Targeted Mass Spectrometry Assays for Specific Quantification of Urinary proPSA Isoforms.

Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. The pre-mature forms of prostate-specific antigen (PSA), proPSA, were shown to be associated with PCa. However, there is a technical challenge in the development of antibody-based immunoassays for specific recognition of each individual proPSA isoform. Herein, we report the development of highly specific, antibody-free, targeted mass spectrometry assays for simultaneous quantification of [-2], [-4], [-5], and [-7] proPSA isoforms in voided urine. The newly developed proPSA assays capitalize on Lys-C digestion to generate surrogate peptides with appropriate length (9-16 amino acids) along with long-gradient liquid chromatography separation. The assay utility of these isoform markers was evaluated in a cohort of 30 well-established clinical urine samples for distinguishing PCa patients from healthy controls. Under the 95% confidence interval, the combination of [-2] and [-4] proPSA isoforms yields the area under curve (AUC) of 0.86, and the AUC value for the combined all four isoforms was calculated to be 0.85. We have further verified [-2]proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples. Validation of proPSA isoforms in large-scale cohorts is needed to demonstrate their potential clinical utility.

Exploring Nucleobase Modifications in Oligonucleotide Analogues for Use as Environmentally Responsive Fluorophores and Beyond.

Over the past two decades, it has become abundantly clear that nucleic acid biochemistry, especially with respect to RNA, is more convoluted and complex than previously appreciated. Indeed, the application and exploitation of nucleic acids beyond their predestined role as the medium for storage and transmission of genetic information to the treatment and study of diseases has been achieved. In other areas of endeavor, utilization of nucleic acids as a probe molecule requires that they possess a reporter group. The reporter group of choice is often a luminophore because fluorescence spectroscopy has emerged as an indispensable tool to probe the structural and functional properties of modified nucleic acids. The scope of this review spans research done in the Hudson lab at The University of Western Ontario and is focused on modified pyrimidine nucleobases and their applications as environmentally sensitive fluorophores, base discriminating fluorophores, and in service of antisense applications as well as tantalizing new results as G-quadruplex destabilizing agents. While this review is a focused personal account, particularly influential work of colleagues in the chemistry community will be highlighted. The intention is not to make a comprehensive review, citations to the existing excellent reviews are given, any omission of the wonderful and impactful work being done by others globally is not intentional. Thus, this review will briefly introduce the context of our work, summarize what has been accomplished and finish with the prospects of future developments.

Chimeric GFP-uracil based molecular rotor fluorophores.

Uracil has been modified at the 5-position to derive a small library of nucleobase-chromophores which were inspired by green fluorescent protein (GFP). The key steps in the syntheses were Erlenmeyer azlactone synthesis followed by amination by use of hexamethyl disilazane (HMDS) to produce the imidazolinone derivatives. The uracil analogues displayed emission in the green region of visible spectrum and exhibited microenvironmental sensitivity exemplified by polarity-based solvatochromism and viscosity-dependent emission enhancement. Solid-state quantum yields of approximately 0.2 and solvent dependent emission wavelengths beyond 500 nm were observed. Select analogues were incorporated into peptide nucleic acid (PNA) strands which upon duplex formation with DNA showed good response ranging from a turn-off of fluorescence in presence of an opposing mismatched residue to a greater than 3-fold turn-on of fluorescence upon binding to fully complementary DNA strand.

Explicating Exact versus Conceptual Replication.

What does it mean to replicate an experiment? A distinction is often drawn between 'exact' (or 'direct') and 'conceptual' replication. However, in recent work, Uljana Feest argues that the notion of replication in itself, whether exact or conceptual, is flawed due to the problem of systematic error, and Edouard Machery argues that, although the notion of replication is not flawed, we should nevertheless dispense with the distinction between exact and conceptual replication. My plan in this paper is to defend the value of replication, along with the distinction between exact and conceptual replication, from the critiques of Feest and Machery. To that end, I provide an explication of conceptual replication, and distinguish it from what I call 'experimental' replication. On the basis, then, of a tripartite distinction between exact, experimental and conceptual replication, I argue in response to Feest that replication is still informative despite the prospect of systematic error. I also rebut Machery's claim that conceptual replication is fundamentally confused and wrongly conflates replication and extension, and in turn raise some objections to his own Resampling Account of replication.

A simple fluorescent assay for the detection of peptide nucleic acid-directed double strand duplex invasion.

Peptide nucleic acid (PNA) is a mimic of nucleic acids that is able to bind complementary oligonucleotides with high affinity and excellent selectivity. As such, the use of PNA has been proposed in numerous applications in biochemistry, medicine, and biotechnology. Sequences of pseudo-complementary PNAs containing diaminopurine (D)-2-thiouracil (S U) base pairs bind to complementary regions within double-stranded DNA targets. This type of binding is termed "double duplex invasion" and involves unwinding of the duplex accompanied by simultaneous hybridization of both DNA strands by the two pseudo-complementary PNAs. In this study, a simple method of assaying DNA strand invasion by pseudo-complementary PNAs was developed. This method is based on the incorporation of a single fluorescent cytidine analog, phenylpyrrolocytidine (PhpC), into the double-stranded DNA target such that upon invasion by PNA, the PhpC is displaced to a single-stranded region resulting in the turn-on of fluorescence emission. This fluorescent assay is applicable to studies both at equilibrium and approach-to-equilibrium (time-dependent) conditions.

Identifying G-Quadruplex-DNA-Disrupting Small Molecules.

The quest for small molecules that strongly bind to G-quadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV-vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4-disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.

Comparison of rectal swab, glove tip, and participant-collected stool techniques for gut microbiome sampling.

BACKGROUND: Studies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method. RESULTS: We prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant's home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (p< 0.001) as measured by Bray-Curtis and unifrac distances. There were no significant differences in between the swab and glove tip samples with regard to beta diversity (p> 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods. CONCLUSION: The glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.

6-phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein.

Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O6 -[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O6 -POB-G) and O6 -methylguanine (O6 -Me-G) adducts in DNA. These adducts can be directly repaired by O6 -alkylguanine-DNA alkyltransferase (AGT). Repair begins by flipping the damaged base out of the DNA helix. AGT binding and base-flipping have been previously studied using pyrrolocytosine as a fluorescent probe paired to the O6 -alkylguanine lesion, but low fluorescence yield limited the resolution of steps in the repair process. Here, we utilize the highly fluorescent 6-phenylpyrrolo-2'-deoxycytidine (6-phenylpyrrolo-C) to investigate AGT-DNA interactions. Synthetic oligodeoxynucleotide duplexes containing O6 -POB-G and O6 -Me-G adducts were placed within the CpG sites of codons 158, 245, and 248 of the p53 tumor suppressor gene and base-paired to 6-phenylpyrrolo-C in the opposite strand. Neighboring cytosine was either unmethylated or methylated. Stopped-flow fluorescence measurements were performed by mixing the DNA duplexes with C145A or R128G AGT variants. We observe a rapid, two-step, nearly irreversible binding of AGT to DNA followed by two slower steps, one of which is base-flipping. Placing 5-methylcytosine immediately 5' to the alkylated guanosine causes a reduction in rate constant of nucleotide flipping. O6 -POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O6 -Me-G at the same position. A similar effect was not observed at other codons.

Fluorescent Biaryl Uracils with C5-Dihydro- and Quinazolinone Heterocyclic Appendages in PNA.

There has been much effort to exploit fluorescence techniques in the detection of nucleic acids. Canonical nucleic acids are essentially nonfluorescent; however, the modification of the nucleobase has proved to be a fruitful way to engender fluorescence. Much of the chemistry used to prepare modified nucleobases relies on expensive transition metal catalysts. In this work, we describe the synthesis of biaryl quinazolinone-uracil nucleobase analogs prepared by the condensation of anthranilamide derivatives and 5-formyluracil using inexpensive copper salts. A selection of modified nucleobases were prepared, and the effect of methoxy- or nitro- group substitution on the photophysical properties was examined. Both the dihydroquinazolinone and quinazolinone modified uracils have much larger molar absorptivity (~4-8×) than natural uracil and produce modest blue fluorescence. The quinazolinone-modified uracils display higher quantum yields than the corresponding dihydroquinazolinones and also show temperature and viscosity dependent emission consistent with molecular rotor behavior. Peptide nucleic acid (PNA) monomers possessing quinazolinone modified uracils were prepared and incorporated into oligomers. In the sequence context examined, the nitro-substituted, methoxy-substituted and unmodified quinazolinone inserts resulted in a stabilization (∆Tm = +4.0/insert; +2.0/insert; +1.0/insert, respectively) relative to control PNA sequence upon hybridization to complementary DNA. All three derivatives responded to hybridization by the "turn-on" of fluorescence intensity by ca. 3-to-4 fold and may find use as probes for complementary DNA sequences.

