomy is not associated with a significant increase in measured or self-reported lymphedema rates. Therefore, SLNB may be performed at the time of prophylactic mastectomy without an increased risk of lymphedema