Prevalence of pretreatment drug resistance in persons initiating and reinitiating antiretroviral therapy in Sri Lanka: results from a national representative survey.

BACKGROUND: We conducted a nationwide cross-sectional study to estimate pretreatment drug resistance (PDR) prevalence in adults initiating ART in Sri Lanka following the WHO's recommendations. METHODS: HIV drug resistance was determined on dried blood spots (DBSs) using population-based sequencing of the protease and reverse transcriptase genes and interpretation was based on Stanford HIVdb v9.0. Analyses were weighted to adjust for multistage sampling and genotypic failure rate. We used logistic regression to assess differences between groups. RESULTS: Overall, in 10% (15 of 150) of patients initiating ART, HIV drug resistance mutations were detected. The prevalence of resistance to NNRTI drugs efavirenz/nevirapine was 8.4% (95% CI 4.6-15.0) but differed among those reporting having prior antiretroviral (ARV) exposure (24.4%, 95% CI 13.8-39.5) compared with 4.6% (95% CI 1.6-12.8) for those reporting as being ARV naive (OR 4.6, 95% CI 1.3-16.6, P = 0.021). PDR to efavirenz/nevirapine was also nearly twice as high among women (14.1%, 95% CI 6.1-29.4) compared with men (7.0%, 95% CI 3.1-14.7) (P = 0.340) and three times high among heterosexuals (10.4%, 95% CI 2.4-35.4) compared with MSM (3.8%, 95% CI 1.1-12.7) (P = 0.028). NRTI PDR prevalence was 3.8% (95% CI 1.1-12.1) and no PI PDR was observed in the study. CONCLUSIONS: A high prevalence of efavirenz/nevirapine PDR was reported, especially in patients with prior ARV exposure, in women and those reporting being heterosexual. These findings highlight the need to fast-track the transition to the WHO-recommended dolutegravir-based first-line ART.

Signal amplification through nucleotide extension and excision on a dendritic DNA platform.

Techniques that provide strong signal amplification are useful in diagnostic applications, especially in detecting low concentrations of non-amplifiable target molecules. A versatile and strong signal amplification method based on activities of a DNA polymerase to generate high concentrations of pyrophosphate (PPi) is described. The generation of PPi is catalyzed by nucleotide extension and excision activities of a DNA polymerase on an oligonucleotide cassette. The signal is generated upon enzymatic conversion of PPi to ATP and ATP levels subsequently detected with firefly luciferase. Bioluminescence produced by an oligonucleotide cassette consisting of just two polymerase reaction sites is sufficient to detect them at low attomole levels. The attachment of a large number of these oligonucleotide cassettes to DNA dendrimers enabled the detection of such poly-valent substrate molecules at low zeptomole (10(-21)mol) concentrations. The extent of signal amplification obtained with dendrimer substrates is comparable to exponential target amplifications provided by nucleic acid amplification methods. The attachment of such PPi-generating dendritic DNA platforms to ligands that mediate target recognition would potentially permit detection of extremely low concentrations of analytes in diagnostic assays.

Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR.

A random sequence library of single stranded DNA was screened to isolate sequences with high affinity for Thermus aquaticus DNA polymerase (Taq pol), a thermostable enzyme commonly used in the polymerase chain reaction (PCR). Selected oligonucleotide sequences bound Taq pol with dissociation constants in the low picomolar range, and efficiently inhibited polymerase activity at room temperature (20 to 25 degrees C), but did not inhibit at temperatures above 40 degrees C. Moreover, inhibition was thermally reversible. A process called "hot start" PCR is commonly used to prevent non-specific PCR products in amplification of low copy number targets. We show that the addition of oligonucleotide inhibitors eliminated the need for "hot start" conditions and improved the efficiency of detection of a low copy number target in PCR.

Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer.

Single-stranded DNA aptamers that recognize DNA polymerase from Thermus acquaticus (Taq pol) with high affinity have been described recently. These aptamers have been shown to efficiently inhibit the polymerase activity of Taq pol and are useful in enhancing the amplification efficiency of low copy number targets by the polymerase chain reaction (PCR). Aptamers selected to bind to Taq pol fell into two different sequence families and inhibited several DNA polymerases isolated from the Thermus species, including that from Thermus thermophilus (Tth pol). Aptamers from one sequence family inhibited the Stoffel fragment of Taq pol efficiently, whereas those from the other family did not. Truncated aptamers derived from two parent ligands from both families were combined to form a heterodimeric aptamer that effectively inhibited all three polymerases and were shown to be useful in detecting a low copy number target by PCR amplification. These data demonstrate that the combination of aptamers with different properties into a single molecule broadens their spectrum of utility.

Competitive nucleosome reconstitution of polydeoxynucleotides containing oligoguanosine tracts.