On the Necessity of Nucleobase Protection for 2-Thiouracil for Fmoc-Based Pseudo-Complementary Peptide Nucleic Acid Oligomer Synthesis.

A selection of benzyl-based protecting groups for thiouracil (SU) for the synthesis of pseudo-complementary peptide nucleic acid (PNA) has been evaluated. The 4-methoxybenzyl-protecting group that has found use for SU during Boc-based oligomerization is also suitable for Fmoc-based oligomerization. Furthermore, it is demonstrated that SU protection is unnecessary for the successful synthesis of thiouracil-containing PNA. The new 2-thiothymine (ST) PNA monomer has also been prepared and incorporated into an oligomer and its binding to complementary PNA evaluated.

A fluorescent 3,7-bis-(naphthalen-1-ylethynylated)-2'-deoxyadenosine analogue reports thymidine in complementary DNA by a large emission Stokes shift.

The new environmentally responsive fluorescent nucleosides, 3,7-bis-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2'-deoxyadenosine (3n7nzA, 1) and 7-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2'-deoxyadenosine (37nzA, 2), have been synthesized. Both 3n7nzA (1) and 37nzA (2) possess large π-conjugated systems which extend into both the minor and major grooves or the major groove alone, respectively. The nucleosides exhibited large solvatochromic shifts (3n7nzA: Δλ = 45 nm, 37nzA: Δλ = 78 nm) and were examined for their ability to fluorimetrically report hybridization events. When incorporated into ODN probes, the bis-substituted 3n7nzA (1) selectively recognized thymidine on target strands which was reported by a distinct change in its emission wavelength in the long wavelength region, whereas 37nzA (2) showed a preference for pairing to cytidine and a smaller wavelength shift. Thus, 3n7nzA (1) has the potential for use as a fluorescent probe for structural studies of DNAs/RNAs including the detection of single-base alterations in target DNA sequences.

Draining the swamp while making America great again: senior dissonance in the age of Trump.

In his surprise election as president, Donald Trump enjoyed disproportionate electoral support from older voters, many of whom saw in Trump a person who would work to reverse demographic, economic, and cultural forces that had transformed American life as they had long seen it. Yet, Trump's campaign and incumbency has also been very much about gutting the Washington policy establishment of officials, bureaucrats, and lobbyists (aka, "the swamp") which, for more than half a century, has been instrumental in enacting and expanding legislation that has benefitted older Americans far more than any other social policy constituency in the country. This article contrasts the value-oriented electoral support Trump enjoyed from older Americans with their interest concerns centered on policies such as the Affordable Care Act, Medicaid, and a host of smaller grant-in-aid programs. It then reviews the strong institutional base seniors and their advocates have in Washington, posing whether interest-oriented concerns may outweigh ideological ones as policy options emerge from a Republican-controlled government prior to the 2018 elections.

The fluorescently responsive 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine discriminates cytidine via the DNA minor groove.

A new environmentally responsive fluorescent nucleoside, 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine (3nzG), has been synthesized. The nucleoside, 3nzG, exhibited solvatochromic properties and when introduced into ODN probes it was able to recognize 2'-deoxycytidine in target strands by a distinct change in its emission wavelength through probing microenvironmental changes in the DNA minor groove. Thus, 3nzG has the potential for use as a fluorescent probe molecule for micro-structural studies of nucleic acids including the detection of single-base alterations in target DNA sequences.

Unusual C7- versus Normal 5'-O-Dimethoxytritylation of 6-Arylpyrrolocytidine Analogs.

Fluorescent deoxynucleosides possessing the modified bases 6-(2-benzo[b]furyl)- and 6-(2-furyl)pyrrolocytosine (BFpC and FpC) have been synthesized along with the quencher nucleosides possessing 6-{4-[(4-dimethylamino)azo]phenyl}pyrrolocytosine (DABCYLpC) and 6-(p-nitrophenyl)pyrrolocytosine (p-NO2-PhpC) nucleobase analogs. Standard treatment of BFpC, FpC, DABCYLpC, and p-NO2-PhpC with dimethoxytrityl chloride (DMT-Cl) led to the unusual substitution on the C7 of the pyrrolocytosine skeleton. The desired 5'-O-DMT-protected nucleoside analogs were synthesized from suitably protected 5'-O-DMT cytidines. Subsequent phosphitylation smoothly afforded BFpC-, FpC-, DABCYLpC-, and p-NO2-PhpC-derived monomers suitable for standard oligonucleotide synthesis.

