Protocol, rationale and design of PEOPLE (Post ExpOsure Prophylaxis for LEprosy in the Comoros and Madagascar): A cluster randomized trial on effectiveness of different modalities of implementation of post-exposure prophylaxis of leprosy contacts

Background: Leprosy is an ancient infectious disease with a global annual incidence that has plateaued above 200,000 new cases since over a decade. New strategies are required to overcome this stalemate. Post-exposure prophylaxis (PEP) with a single dose of Rifampicin (SDR) has conditionally been recommended by the World Health Organization (WHO), based on a randomized-controlled-Trial in Bangladesh. More evidence is required. The Post ExpOsure Prophylaxis for Leprosy (PEOPLE) trial will assess effectiveness of different modalities of PEP on the Comoros and Madagascar. Methods: PEOPLE is a cluster-randomized trial with villages selected on previous leprosy-incidence and randomly allocated to four arms. Four annual door-To-door surveys will be performed in all arms. All consenting permanent residents will be screened for leprosy. Leprosy patients will be treated according to international guidelines and eligible contacts will be provided with SDR-PEP. Arm-1 is the comparator in which no PEP will be provided. In arms 2, 3 and 4, SDR-PEP will be provided at double the regular dose (20 mg/kg) to eligible contacts aged two years and above. In arm 2 all household-members of incident leprosy patients are eligible. In arm 3 not only household-members but also neighbourhood contacts living within 100-m of an incident case are eligible. In arm 4 such neighbourhood contacts are only eligible if they test positive to anti-PGL-I, a serological marker. Incidence rate ratios calculated between the comparator arm 1 and each of the intervention arms will constitute the primary outcome. Discussion: Different trials on PEP have yielded varying results. The pivotal COLEP trial in Bangladesh showed a 57% reduction in incidence over a two-year period post-intervention without any rebound in the following years. A study in a high-incidence setting in Indonesia showed no effect of PEP provided to close contacts but a major effect of PEP provided as a blanket measure to an entire island population. High background incidence could be the reason of the lack of effect of PEP provided to individual contacts. The PEOPLE trial will assess effectiveness of PEP in a high incidence setting and will compare three different approaches, to identify who benefits most from PEP. Trial registration: Clinicaltrials.Gov. NCT03662022. Initial Protocol Version 1.2, 27-Aug-2018

Probing the urea dependence of residual structure in denatured human α-lactalbumin

Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. 15N R2 values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 310 helices in native α-LA show R2 values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant

Local Cooperativity in an Amyloidogenic State of Human Lysozyme Observed at Atomic Resolution

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein

Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein

We present a new methodology for site-specific sensing of peptide–oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide–ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide–ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide–ODN interactions

Architectures and biogenesis of non-flagellar protein appendages in Gram-negative bacteria

Bacteria commonly expose non-flagellar proteinaceous appendages on their outer surfaces. These extracellular structures, called pili or fimbriae, are employed in attachment and invasion, biofilm formation, cell motility or protein and DNA transport across membranes. Over the past 15 years, the power of molecular and structural techniques has revolutionalized our understanding of the biogenesis, structure, function and mode of action of these bacterial organelles. Here, we review the five known classes of Gram-negative non-flagellar appendages from a biosynthetic and structural point of view

Structural characterisation of the human alpha-lactalbumin molten globule at high temperature.

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments

Probing the effect of temperature on the backbone dynamics of the human alpha-lactalbumin molten globule.

Molten globules are compact, partially folded proteins postulated to be general intermediates in protein folding. Human alpha-lactalbumin (alpha-LA) is a two-domain Ca(2+)-binding protein that partially unfolds at low pH to form a molten globule. NMR spectra of molten globules are characterized by broadened resonances due to conformational fluctuations on microsecond to millisecond time scales. These species are often studied at high temperature where NMR resonances are observed to sharpen. The effect of higher temperatures on fast time-scale backbone dynamics of molten globules has not been investigated previously. Here, 1D (15)N direct-detection and 2D indirect-detection (1)H-(15)N heteronuclear NOE experiments have been used to probe fast time-scale dynamics at low and high temperatures for three disulfide-bond variants of human alpha-LA that form molten globules. Disulfide bonds are found to have a significant effect on backbone dynamics within the beta-domain of the molten globule; within the alpha-domain, dynamics are not significantly influenced by these bonds. At 20 degrees C, backbone mobility is significantly decreased in both domains of the molten globule compared to the mobility at 40-50 degrees C. Heteronuclear NOE values determined at 20 degrees C for the alpha-domain are closely similar to those observed for native alpha-LA, indicating that the alpha-LA molten globule has even more native-like character than suggested by studies conducted at higher temperature. Our results highlight the importance of considering the temperature dependence of the molten globule ensemble when making comparisons between experimental data obtained under different conditions

Target Specificity of Human Immunodeficiency Virus Type 1 NCp7 Requires an Intact Conformation of Its CCHC N-Terminal Zinc Finger

The modification of zinc-binding residues inside the conserved CCHC motif of human immunodeficiency virus type 1 NCp7, in particular into CCHH, induces a complete loss of infectivity. Since the mutant His28NCp7 has been shown to be devoid of infectivity in vivo, the structure-function relationships of the mutant His28(12-53)NCp7 were investigated by nuclear magnetic resonance and surface plasmonic resonance. Although the Cys28→His mutation modifies drastically the structure of the core domain (residues 12 to 53) of NCp7, His28(12-53)NCp7 still interacts with a 10-fold-lower affinity to specific nucleic acid targets, such as SL3, a stem-loop critically involved in viral RNA packaging, and without affinity change with the nonspecific, single-stranded nucleic acid poly(T). Moreover, His28(12-53)NCp7 and native (12-53)NCp7 displayed the same affinity with reverse transcriptase, but the natures of the complexes are probably different, accounting for the drastic reduction in the amount of RNA packaged in the mutated virus. We propose a structural model of His28(12-53)NCp7 that provides insights into the NCp7 structural features necessary for target recognition and that shows that the specific native structure of the zinc finger domain is strictly required for the optimal target selectivity of NCp7