Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate

Validation of the FAMACHA© eye colour chart using sensitivity/specificity analysis on two South African sheep farms

A validation study of the FAMACHA© system for clinical evaluation of anaemia due to Haemonchus contortus was conducted on two commercial sheep farms in the summer rainfall region of South Africa. In this region, the Haemonchus season lasts from October to April. On Farm 1 the system was tested over a period of five successive years in consecutive sets of young stud Merino replacement rams and ewes examined at intervals of 3–5 weeks over each Haemonchus season, under routine farming conditions. When FAMACHA© scores of 3, 4, and 5 and haematocrit values of ≤22%, ≤19%, and ≤15% were separately considered to be anaemic, sensitivity on Farm 1 ranged from a maximum of 83% for a haematocrit cut-off of ≤15%, to 40% for a haematocrit cut-off of ≤22%. Sensitivity increased to 93% when FAMACHA© scores of 2, 3, 4, and 5 were considered anaemic at a cut-off value of ≤19%, but the positive predictive value decreased to 0.43, indicating that many non-anaemic animals would be treated. The analysis indicated a high level of classification bias on Farm 1, with the animals consistently being classified one FAMACHA© category lower (i.e. less anaemic) than reality.\ud \ud On Farm 2 the test was conducted over two successive years in yearling rams evaluated at weekly to fortnightly intervals during each worm season. Every ram judged to be in FAMACHA© category 4 or 5 was bled for haematocrit determination, and it was only dewormed with effective anthelmintics if the haematocrit was 15% or lower. When FAMACHA© scores of 3, 4, and 5 and haematocrit values of ≤22% and ≤19% were separately considered to be anaemic on Farm 2, sensitivity ranged from 64% for a haematocrit cut-off of ≤22%, to 80% for a cut-off of ≤19%.\ud \ud For identical haematocrit cut-off values and proportions of the sampled flock considered to be diseased as for Farm 1, sensitivity was always higher for Farm 2. On the other hand, further analysis of the data indicated that the magnitude of the error on Farm 1 was very consistent on average over the entire trial period.\ud \ud The results of this study indicate that (i) persons introduced to the system should not only be trained, but also be evaluated for accuracy of application; (ii) the sensitivity of the FAMACHA© diagnostic system should ideally be evaluated at shorter intervals to avoid production losses due to failure to detect anaemic animals which may be at risk of death; (iii) that calibration of the FAMACHA© scoring is essential per individual evaluator, and (iv) that animals should be examined at weekly intervals during periods of the highest worm challenge

Supplementary Material for: Familiar Papillary Thyroid Carcinoma in a Large Brazilian Family Is Not Associated with Succinate Dehydrogenase Defects

<b><i>Background:</i></b> Thyroid cancer is the most common endocrine gland malignancy. Advances in understanding the genetic basis for thyroid cancer revealed the potential involvement of several genes in the formation of thyroid tumors. Mutations in the gene coding for succinate dehydrogenase subtype B <i>(SDHB)</i> have been implicated in papillary thyroid cancer (PTC). Succinate dehydrogenase (SDH) is a heterotetrameric protein composed of four subunits, <i>SDHA,</i><i>SDHB,</i><i>SDHC,</i> and <i>SDHD,</i> and participates in both the electron transport chain and the tricarboxylic acid cycle. The aim of the study was to evaluate the association between variants in the <i>SDHA,</i><i>SDHB,</i><i>SDHC,</i> and <i>SDHD </i>genes and familiar PTC in a large Brazilian family. <b><i>Method:</i></b> Four patients with PTC, 1 patient with PTC and gastrointestinal stromal tumor (GIST), 1 patient with GIST, and their relatives - several of them with different thyroid problems - from a large Brazilian family were screened for genetic variations of <i>SDHx</i> genes with the use of polymerase chain reaction-single-stranded conformational polymorphism and direct sequencing. <b><i>Results:</i></b> Only one rare variation in <i>SDHA</i> was found in some of the family members, but not segregating with the disease. No other genetic variants of these genes were detected in the family members that presented with PTC and/or GIST. <b><i>Conclusion:</i></b> Familiar PTC and a GIST were not associated with <i>SDHx </i>mutations; additional genetic defects, yet unknown, may be responsible for the development of tumor