8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.

A conserved uORF in the ilvBNC mRNA of Corynebacterium species regulates ilv operon expression.

Computational methods can be used to identify putative structured noncoding RNAs (ncRNAs) in bacteria, which can then be validated using various biochemical and genetic approaches. In a search for ncRNAs in Corynebacterium pseudotuberculosis, we observed a conserved region called the ilvB-II motif located upstream of the ilvB gene that is also present in other members of this genus. This gene codes for an enzyme involved in the production of branched-chain amino acids (BCAAs). The ilvB gene in some bacteria is regulated by members of a ppGpp-sensing riboswitch class, but previous and current data suggest that the ilvB-II motif regulates expression by a transcription attenuation mechanism involving protein translation from an upstream open reading frame (uORF or leader peptide). All representatives of this RNA motif carry a start codon positioned in-frame with a nearby stop codon, and the peptides resulting from translation of this uORF are enriched for BCAAs, suggesting that expression of the ilvB gene in the host cells is controlled by attenuation. Furthermore, recently discovered RNA motifs also associated with ilvB genes in other bacterial species appear to carry distinct uORFs, suggesting that transcription attenuation by uORF translation is a common mechanism for regulating ilvB genes.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2'-deoxyguanosine.

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2'-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 2'-deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA.

The case of the missing allosteric ribozymes.

The RNA World theory encompasses the hypothesis that sophisticated ribozymes and riboswitches were the primary drivers of metabolic processes in ancient organisms. Several types of catalytic RNAs and many classes of ligand-sensing RNA switches still exist in modern cells. Curiously, allosteric ribozymes formed by the merger of RNA enzyme and RNA switch components are largely absent in today's biological systems. This is true despite the striking abundances of various classes of both self-cleaving ribozymes and riboswitch aptamers. Here we present the known types of ligand-controlled ribozymes and riboswitches and discuss the possible reasons why fused ribozyme-aptamer constructs have been disfavored through evolution.

A second riboswitch class for the enzyme cofactor NAD.

A bacterial noncoding RNA motif almost exclusively associated with pnuC genes was uncovered using comparative sequence analysis. Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found "pnuC motif" RNAs might function as aptamers for a novel class of NAD+-sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD+, nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD+ binding.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Growth characteristics underlying the lack of a chin in pigs: a histomorphometric study.

OBJECTIVES: Despite similar mandibular growth to that of humans, pigs lack a chin projection as shown in most humans. To understand whether this divergence is contributed to differences in local symphyseal growth, this project characterized bone modeling activities at the symphyseal surfaces of juvenile pigs. MATERIAL AND METHODS: Symphyseal specimens from two age groups (4- and 6-month-old, n = 10) were processed into histological sections with and without decalcification, which were assessed for surface mineral apposition and bone resorption, respectively. In a blinded fashion, measurements of four parameters (MAR: mineral apposition rate, MAZ: mineral apposition zone, ES/BS: eroded surface and OC.N/BS: osteoclast number) were obtained and tested by a multivariate two-way mixed-model analyses of variance (manova) for the differences between symphyseal regions and ages. RESULTS: Qualitatively, pig symphyseal labial and lingual surfaces were horizontally oriented and characterized by mineral apposition and bone resorption, respectively. Quantitatively, labial mineral apposition tended to be greater rostrally than caudally at 4 months, which became greater caudally than rostrally at 6 months (region/age interactions: p = 0.127 for MAR, p = 0.012 for MAZ). Lingual bone resorption tended to be greater caudally than rostrally, but only ES/BS measurements were significant (p = 0.039) regardless of age, while OC.N/BS measurements varied with ages and regions (age/region interaction, p = 0.087). CONCLUSIONS: Insufficient differential in symphyseal surface modeling between the labial-caudal and labial-rostral regions contributes to the lack of chin projection in the pig.

Nemo promotes Notch-mediated lateral inhibition downstream of proneural factors.

During neurogenesis, conserved tissue-specific proneural factors establish a cell's competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of 'core' and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. Here, we identify nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and find that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. We find that nmo loss of function leads to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.

Characterizing cognitive deficits and dementia in an aging urban population in India.

Rapid rise in the population of older adults in India will lead to the need for increased health care services related to diagnosis, management, and long-term care for those with dementia and cognitive impairment. A direct approach for service provision through memory clinics can be an effective, successful, and sustaining means of delivering specialized health care services. We have established a memory clinic in Mumbai, India by employing the diverse clinical skills available in Indian academic institutions, diagnostic and research expertise of clinicians and psychologists, and the support of the U.S. National Institutes of Health. Our project involved recruitment of patients, clinical and neuropsychological assessment, and standardized diagnostic procedures, demonstrating the feasibility of using research methods to develop a memory clinic. In this paper, we describe the development of a community-based memory clinic in urban India, including linguistic and cultural factors and present detailed results, including diagnostic characterization, on 194 subjects with various stages of cognitive deficits. Our findings support the feasibility of developing a memory clinic in a public hospital and successful use of research diagnostic criteria to categorize cognitive deficits observed in this population, which may be used to inform the development of other such clinics.

