Design of a sequence-specific DNA bisintercalator

Programmable bisintercalators: Symmetric synthetic DNA bisintercalators (see picture) based on the H-pin polyamide motif afford high affinity and programmable sequence specificity

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation

Allosteric inhibition of protein--DNA complexes by polyamide--intercalator conjugates

The sequence-specific inhibition of essential protein-DNA contacts in the promoter of a gene is a central issue for the regulation of gene expression by chemical methods. Hairpin polyamides have been shown to inhibit protein-DNA complexes in some but not all cases. For example, polyamides co-occupy the same DNA sequence in the minor groove in the presence of major-groove binding bZip proteins. Four hairpin polyamide-acridine conjugates were synthesized and shown to bind the minor groove of DNA with high affinity in a sequence-specific manner. The polyamide-acridine conjugates were shown to unwind DNA (phi = 14-15 degrees), evidence for intercalation by the acridine moiety. Importantly, the polyamide-intercalator conjugates, which combine sequence-specific groove binding with proximal local unwinding, inhibit major-groove DNA binding by the GCN4 bZip protein. This class of DNA binding molecules creates a sequence-specific allosteric change in DNA structure and has the potential to be a general inhibitor of transcription factor binding independent of the specific protein-DNA structure

Allosteric inhibition of protein--DNA complexes by polyamide--intercalator conjugates

The sequence-specific inhibition of essential protein-DNA contacts in the promoter of a gene is a central issue for the regulation of gene expression by chemical methods. Hairpin polyamides have been shown to inhibit protein-DNA complexes in some but not all cases. For example, polyamides co-occupy the same DNA sequence in the minor groove in the presence of major-groove binding bZip proteins. Four hairpin polyamide-acridine conjugates were synthesized and shown to bind the minor groove of DNA with high affinity in a sequence-specific manner. The polyamide-acridine conjugates were shown to unwind DNA (phi = 14-15 degrees), evidence for intercalation by the acridine moiety. Importantly, the polyamide-intercalator conjugates, which combine sequence-specific groove binding with proximal local unwinding, inhibit major-groove DNA binding by the GCN4 bZip protein. This class of DNA binding molecules creates a sequence-specific allosteric change in DNA structure and has the potential to be a general inhibitor of transcription factor binding independent of the specific protein-DNA structure

Selective Vulnerability of the Cochlear Basal Turn to Acrylonitrile and Noise

Exposure to acrylonitrile, a high-production industrial chemical, can promote noise-induced hearing loss (NIHL) in the rat even though this agent does not itself produce permanent hearing loss. The mechanism by which acrylonitrile promotes NIHL includes oxidative stress as antioxidant drugs can partially protect the cochlea from acrylonitrile + noise. Acrylonitrile depletes glutathione levels while noise can increase the formation of reactive oxygen species. It was previously noted that the high-frequency or basal turn of the cochlea was particularly vulnerable to the combined effects of acrylonitrile and noise when the octave band noise (OBN) was centered at 8 kHz. Normally, such a noise would be expected to yield damage at a more apical region of the cochlea. The present study was designed to determine whether the basal cochlea is selectively sensitive to acrylonitrile or whether, by adjusting the frequency of the noise band, it would be possible to control the region of the auditory impairment. Rats were exposed to one of three different OBNs centered at different frequencies (4 kHz, 110 dB and 8 or 16 kHz at 97 dB) for 5 days, with and without administration of acrylonitrile (50 mg/kg/day). The noise was set to cause limited NIHL by itself. Auditory function was monitored by recording distortion products, by compound action potentials, and by performing cochlear histology. While the ACN-only and noise-only exposures induced no or little permanent auditory loss, the three exposures to acrylonitrile + noise produced similar auditory and cochlear impairments above 16 kHz, despite the fact that the noise exposures covered 2 octaves. These observations show that the basal cochlea is much more sensitive to acrylonitrile + noise than the apical partition. They provide an initial basis for distinguishing the pattern of cochlear injury that results from noise exposure from that which occurs due to the combined effects of noise and a chemical contaminant

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation

WaSH CQI: Applying continuous quality improvement methods to water service delivery in four districts of rural northern Ghana

Continuous, safely managed water is critical to health and development, but rural service delivery faces complex challenges in low- and middle-income countries (LMICs). We report the first application of continuous quality improvement (CQI) methods to improve the microbial quality of household water for consumption (HWC) and the functionality of water sources in four rural districts of northern Ghana. We further report on the impacts of interventions developed through these methods. A local CQI team was formed and trained in CQI methods. Baseline data were collected and analyzed to identify determinants of service delivery problems and microbial safety. The CQI team randomized communities, developed an improvement package, iteratively piloted it in intervention communities, and used uptake survey data to refine the package. The final improvement package comprised safe water storage containers, refresher training for community WaSH committees and replacement of missing maintenance tools. This package significantly reduced contamination of HWC (p<0.01), and significant reduction in contamination persisted two years after implementation. Repair times in both intervention and control arms decreased relative to baseline (p<0.05), but differences between intervention and control arms were not significant at endline. Further work is needed to build on the gains in household water quality observed in this work, sustain and scale these improvements, and explore applications of CQI to other aspects of water supply and sanitation

An evaluation of the SWATRER and, CERES-Millet models for southwest Niger

The SWATKER (Dierckx el aL, 1986) soil water and CERES-Millet (Godwin el ol.. 1984; Jones el al. 1984) growth models wcre evaluated for millet (PennisNwn arnuicmun) during the 1989 growing season at Tara, Niger (300 km, south of Niamey). The required minimum data sets for the plant and soil components of each modcl were either f~ld determined or obtained from the literature. A field experiment was carried out in order to validate the soil water balance and plant growth subroutine6 Soil water content. leaf area and dry matter (DM) were measured weekly. Good agreement between simulated and measured soil water content was obtained with SWATRER. The CERES-Millet modcl overestimated leaf area index (LAI). DM and yield by 46%, 40% and 101% respectively. The CERES-Millet modcl consistently overestimated soil water content by 5% throughout the growing season. The SWATRER model showed good promise for evaluating waterilertilizcr management strategies. Once the CERES-Millet modcl is calibrated. it can be utilized to evaluate new production zonq new cultivara and numerous cultural practices

Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation