A novel sequencing-based vaginal health assay combining self-sampling, HPV detection and genotyping, STI detection, and vaginal microbiome analysis.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman's reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive vaginal health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 31 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 3 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, reference ranges were determined in a group of 50 self-reported healthy women. The HPV sequencing portion of the test was evaluated against the digene High-Risk HPV HC2 DNA test. For hrHPV genotyping, agreement was 95.3% with a kappa of 0.804 (601 samples); after removal of samples in which the digene hrHPV probe showed cross-reactivity with lrHPV types, the sensitivity and specificity of the hrHPV genotyping assay were 94.5% and 96.6%, respectively, with a kappa of 0.841. For lrHPV genotyping, agreement was 93.9% with a kappa of 0.788 (148 samples), while sensitivity and specificity were 100% and 92.9%, respectively. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

Synthesis and Enzymatic Studies of Isoprenoid Thiolo Bisubstrate Analogues.

Chain elongation prenyltransferases catalyze the addition of the hydrocarbon moiety of allylic isoprenoid diphosphates to the carbon-carbon double bond in isopentenyl diphosphate (IPP) in the primary building reactions in the isoprenoid biosynthetic pathway. Bis-O-diphosphate analogues 3-OPP/OPP, 4-OPP/OPP, and 5-OPP/OPP and bis-thiolodiphosphate bisubstrate analogues 3-SPP/SPP, 4-SPP/SPP, and 5-SPP/SPP were synthesized. The analogues 4-OPP/OPP, 5-OPP/OPP, 4-SPP/SPP, and 5-SPP/SPP were excellent competitive inhibitors of avian farnesyl diphosphate synthase with KI = 1.0 ± 0.12 µM, KI = 0.5 ± 0.2 µM, KI = 0.7 ± 0.3 µM, and KI = 2.9 ± 0.27 µM, respectively, whereas, analogues 3-OPP/OPP and 3-SPP/SPP displayed mixed type inhibition with KI = 1.4 µM and KI = 5.5 µM, respectively.

Synthesis and enzymatic studies of bisubstrate analogues for farnesyl diphosphate synthase.

Farnesyl diphosphate synthase catalyzes the sequential chain elongation reactions between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP) and between IPP and GPP to give farnesyl diphosphate (FPP). Bisubstrate analogues containing the allylic and homoallylic substrates were synthesized by joining fragments for IPP and the allylic diphosphates with a C-C bond between the methyl group at C3 in IPP and the Z-methyl group at C3 in DMAPP (3-OPP) and GPP (4-OPP), respectively. These constructs placed substantial limits on the conformational space available to the analogues relative to the two substrates. The key features of the synthesis of bisubstrate analogues 3-OPP and 4-OPP are a regioselective C-alkylation of the dianion of 3-methyl-3-buten-1-ol (5), a Z-selective cuprate addition of alkyl groups to an α,ß-alkynyl ester intermediate, and differential activation of allylic and homoallylic alcohols in the analogues, followed by a simultaneous displacement of the leaving groups with tris(tetra-n-butylammonium) hydrogen diphosphate to give the corresponding bisdiphosphate analogues. The bisubstrate analogues were substrates for FPP synthase, giving novel seven-membered ring analogues of GPP and FPP. The catalytic efficiencies for cyclization of 3-OPP and 4-OPP were similar to those for chain elongation with IPP and DMAPP.

Global transcriptional, physiological, and metabolite analyses of the responses of Desulfovibrio vulgaris hildenborough to salt adaptation.

The response of Desulfovibrio vulgaris Hildenborough to salt adaptation (long-term NaCl exposure) was examined by performing physiological, global transcriptional, and metabolite analyses. Salt adaptation was reflected by increased expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). The expression of genes involved in carbon metabolism, cell growth, and phage structures was decreased. Transcriptome profiles of D. vulgaris responses to salt adaptation were compared with transcriptome profiles of D. vulgaris responses to salt shock (short-term NaCl exposure). Metabolite assays showed that glutamate and alanine accumulated under salt adaptation conditions, suggesting that these amino acids may be used as osmoprotectants in D. vulgaris. Addition of amino acids (glutamate, alanine, and tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model that links the observed results to currently available knowledge is proposed to increase our understanding of the mechanisms of D. vulgaris adaptation to elevated NaCl levels.

Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris hildenborough using gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry.

Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.

Synthesis of deuterium-labeled derivatives of dimethylallyl diphosphate.

Short practical syntheses for five deuterium-labeled derivatives of dimethylallyl diphosphate (DMAPP) useful for enzymological studies are reported. These include the preparation of the C1-labeled derivatives (R)-[1-2H]3-methylbut-2-enyl diphosphate ((R)-[1-2H]1-OPP) and (S)-[1-2H]3-methylbut-2-enyl diphosphate ((S)-[1-2H]1-OPP), the C2-labeled derivative [2-2H]3-methylbut-2-enyl diphosphate ([2-2H]1-OPP), and the methyl-labeled derivatives (E)-[4,4,4-2H3]3-methylbut-2-enyl diphosphate ((E)-[4,4,4-2H3]1-OPP) and (Z)-[4,4,4-2H3]3-methyl-but-2-enyl diphosphate ((Z)-[4,4,4-2H3]1-OPP).