Epigenetic regulation and downstream targets of the Rhox5 homeobox gene.

The discovery of the Rhox homeobox gene cluster on the X chromosome opens up new vistas in the regulation of reproductive processes in mammals. In mice, this cluster comprises more than 30 genes that are selectively expressed in reproductive tissues. A subset of Rhox genes are androgen and AR regulated in postnatal and adult Sertoli cells, making them candidates to mediate androgen-dependent steps during spermatogenesis. The best characterized of these androgen/AR-regulated genes is Rhox5 (Pem), the founding member of the Rhox gene cluster. Targeted deletion of Rhox5 in mice causes male subfertility marked by increased germ-cell apoptosis and decreased sperm count and motility. Microarray analyses identified a wide variety of genes regulated by Rhox5 in Sertoli cells. One of them is the tumour suppressor UNC5C, a pro-apoptotic molecule previously only known to be involved in brain development. Targeted deletion of Unc5c causes decreased germ-cell apoptosis in postnatal and adult testes, indicating that it also has a role in spermatogenesis and supporting a model in which Rhox5 promotes germ-cell survival by downregulating Unc5c. Rhox5 has two independently regulated promoters that have distinct expression patterns. The unique tissue-specific and developmentally regulated transcription pattern of these two promoters appear to be controlled by DNA methylation. Both promoters are methylated in tissues in which they are not expressed, suggesting that DNA methylation serves to repress Rhox5 expression in inappropriate cell types and tissues. In summary, the Rhox gene cluster is an epigenetically regulated set of genes encoding a large number of transcription factors that are strong candidates to regulate gametogenesis and other aspects of reproduction.

Semiclassical statistical mechanics of two-dimensional hard-body fluids.

The problem of calculating the thermodynamic properties of two-dimensional semiclassical hard-body fluids is studied. Explicit expressions are given for the first-order quantum corrections to the free energy, equation of state, and virial coefficients. The numerical results are calculated for the planar hard dumbbell fluid. Significant features are the increase in quantum corrections with increasing eta and increasing L*=L/sigma(0).

Evaluation of a combined immunomagnetic separation/flow cytometry technique for epidemiological investigations of Cryptosporidium in domestic and Australian native animals.

A combined immunomagnetic separation (IMS) and flow cytometry (FC) technique was developed for the sensitive detection of Cryptosporidium in faecal samples. The IMS/FC technique was found to be approximately 50-fold more sensitive than formol-ether concentration, which is commonly used for Cryptosporidium epidemiological investigations. Of 31 faecal samples from captive animals 16 were found to contain Cryptosporidium oocysts when analysed using the IMS/FC compared to four when using formol-ether concentration (FEC). In a wild population of eastern grey kangaroos Macropus giganteus 66.3% of infected animals were shedding <500oocysts/gfaeces when analysed using IMS/FC. This is below the detection limit for the FEC method. The dispersal of Cryptosporidium in host populations is aggregated, with many individuals shedding low numbers of oocysts and few individuals shedding numbers of oocysts sufficiently high to be detected by FEC. This research demonstrates that the prevalence and oocyst shedding intensity of Cryptosporidium in animal populations will be significantly underestimated using standard detection methods.

Laparoscopic anatrophic nephrolithotomy: feasibility study in a chronic porcine model.

PURPOSE Anatrophic nephrolithotomy performed via open surgery involves incising the renal parenchyma along an avascular plane to remove a large, complex renal stone. We determined the feasibility of performing laparoscopic anatrophic nephrolithotomy in a survival porcine model. Furthermore, we present a novel technique of creating a staghorn calculus in the porcine model. MATERIALS AND METHODS After developing the technique in 3 pigs the survival study was performed in 10 consecutive animals. The procedure comprised 2 aspects. 1) We developed an animal model for staghorn calculi by retrograde injection of polyurethane (Fomo Products, Inc., Norton, Ohio) into the renal pelvis through a ureteral catheter. For a 2-week period the staghorn calculus was allowed to create hydronephrosis. 2) Laparoscopic anatrophic nephrolithotomy was done, involving control of the renal artery and vein, in situ renal hypothermia with ice slush in 1 animal, lateral renal parenchymal incision, stone extraction and suture repair of the incised collecting system and renal parenchyma. RESULTS Synthetic stone formation and laparoscopic anatrophic nephrolithotomy were successful in all 10 animals, including 1 that underwent staged bilateral anatrophic nephrolithotomy. Mean operative time for anatrophic nephrolithotomy was 125 minutes. Mean blood loss was 68 cc and mean warm ischemia time was 30 minutes (range 23 to 39). A residual small pelvicaliceal calculus was noted postoperatively in the initial 3 cases only. Thereafter, routine intraoperative ultrasonography and flexible endoscopy were done for stone localization, resulting in a stone-free rate of 100% in all 7 remaining animals. Diethylenetriamine pentaacetic acid renal scans documented improvement in the glomerular filtration rate from a mean of 26.4 ml. per minute after stone creation and hydronephrosis to 54.8 ml. per minute 4 to 5 weeks after laparoscopic anatrophic nephrolithotomy. CONCLUSIONS Laparoscopic techniques can be applied to complex stone surgery such as anatrophic nephrolithotomy with encouraging surgical and functional outcomes. To our knowledge this report represents the initial study of in situ creation of experimental staghorn calculi and laparoscopic anatrophic nephrolithotomy performed completely intracorporeally in a chronic porcine model.

Percutaneous endopyeloplasty: a novel technique.

BACKGROUND AND PURPOSE: Despite a 10% to 15% failure rate, endopyelotomy remains the treatment of choice for most patients with ureteropelvic junction (UPJ) obstruction. We present a novel technique of percutaneous endopyeloplasty, wherein a precise, full-thickness approximation of a standard longitudinal endopyelotomy incision is performed in a horizontal Heineke-Mikulicz fashion through the conventional solitary percutaneous tract via a nephroscope. We assess the feasibility and efficacy of percutaneous endopyeloplasty in a chronic porcine bilateral UPJ obstruction model and compare outcome data with those#10; of conventional endopyelotomy and laparoscopic pyeloplasty. MATERIALS AND METHODS: Partial UPJ obstruction was created in 20 kidneys (11 pigs) by laparoscopic ligation of the upper ureter over a 5F ureteral catheter. After development of hydronephrosis over a period of 4 to 6 weeks, percutaneous endopyeloplasty (N = 10), conventional percutaneous endopyelotomy (N = 5), or laparoscopic pyeloplasty (N = 5) was performed. The essential steps of percutaneous endopyeloplasty include retrograde ureteral catheterization, standard percutaneous endopyelotomy incision, mobilization of the distal ureteral lip, horizontal suturing of the endopyelotomy incision through the nephroscope, and nephrostomy drainage and ureteral stenting. Suturing was performed using a modified 5-mm laparoscopic device (Sew Right 5 SR; LSI Solutions, Rochester, NY), which was passed through the nephroscope. RESULTS: Percutaneous endopyeloplasty was technically successful in all 10 kidneys with a mean total operative time of 81.4 minutes (range 51-117 minutes). The mean endopyeloplasty suturing time was 29.4 minutes (range 20-64 minutes). Three kidneys required two sutures, while seven kidneys required three sutures to complete the endopyeloplasty. The solitary complication was a lower-pole infundibular stenosis. Over a mean follow-up of 7.7 weeks, all renal units showed relief of obstruction, as evidenced by regression of hydronephrosis,#10; improvement in T(1/2) and glomerular filtration rate on renogram, and a low intrapelvic pressure on Whitaker test. At autopsy, the endopyeloplasty site showed a fine, well-healed transverse scar with no evidence of residual suture on the mucosal surface. The mean caliber of the UPJ following endopyeloplasty (13.8F +/- 2.2F) was significantly greater (P = 0.01) than that following endopyelotomy (7.5F +/- 1.9F). Intraoperative extravasation on completion of endopyeloplasty was absent (N = 6) or mild (N = 4) compared with that seen in all five kidneys following endopyelotomy. CONCLUSION: Percutaneous endopyeloplasty is feasible, simple, reproducible, and effective. Its advantages over conventional endopyelotomy include transrenal performance of a Fenger-plasty, wider caliber of the UPJ, absence of extravasation, and shorter duration of ureteral stenting.

Identification of a cDNA clone encoding for a galactose-binding lectin from peanut (Arachis hypogaea) seedling roots.

A cDNA clone obtained from developing peanut (Arachis hypogaea) seedling roots, when expressed in Escherichia coli and insect cells (Sf9) gave a 29 kDa subunit protein. The native recombinant protein agglutinates neuraminidase treated human erythrocytes and the agglutination is inhibited by galactose. Nucleotide sequence and predicted amino acid sequence analyses indicate that it is different from peanut seed (PNA and SGL) and nodule (NGLa and NGLb) galactose-binding lectins.

Interaction between Cryptosporidium oocysts and water treatment coagulants.

