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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A rapid slid coagglutination test-an alternative to the fluorescent antibody test for the identification of Neisseria gonorrhoeae.

The Phadebact(R) Gonococcus Test, a slide coagglutination test, was compared with the Difco fluorescent antibody test for the identification of Neisseria gonorrhoeae isolated from 18- to 24-hour primary plates. A total of 316 morphologically characteristic, oxidase-positive, Gram-negative diplococci were tested. Altogether 298 isolates were identified definitively as N. gonorrhoeae by a rapid carbohydrate utilisation test; 287 of the 298 isolates of N. gonorrhoeae were identified by the coagglutination test, a sensitivity of 96%. The sensitivity of the fluorescent antibody test was 85% (254 of 298 isolates). False-positive results due to cross-reactions with non-gonococcal Neisseria were uncommon (1 of 18 non-gonococcal isolates in the coagglutination test, a specificity of 94%; 2 of 18 in the fluorescent antibody test, a specificity of 88%). None of the 14 other contaminant organisms seen frequently on primary isolation media gave positive reactions. Interpretation of the coagglutination test proved to be difficult initially. Thirty-two (10%) coagglutination tests had to be repeated; 3 of the 32 (1% of the total isolates tested) remained uninterpretable.

Course of bipolar disorder in eastern India.

The life course of affective episodes was determined for 95 consecutively admitted patients from eastern India fulfilling RDC criteria for definite mania during the current episode, using SADS-L interviews. There was a significantly greater frequency of manic compared to depressive relapses. Presentation as recurrent mania was very common. The total numbers of affective and manic episodes were significantly higher among those with recurrent mania and in cases where the first illness episode was manic.

Susceptibility of Campylobacter jejuni to twenty-three antimicrobial agents.

Twenty-three antimicrobial agents, including 4 new broadspectrum beta-lactam antibiotics were tested against 50 clinical isolates of Campylobacter jejuni. The activity of metabolites of metronidazole and tinidazole was also tested. Minimum inhibitory concentrations (MIC) were determined by agar dilution. beta-lactamase production was detected by a chromogenic cephalosporin method. All strains were susceptible to erythromycin, clindamycin, chloramphenicol, furazolidone, aminoglycosides (including gentamicin), cefotaxime and NF-thienamycin. All isolates were resistant to penicillin, cephalothin, cefoperazone, vancomycin, rifampicin and trimethoprim; beta-lactamase was detected in 2% of isolates. Some strains were resistant and others sensitive to the other drugs tested, which included ampicillin, moxalactum tetracycline, metronidazole and tinidazole. The 'hydroxy' metabolites of metronidazole and tinidazole were more active than the parent compounds.

In vitro antibacterial activity of enoxacin (CI-919).

Antibacterial activity of enoxacin was evaluated against more than 3,700 clinical isolates using the agar-dilution method and an inoculum of 10(4)-10(5) cells per site. For comparison other antibiotics appropriate for each species were also included. For most enterobacteria and for Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the MIC90 of enoxacin was below 2 mg/l. Serratia marcescens was more resistant; the MIC90 being 4 mg/ml. Enoxacin also showed high activity against Campylobacter jejuni and Neisseria gonorrhoeae. Streptococci were comparatively resistant, 32 mg/l to 64 mg/l of the compound being required to inhibit 90% of strains.

Flow cytometry and RT-PCR for rotavirus detection in artificially seeded oyster meat.

A flow cytometry (FC)-based method was developed for the detection of rotavirus in oyster meat using simian rotavirus SA11 as a model. To study virus recovery, oyster meat was injected with rotavirus and the oyster extract used to infect MA104 cell monolayers. Following varying periods of infection, the cells were recovered and reacted with the monoclonal antibody M60 which is specific for the rotavirus group A serotypes 1-4 outer capsid protein, VP7, followed by a second antibody (anti mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells as well as the level of infection. To evaluate the sensitivity of the method, non-inoculated oysters were processed following the same extraction protocol and, at the end, they were seeded with the same amount of virus used for oyster inoculation. This seeded oyster extract was then used to infect MA104 cells and the number of infected cells determined using the same FC procedure. A semi-nested two-step PCR for detection of rotavirus nucleic acid was undertaken to compare the sensitivity of FC with RT-PCR. Using FC, as little as 0.02 flow cytometry units (fcu) (number of infected cells counted by FC) could be detected after 72 h of cell infection. This is a very similar limit of sensitivity to that obtained with RT-PCR. Both methods are approximately 100 times more sensitive than the plaque-forming units (pfu) assay.

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.

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.

Ictal confusion--an electroclinical syndrome report of three adult cases with unusual features.

Ictal confusion--an electro-clinical syndrome consisting of varying degrees of confusion with an EEG correlate of seizure activity, abolished by intravenous diazepam, is reported in three adult patients. The unusual features were 'de novo' occurrence in adults, a chronic course, focal neurological deficits and a poor response to anticonvulsants.

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.

Prevalence of atypical mycobacteria in sputum of patients undergoing treatment at a tuberculosis clinic.

A total of 4,554 sputum specimens of 3,943 patients were examined with a view to isolate atypical mycobacteria. Of these 604 (13.3%) specimens were culture positive for AFB, out of which 48 cultures (7.9%) from 47 patients were identified as atypical mycobacteria. Two patients were considered as probable cases of atypical mycobacterial disease while in the remaining 45 patients, atypical mycobacteria appeared to colonize the lungs without producing any disease.