Effects of exercise amount and intensity on abdominal obesity and glucose tolerance in obese adults: a randomized trial.

BACKGROUND: Exercise reduces obesity and related glucose tolerance, but whether increasing exercise intensity offers additional benefit at fixed exercise amounts is unknown. OBJECTIVE: To determine the separate effects of exercise amount and intensity on abdominal obesity and glucose tolerance. DESIGN: 24-week, single-center, parallel-group trial from 2009 to 2013. (ClinicalTrials.gov: NCT00955071). SETTING: Kingston, Ontario, Canada. PARTICIPANTS: 300 abdominally obese adults. INTERVENTION: Control (no exercise) (n = 75) or 5 weekly sessions of low-amount, low-intensity exercise (LALI) (180 and 300 kcal/session for women and men, respectively, at 50% of maximum oxygen consumption [V̇o2peak]) (n = 73); high-amount, low-intensity exercise (HALI) (360 and 600 kcal/session, respectively, at 50% of V̇o2peak) (n = 76); or high-amount, high-intensity exercise (HAHI) (360 and 600 kcal/session, respectively, at 75% of V̇o2peak) (n = 76). Daily unsupervised physical activity and sedentary time were measured by accelerometer. MEASUREMENTS: Waist circumference and 2-hour glucose level (primary outcomes) and cardiorespiratory fitness and measures of insulin action (secondary measurements). RESULTS: 217 participants (72.3%) completed the intervention. Mean exercise time in minutes per session was 31 (SD, 4.4) for LALI, 58 (SD, 7.6) for HALI, and 40 (SD, 6.2) for HAHI. Daily unsupervised physical activity and sedentary time did not change in any exercise group versus control (P > 0.33). After adjustment for age and sex in a linear mixed model, reductions in waist circumference were greater in the LALI (-3.9 cm [95% CI, -5.6 to -2.3 cm]; P < 0.001), HALI (-4.6 cm [CI, -6.2 to -3.0 cm]; P < 0.001), and HAHI (-4.6 cm [CI, -6.3 to -2.9 cm]; P < 0.001) groups than the control group but did not differ among the exercise groups (P > 0.43). After adjustment for covariates, reductions in 2-hour glucose level were greater in the HAHI group (-0.7 mmol/L [-12.5 mg/dL] [CI, -1.3 to -0.1 mmol/L {-23.5 to -1.5 mg/dL}]; P = 0.027) than the control group but did not differ for the LALI or HALI group versus the control group (P > 0.159). Weight loss was greater in all exercise groups than the control group (P < 0.001); however, reduction in body weight did not differ among the exercise groups (P > 0.182). LIMITATION: The clinical importance of reducing 2-hour glucose level in nondiabetic adults remains undetermined. CONCLUSION: Fixed amounts of exercise independent of exercise intensity resulted in similar reductions in abdominal obesity. Reduction in 2-hour glucose level was restricted to high-intensity exercise.

PNA Molecular Beacons Assembled by Post-Synthetic Click Chemistry Functionalization.

To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post-synthetic on-resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide-containing monomers into the oligomer by automatic solid-phase peptide synthesis and subsequent derivatization with pyrene moieties by copper-catalyzed azide-alkyne cycloaddition. Two pyrene-based quencher-free PNA molecular beacons, a stemless MB and one possessing a stem-loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem-loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.

Small gold nanoparticles for interfacial Staudinger-Bertozzi ligation.

