Perceived stigma related to TB preventive therapy.

BACKGROUND: TB preventative therapy (TPT) is crucial for reducing the burden of TB in endemic settings. We assessed stigma associated with TPT and the social groups from whom stigma was anticipated.METHODS: We conducted an anonymous cross-sectional survey of community-dwelling adults in rural South Africa. Descriptive statistics, exploratory factor analysis, χ² tests, Kruskal-Wallis tests, and Poisson regression were used to identify factors associated with TPT stigma.RESULTS: The mean age of the 104 participants was 35 years, 65% were female, and 26% had completed secondary school. The vast majority perceived stigma associated with TPT (71%; mean score 1.7, SD ± 1.4). Factor analysis identified a two-factor solution that explained 61.9% of the variance. Being single (P < 0.001), previously screened for TB (P = 0.04), worried about being infected by TB (P = 0.006), and interested in taking TPT (P = 0.01) were associated with higher perceived stigma scores. TPT stigma was perceived among 8%, 16%, and 66% of their family, friends, and other community members, respectively.CONCLUSION: The prevalence of TPT-related stigma in a rural South African community was high. Community members anticipated less stigma from family members compared to other social groups. Global expansion and implementation of TPT will require novel interventions, such as engaging patients´ families to support uptake and promote adherence.

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal.

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.

Conformational variability of the Cu site in one subunit of bovine CuZn superoxide dismutase: the importance of mobility in the Glu119-Leu142 loop region for catalytic function.

The structure of the catalytic site in one subunit of bovine CuZn superoxide dismutase is shown to be highly variable. A series of crystal structures at approximately 1.7 A have been determined using data collected from different crystals. Several conformations are observed for the copper site from one of the subunits. These conformations lie between those expected for the Cu(II) and Cu(I) forms of the enzyme and may represent a slow positional rearrangement of the Cu site during the crystallisation process due to the presence of a trace reductant in the mother liquor. These states perhaps indicate some functionally relevant structural steps that ultimately result in the breakage of the imidazolate bridge between the two metal sites. This behaviour is not observed for the second subunit of the dimeric enzyme, which remains in the five-coordinate, distorted square planar geometry in all cases. We suggest that this asymmetric behaviour may be caused by the lack of mobility for the Glu119-Leu142 loop region in the second subunit caused by crystal contacts. This region forms one wall of the active-site cavity, and its mobility has been suggested, via molecular dynamics studies, to be important for the catalytic mechanism.

Implementation of cluster analysis for ab initio phasing using the molecular envelope from solution X-ray scattering.

Solution of the phase problem is central to crystallographic structure determination. The conventional methods of isomorphous replacement (MIR or SIR) and molecular replacement are ineffective in the absence of a suitable isomorphous heavy-atom derivative or knowledge of the structure of a homologous protein. A recent method utilizing the low-resolution molecular shape determined from solution X-ray scattering data has shown to be successful in locating the molecular shape within the crystallographic unit cell in the case of the trimer nitrite reductase (NiR, 105 kDa) [Hao et al. (1999), Acta Cryst. D55, 243-246]. This was achieved by performing a direct real-space search for orientation and translation using knowledge of the orientation of the polar angles of the non-crystallographic axis obtained by performing a self-rotation on crystallographic data. This effectively reduces the potential six-dimensional search to a four-dimensional one (Eulerian angle gamma and three translational parameters). In the case of NiR, the direct four-dimensional search produced a clear solution that was in good agreement with the known structure. The program FSEARCH incorporating this method has been generalized to handle molecules from all space groups and in particular those in possession of non-crystallographic symmetry. However, the method employed was initially unsuccessful when applied to the small dimeric molecule superoxide dismutase (SOD, 32 kDa) owing to the absence of strong reflections at low resolution caused by saturation at the detector. The determined solution deviated greatly from that of the known structure [Hough & Hasnain (1999), J. Mol. Biol. 287, 579-592]. It was found that once these absent reflections were replaced by a series of randomly generated intensity values and cluster analysis was performed on the output, the signal-to-noise ratio was improved and a most probable solution was found. The electron-density map of the stochastically determined solution agrees well with the known structure; the phase error calculated from this map was 67 degrees within 14 A resolution.