The electrokinetic properties of gamma-irradiated Cryptosporidium oocysts in the presence of coagulants (ferric chloride and alum) and coagulant aids (DADMAC based cationic polyelectrolytes) have been studied. The zeta potential of the oocysts was unaffected by the addition of ferric chloride at all pH values (3-10) studied. Addition of alum resulted in reversal of the oocysts charge, which suggests that the initial stage in the coagulation process leading to floc formation proceeds via the adsorption of hydrolysed aluminium species. The cationic polyelectrolyte Magnafloc LT35 was adsorbed onto iron flocs at doses of 0.1 mg/L even against an electrostatic barrier. The cationic polyelectrolyte only adsorbed and caused charge reversal at the oocyst surface at around 0.4 mg/L, suggesting a lower affinity for this surface. These results indicate that the oocysts, unlike inorganic colloidal materials such as metal oxides, appear to possess a lower surface density of active or charged sites. The lower density of sites, combined with the rapid precipitation of iron salts, may be responsible for the lack of specific adsorption of either hydroxylated ferric species or primary iron hydroxide particles on the oocysts. Further, this suggests that a process of sweep flocculation, where oocysts are engulfed in flocs during coagulation and floc formation, is the more likely mechanism involved. By comparison, it is likely that the specific interaction of hydrolysed aluminium species with the oocysts surface would result in a stronger link at the oocyst-floc interface and that the flocculation process may initially proceed via charge neutralisation.

Characterization of the antiapoptotic (p35) gene homologue of Spodoptera litura nucleopolyhedrosis virus (SlNPV).

Antiapoptotic genes of baculoviruses have been shown to prevent virus induced apoptosis in insect cells. Dot blot and Southern hybridizations of EcoRI genomic library and genomic digests of Spodoptera litura nucleopolyhedrosis virus (SlNPV) respectively give strong hybridization signals with antiapoptotic DNA (p35 gene) probe of the prototype Autographa californica nucleopolyhedrosis virus (AcNPV). Both the hybridizations indicate the presence of a homologous gene in the 1.8 kb EcoRI-Y fragment of SlNPV. The sequence of 1.244 kb region of this fragment encompasses an open reading frame coding for a polypeptide of 296 amino acids under sequential early (TATA) and late (TAAG) promoter motifs like that in other baculovirus p35 genes. The putative SlNPV p35 ORF expresses abundantly as a 35 kDa protein in Spodopterafrugiperda (Sf9) cells when allowed to express under the polyhedrin promoter of AcNPV.

Sequences of Citrus tristeza virus separated in time and space are essentially identical.

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

A point mutation at the Miniature1 seed locus reduces levels of the encoded protein, but not its mRNA, in maize.

We report here on the molecular nature of an EMS-induced mutant, mn1-89, a leaky semidominant allele of the Miniature1 (Mn1) seed locus that encodes a seed-specific cell wall invertase, INCW2. The mn1-89 locus specifies normal levels of the Incw2 transcript but extremely low levels (about 6% of normal) of the protein and enzyme activity are expressed. Sequence analysis of Incw2 clones derived from the parental Mn1 and the mutant genotypes shows a C to T transition in the mn1-89 allele, leading to a single amino acid alteration (proline to leucine) near the C-terminus of the mutant INCW2 protein. Although this change is not in the catalytic domain, putative N-glycosylation sites, or the beta-fructosidase motif, it does lie in a motif that is well conserved among all plant invertases and related fructosyltransferases. On the basis of these genetic in planta data, we believe we have identified a proline residue in a hitherto unknown GPFG motif as critical for the stability of such proteins. The single base change (C to T) also leads to the elimination of a BglI restriction site in the mutant allele. Indeed, BglI restriction digests of genomic DNAs from mn1-89 and Mn1 genotypes show one and two fragments, respectively. Sequence analysis of RT-PCR-derived endosperm Incw clones from mn1-1 (the reference allele) seeds predict five amino acid substitutions relative to Mn1. Whether or not these sequences are encoded by the mn1-1 locus or another non-allelic Incw gene in the maize genome remains to be elucidated.

Dentofacial morphology and upper respiratory function in 8-10-year-old children.

OBJECTIVES: The aim of this preliminary study was to assess the nature of associations between selected dentofacial morphological variables and respiratory mode as measured by percent nasality (%N) as part of an ongoing longitudinal study. DESIGN: Cross-sectional cohort study. SETTING AND SAMPLE POPULATION: The Pediatric Clinical Study Center, Children's Hospital, Columbus, OH. Ninety-eight normal children were tested. EXPERIMENTAL VARIABLE: Normal variation in %N. OUTCOME MEASURE: Selected dentofacial morphological variables including total and lower anterior face heights, face width, and palatal arch width and %N were estimated. RESULTS: Small associations between morphologic features and respiratory mode were found, but none were statistically significant. CONCLUSION: No evidence exists for the classic association between 'mouth breathing' and the stereotype of the 'adenoid facies'.