Small gold nanoparticles (AuNPs) that possess interfacial methyl-2-(diphenylphosphino)benzoate moieties have been successfully synthesized (Staudinger-AuNPs) and characterized by multi-nuclear MR spectroscopy, transmission electron microscopy (TEM), UV-Vis spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy (XPS). In particular, XPS was remarkably sensitive for characterization of the novel nanomaterial, and in furnishing proof of its interfacial reactivity. These Staudinger-AuNPs were found to be stable to the oxidation of the phosphine center. The reaction with benzyl azide in a Staudinger-Bertozzi ligation, as a model system, was investigated using (31)P NMR spectroscopy. This demonstrated that the interfacial reaction was clean and quantitative. To showcase the potential utility of these Staudinger-AuNPs in bioorganic chemistry, a AuNP bioconjugate was prepared by reacting the Staudinger-AuNPs with a novel azide-labeled CRGDK peptide. The CRGDK peptide could be covalently attached to the AuNP efficiently, chemoselectively, and with a high loading.

Characterizing the Performance of Denitrifying Bioreactors during Simulated Subsurface Drainage Events.

The need to mitigate nitrate export from corn and soybean fields with subsurface (tile) drainage systems, a major environmental issue in the midwestern United States, has made the efficacy of field-edge, subsurface bioreactors an active subject of research. This study of three such bioreactors located on the University of Illinois South Farms during their first 6 mo of operation (July-Dec. 2012) focused on the interactions of seasonal temperature changes and hydraulic retention times (HRTs), which were subject to experimental manipulation. Changes in nitrate, phosphate, oxygen, and dissolved organic carbon were monitored in influent and effluent to assess the benefits and the potential harmful effects of bioreactors for nearby aquatic ecosystems. On average, bioreactors reduced nitrate loads by 63%, with minimum and maximum reductions of 20 and 98% at low and high HRTs, respectively. The removal rate per unit reactor volume averaged 11.6 g NO-N m d (range, 5-30 g NO-N m d). Multiple regression models with exponential dependencies on influent water temperature and on HRT explained 73% of the variance in NO-N load reduction and 43% of the variance in its removal rate. Although concentrations of dissolved reactive phosphorus and dissolved organic carbon in the bioreactor effluent increased relative to the influent by an order of magnitude during initial tests, within 1 mo of operation they stabilized at nearly equal values.

Hereditary hyperplastic gingivitis in North American farmed silver fox (Vulpes vulpes).

Hereditary hyperplastic gingivitis is a progressive growth of gingival tissues in foxes resulting in dental encapsulation. It is an autosomal recessive condition displaying a gender-biased penetrance, with an association with superior fur quality. This disease has been primarily described in European farmed foxes. Here we document its emergence in Canada.

Gingivite hyperplasique héréditaire chez le renard argenté d'élevage d'Amérique du Nord(Vulpes vulpes). La gingivite hyperplasique héréditaire est une croissance progressive des tissus gingivaux chez les renards qui produit une encapsulation dentaire. Il s'agit d'une affection récessive autosomique qui manifeste une pénétration privilégiant un sexe et qui présente une association avec une qualité de fourrure supérieure. Cette maladie a été principalement décrite chez les renards d'élevage européen. Nous documentons ici son émergence au Canada.(Traduit par Isabelle Vallières).

Genome-wide expression analysis of hereditary hyperplastic gingivitis in silver foxes (Vulpes vulpes) using canine microarrays.

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive condition found predominantly in farmed silver foxes, first documented in Europe in the 1940s. Hereditary gingival fibromatosis (HGF) is an analogous condition occurring in humans. HGF has a heterogeneous aetiology with emphasis placed on the autosomal dominant forms of inheritance for which there are three known loci: HGF1, HGF2, and HGF3. Among these, only one causative mutation has been determined, in the Son of sevenless homolog 1 (SOS1) gene. The goal of this study was to explore potential molecular or cellular mechanisms underlying HHG by analysis of global gene expression patterns from Affymetrix Canine 2.0 microarrays cross-referenced against candidate genes within the human loci. We conclude that the SOS1 gene involved in HGF1 is not significantly up-regulated in HHG. However, the structurally and functionally similar SOS2 gene is up-regulated in affected foxes, and we propose this as a candidate gene for HHG. At HGF2 we identify RASA1 (rat sarcoma viral p21 protein activator 1) as a candidate gene for HHG, as it is up-regulated in affected foxes and is involved in MAPK signalling. From comparison to the genes within the HGF3 locus, we find evidence for a role of androgens in HHG phenotype severity by differential up-regulation of SRD5A2 in HHG-affected foxes. We hypothesize that the putative mutation occurs upstream of RAS in the extracellular signal-regulated kinase component of MAPK signalling.