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Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2'-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of ∼50-100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5' DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2'-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.

Although many new assays for HIV have been developed, several labs still use simple and reliable radioactivity-based reverse transcriptase (RT) nucleotide incorporation assays for detection and quantification. We describe here a new assay for detection and quantitation of HIV RT activity that is based on a high affinity DNA aptamer to RT. The aptamer is sequestered on 96-well plates where it can bind to RT and other constituents can be removed by extensive washing. Since the aptamer mimics a primer-template, upon radiolabeled nucleotide addition, bound RT molecules can extend the aptamer and the radioactive signal can be detected by standard methods. In addition to being procedurally simple, the assay demonstrated high sensitivity (detection limits for RT and virions were ≤6400 molecules (∼4 × 10−8 units) and ∼100–300 virions, respectively) and was essentially linear over a range of at least 104. Both wild type and drug-resistant forms of HIV-1 RT were detectable as was HIV-2 RT, although there were some modest differences in sensitivity.

B chromosomes are extra chromosomes found in many species of plants, animals, and fungi. B chromosomes often manipulate common cellular processes to increase their frequency, sometimes to the detriment of organismal fitness. Here, we characterize B chromosomes in several species of Lake Malawi cichlid fish. Whole genome sequencing of Metriaclima zebra "Boadzulu" individuals revealed blocks of sequence with unusually high sequence coverage, indicative of increased copy number of those sequences. These regions of high sequence coverage were found only in females. SNPs unique to the high copy number sequences permitted the design of specific amplification primers. These primers amplified fragments only in Metriaclima lombardoi individuals that carried a cytologically identified B chromosome (B-carriers), indicating these extra copies are located on the B chromosome. These same primers were used to identify B-carrying individuals in additional species from Lake Malawi. Across 7 species, a total of 43 B-carriers were identified among 323 females. B-carriers were exclusively female; no B chromosomes were observed in the 317 males surveyed from these species. Quantitative analysis of the copy number variation of B-specific sequence blocks suggests that B-carriers possess a single B chromosome, consistent with previous karyotyping of M. lombardoi A single B chromosome in B-carriers is consistent with 2 potential drive mechanisms: one involving nondisjunction and preferential segregation in a mitotic division prior to the germ-line, and the other involving preferential segregation during meiosis I.

Freshness is the main concern of seafood quality, and the principal method to evaluate seafood freshness is sensory evaluation. The aim of this work was to study the quality changes of cobia, Rachycentron canadum, under ice storage through sensory and physical analysis as well as bacterial counting of specific spoilage organisms (SSOs). In particular, the utilization of a quality index method (QIM) scheme was proposed. Samples stored for 0–30 d were analyzed with the QIM. Ten panelists observed and registered the changes occurring in the fish starting on day zero and ending when the fish were spoiled. After developing the scheme, 11 sensory attributes were described in 23 points, which detailed the appearance of skin, eyes, abdomen, gills, and flesh deterioration. The volatile nitrogen compound measurements and microbiological data of the SSOs determined a shelf-life of 19 d for the samples. Sensory analysis showed a rejection point at 15.67 d of ice storage. The natural degradation of myofibrillar proteins and collagen was observed during ice storage. In conclusion, a shelf-life of 15 d was defined for raw cobia stored on ice.

Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5' DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5' DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding.

Freshness is the main concern of seafood quality, and the principal method to evaluate seafood freshness is sensory evaluation. The aim of this work was to study the quality changes of cobia, Rachycentron canadum, under ice storage through sensory and physical analysis as well as bacterial counting of specific spoilage organisms (SSOs). In particular, the utilization of a quality index method (QIM) scheme was proposed. Samples stored for 0-30 d were analyzed with the QIM. Made available in DSpace on 2017-10-27T09:24:46Z (GMT). No. of bitstreams: 1 Fogaaetal2017JournaloftheWorldAquacultureSociety.pdf: 961211 bytes, checksum: 0a4c5e9c14d4e5b202a08e10772e350c (MD5) Previous issue date: 2016-08-03 Autoria: PEREIRA, A. [i.e. M.] L.; Alitiene [i.e. Moura] Lemos Pereira. Version of record online: 21 jul. 2016.

Made available in DSpace on 2018-03-29T00:38:44Z (GMT). No. of bitstreams: 1 PubVet2013Costa1591.pdf: 305252 bytes, checksum: 2c660d147cee59c4afbb80dbc5461e4a (MD5) Previous issue date: 2018-03-28