The ability of synthetic polydeoxynucleotides composed of oligoguanosine tracts of increasing length to form nucleosomes has been determined by several reconstitution procedures. When the presence of nucleosomes is determined by resistance to nuclease digestion, a protected band of approximately 150 base-pairs is detected only with difficulty for polymers containing long tracts of contiguous guanosines. However, when assayed by a shift in the electrophoretic mobility of radiolabeled polymers exchanged at 0.7 M-NaCl with authentic nucleosomes, all polymers tested are seen to form nucleosomes. Quantitative competitive reconstitution shows that the length of the tracts per se does not adversely affect their propensity to form nucleosomes, since even 150 base-pair poly(dG).poly(dC) forms nucleosomes as well as heterogeneous-sequence DNA. However, the ability to form nucleosomes does depend on the length of the polymer repeating unit.

Molecular characterisation of a hsp70 gene from the filarial parasite Setaria digitata.

The filarial parasite Setaria digitata is the causative agent of cerebrospinal nematodiasis in its abnormal hosts such as sheep, goats and horses, and therefore is of significant veterinary importance. Since very little is currently known about the biology of this parasite at molecular level, we have cloned and characterised a hsp70 gene, the first gene to be reported from this parasite. The genomic clone isolated contained sequences from two hsp70 genes. One gene, hsp70-2, was completely sequenced and found to contain nine introns ranging in size from 78 to 195 bp. The region upstream of the initiation codon contained a putative TATA box, two CAAT box elements and three heat-shock elements. A putative transcription initiation site was also identified. The 5' untranslated region contained a splice acceptor sequence. The gene was typically AT rich, having a GC content of 44.5%. The deduced aa sequence potentially encoded a cytosolic protein of 645 aa, which had three consecutive repeats of a tetrapeptide motif, GGMP, at the carboxyl end. The gene appeared to be constitutively transcribed and was not significantly enhanced in response to heat shock in adult worms. Another hsp70 gene (hsp70-1) was located further upstream, arranged in direct tandem with hsp70-2. Southern blot analysis revealed the presence of two or three additional hsp70-related genes in the S. digitata genome.

Molecular characterisation of the actin gene of the filarial parasite Wuchereria bancrofti.

Wuchereria bancrofti is the major cause of lymphatic filariasis in humans. Although it is responsible for this immensely morbid and debilitating disease, very little is known of the basic molecular biology of this parasite, and there is a vast lack of knowledge on its gene organisation. In this study, the actin gene of W. bancrofti has been characterised by sequencing a clone isolated from a genomic DNA library of this parasite. The 5' flanking region had a potential TATA box and a putative mRNA initiation site. The gene had five exons encoding 376 amino acids, and four introns ranging in size from 109 to 190bp. The 3' flanking region had a potential polyadenylation signal with the sequence ATTAAA which is a common natural variant of the conventional sequence AATAAA. The gene was AT-rich, with a GC content of 37.2%. Southern blot analysis of W. bancrofti genomic DNA indicated that the gene is possibly found as a single copy. The actin amino acid sequence of W. bancrofti showed a high degree of homology to the actin of many organisms of different taxonomic groups, but the highest homology was observed with the free-living nematode Plectus acuminatus. This suggests that P. acuminatus may bear a close evolutionary relationship to W. bancrofti.

Restriction fragment length polymorphism analysis rules out cross-infection among renal patients with tuberculosis.

A cluster of five cases of tuberculosis occurred on a renal unit in 1993. The initial impression was that this was an outbreak, and cross-infection was suspected. Restriction fragment length polymorphism analysis was carried out on the strains of Mycobacterium tuberculosis isolated from these cases, using a DNA probe directed against the insertion sequence IS6110. DNA fingerprints obtained by this method differed for all the strains tested, ruling out cross-infection as a cause of the outbreak. This technique is a useful adjunct to standard epidemiological investigations in outbreaks of tuberculosis.

The use of aptamers in large arrays for molecular diagnostics.

BACKGROUND: Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM. METHODS AND RESULTS: The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. CONCLUSIONS: Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.

Large-cell calcifying sertoli cell tumour of the testis detected at screening of a family with Carney syndrome.

We report the detection of a large-cell calcifying Sertoli cell tumour (LCCSCT) in a 34-year-old male during screening of a family with Carney syndrome. The patient had ignored the testicular swelling for 7 years. He also had a cardiac myxoma. The LCCSCT in this patient had prognostically unfavourable features such as large size (>6 cm) and a high mitotic rate. There is only one previous report of a malignant LCCSCT in a patient with Carney syndrome.

Aptamers: an emerging class of molecules that rival antibodies in diagnostics.

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Influence of tetraalkyl ammonium ions on the structure of poly (rG-dC).poly (rG-dC): unexpected transitions among the Z, A and B conformations.

