Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes.

Joining Natural and Synthetic DNA Using Biversal Nucleotides: Efficient Sequencing of Six-Nucleotide DNA.

By rearranging hydrogen bond donor and acceptor groups within a standard Watson-Crick geometry, DNA can add eight independently replicable nucleotides forming four additional not found in standard Terran DNA. For many applications, the orthogonal pairing of standard and nonstandard pairs offers a key advantage. However, other applications require standard and nonstandard nucleotides to communicate with each other.

Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems.

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how "self-avoiding molecular recognition systems" (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases.

Hydroxymethanesulfonate from Volcanic Sulfur Dioxide: A "Mineral" Reservoir for Formaldehyde and Other Simple Carbohydrates in Prebiotic Chemistry.

While formaldehyde (HCHO) was likely generated in Earth's prebiotic atmosphere by ultraviolet light, electrical discharge, and/or volcano-created lightning, HCHO could not have accumulated in substantial amounts in prebiotic environments, including those needed for prebiotic processes that generate nucleosidic carbohydrates. HCHO at high concentrations in alkaline solutions self-reacts in the Cannizzaro reaction to give methanol and formate, neither having prebiotic value. Here, we explore the possibility that volcanic sulfur dioxide (SO) might have generated a reservoir for Hadean HCHO by a reversible reaction with HCHO to give hydroxymethanesulfonate (HMS).

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms.

Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts.

Intersecting pathways to neurodegeneration in Parkinson's disease: effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-proteasome system.

Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone.

The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.

Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1.

The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson's disease.

Two mutations in the DJ-1 gene on chromosome1p36 have been identified recently to cause early-onset, autosomal recessive Parkinson's disease. As no information is available regarding the distribution of DJ-1 protein in the human brain, in this study we used a monoclonal antibody for DJ-1 to map its distribution in frontal cortex and substantia nigra, regions invariably involved in Parkinson's disease. Western blotting of human frontal cortex showed DJ-1 to be an abundant protein in control, idiopathic Parkinson's disease, cases with clinical and pathological phenotypes of Parkinson's disease with R98Q polymorphism for DJ-1, and in progressive supranuclear palsy (PSP) brains.

RING finger 1 mutations in Parkin produce altered localization of the protein.

The Parkin gene (PRKN) encodes an E3 protein-ubiquitin ligase for which loss of function is associated with autosomal-recessive juvenile (<20 years) and early-onset Parkinsonism (<45 years). Although detailed pathological reports are scarce, brains from patients with homozygous exonic deletions demonstrate neuronal loss in the substantia nigra, albeit without the Lewy body pathology characteristic of idiopathic Parkinson's disease. However, there are rare descriptions of more florid pathology, including Lewy bodies and tau positive astrocytes in individuals with compound heterozygous mutations.

L166P mutant DJ-1, causative for recessive Parkinson's disease, is degraded through the ubiquitin-proteasome system.

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system.

Peptide-based, irreversible inhibitors of gamma-secretase activity.

The characterization of the enzymes responsible for amyloid beta-peptide (Abeta) production is considered to be a primary goal towards the development of future therapeutics for the treatment of Alzheimer's disease. Inhibitors of gamma-secretase activity were critical in demonstrating that the presenilins (PSs) likely comprised at least part of the active site of the gamma-secretase enzyme complex, with two highly conserved membrane aspartates presumably acting as catalytic residues. However, whether or not these aspartates are actually the catalytic residues of the enzyme complex or are merely essential for normal PS function and/or maturation is still unknown.

Overexpression of nicastrin increases Abeta production.

Gamma-secretase cleavage is the final proteolytic step that releases the amyloid beta-peptide (Abeta) from the amyloid beta-protein precursor (APP). Significant evidence indicates that the presenilins (PS) are catalytic components of a high molecular weight gamma-secretase complex. The glycoprotein nicastrin was recently identified as a functional unit of this complex based on 1) binding to PS and 2) the ability to modulate Abeta production following mutation of a conserved DYIGS region.

Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains.

Buoyant membrane fractions containing presenilin 1 (PS1), an essential component of the gamma-secretase complex, and APP CTFbeta, a gamma-secretase substrate, can be isolated from cultured cells and brain by several different fractionation procedures that are compatible with in vitro gamma-secretase assays. Analysis of these gradients for amyloid beta protein (Abeta) and CTFgamma production indicated that gamma-secretase activity is predominantly localized in these buoyant membrane microdomains. Consistent with this localization, we find that gamma-secretase activity is cholesterol dependent.