The conformation of the double-stranded, mixed ribodeoxyribo polynucleotide, poly (rG-dC).poly (rG-dC), has been examined in the presence of tetraalkyl ammonium ions. Tetramethyl ammonium ion stabilizes the "low salt" Z conformation (1) of the polymer from submillimolar to molar concentrations of the counterion. In the presence of tetraethyl and tetrapropyl ammonium ions the polymer exists in the low salt Z form up to 2 mM concentration of the counterions and then flips to the right hand helical A form. With tetrabutyl ammonium counterions the polymer is in an A conformation at low ion concentrations and converts to a B form at concentrations greater than thirty millimolar. These results are interpreted in terms of electrostatic and solvent interactions of the polynucleotide.

Sequence limitations of triple helix formation by alternate-strand recognition.

Until recently, oligonucleotide-directed triplex formation has been limited to oligopurine tracts of target DNA. Triplex formation by alternate-strand recognition relaxes this limitation by allowing triplexes to form at 5'-(Pu)m(Py)n-3' and 5'-(Py)m(Pu)n-3' sequences, with the third strand pairing first with purines on one strand and then switching to pair with purines on the other strand. In this study, the interaction of several oligonucleotides with the potential to form triplexes by alternate-strand recognition at the sequence 5'-A8C8A8-3' was studied by chemical probing and affinity cleaving. The results show that triplex formation can be readily accomplished at the 5'-A8C8-3' part of the sequence; however, base triplet formation is disrupted on either side of the strand switch and the Watson-Crick helix is distorted in such a way as to expose the N7 positions of purines adjoining the strand switch. Triplex formation is weak or nonexistent at the 3'-most A8 block, despite the opportunity for recruiting a spacer sequence for the second (C8-A8) strand switch by "slippage". This finding indicates that the C8-A8 strand switch is energetically unfavorable, although pairing at other 5'-(Py)n(Pu)n-3' sequences has been observed, with or without a spacer [Beal, P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478; Jayasena, S. D., & Johnston, B. H. (1992) Nucleic Acids Res. 20, 5279-5288]. Thus, alternate-strand recognition may not be feasible for certain sequences of 5'-(Py)m(Pu)n-3', at least under the conditions examined.

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.

Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.

Intramolecular triple-helix formation at (PunPyn).(PunPyn) tracts: recognition of alternate strands via Pu.PuPy and Py.PuPy base triplets.

Triple-helical DNA shows increasing potential for applications in the control of gene expression (including therapeutics) and the development of sequence-specific DNA-cleaving agents. The major limitation in this technology has been the requirement of homopurine sequences for triplex formation. We describe a simple approach that relaxes this requirement, by utilizing both Pu.PuPy and Py.PuPy base triplets to form a continuous DNA triple helix at tandem oligopurine and oligopyrimidine tracts. [Triplex formation at such a sequence has been previously demonstrated only with the use of a special 3'-3' linkage in the third strand [Horne, D. A., & Dervan, P. B. (1990) J. Am. Chem. Soc. 112, 2435-2437].] Supporting evidence is from chemical probing experiments performed on several oligonucleotides designed to form 3-stranded fold-back structures. The third strand, consisting of both purine and pyrimidine blocks, pairs with purines in the Watson-Crick duplex, switching strands at the junction between the oligopurine and oligopyrimidine blocks but maintaining the required strand polarity without any special linkage. Although Mg2+ ions are not required for the formation of Pu.PuPy base triplets, they show enhanced stability in the presence of Mg2+. In the sequences observed. A.AT triplets appear to be more stable than G.GC triplets. As expected, triplex formation is largely independent of pH unless C+.GC base triplets are required.

Nucleosome reconstitution of core-length poly(dG).poly(dC) and poly(rG-dC).poly(rG-dC).

The double-stranded polypurine.polypyrimidines poly(dG).poly(dC) and poly[d(A-G)].poly[d(T-C)] and the mixed ribose-deoxyribose polynucleotide poly(rG-dC).poly(rG-dC) have been successfully reconstituted into nucleosomes. The radioactively labeled particles comigrate in gel electrophoresis and sucrose density gradient experiments with authentic nucleosomes derived from chicken erythrocyte chromatin. These results show that nucleosomes are able to accommodate a wider variety of polynucleotides than was previously believed.

Nephrotic syndrome, malignant thymoma, and myasthenia gravis. Case report and review of the literature.

We describe a patient with nephrotic syndrome due to focal-segmental glomerulosclerosis, occurring 3 years after thymectomy and myasthenia gravis. Nine other cases of nephrotic syndrome associated with thymoma and myasthenia gravis reported in the literature are reviewed. The nephrotic syndrome may be due to T cell dysfunction associated with thymoma; however, animal models suggest that genetic factors may also be involved.

High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.

Modified RNA sequence pools for in vitro selection.

We report the use of modified RNA, in which the 2'-OH group of pyrimidines is replaced by a 2'-amino (2'-NH2) group to identify high affinity ligands specific for human neutrophil elastase (HNE) by in vitro selection. Compared to unmodified RNA the 2'-NH2-modified RNA ligands show enhanced stability in human serum and urine. Use of RNase T1 cleavage data in the presence of K+ and Li+ ions suggests that the modified RNA ligands selected for HNE form an intermolecular G-quartet structure.