Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase

DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D211) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain. The novel properties of this amino acid substitution led us to express the WT and D211N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the kpol was reduced by a factor of 223 in the mutant, although the Kd for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn2+ over Mg2+. To better understand the functions of this residue within the Ty1 RT active site, we have now examined the in vitro properties of WT and D211N mutant Ty1 RTs in carrying out pyrophosphorolysis, the reverse reaction to polymerization, where pyrophosphate is the substrate and dNTPs are the product. We find that pyrophosphorolysis is efficient only when the base-paired primer template region is >14 bases, and that activity increases when the primer end is blunt-ended or recessed by only a few bases. Using pre-steady state kinetic analysis, we find that the rate of pyrophosphorolysis (kpyro) in the D211N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent Kd for pyrophosphate. These findings indicate that subtle substrate differences can strongly affect the enzyme's ability to properly position the primer-end to carry out pyrophosphorolysis. Further the kinetic data suggests that the D211 residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D211N mutant's transposition defect

Microdroplet-Enabled Highly Parallel Co-Cultivation of Microbial Communities

Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying “unculturable” microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to ∼1,400 in a 10 mm×5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community

Assessment of the medicinal potentials of the methanol extracts of the leaves and stems of Buddleja saligna

<p>Abstract</p> <p>Background</p> <p><it>Buddleja saligna </it>Willd (Loganiaceae) is a small to medium-sized evergreen tree; trunk short, often gnarled and crooked; crown dense, rounded or domed-shaped; foliage greyish green. The wild olives are traditionally used to lower blood pressures in many parts of the world. In southern Africa, bark and leaf decoctions are used to treat colic, coughs, colds, sore eyes, urinary problems and as purgatives.</p> <p>Methods</p> <p>The antibacterial, antioxidant activities and phenolic contents of the methanol extracts of the leaves and stems of <it>Buddleja saligna </it>were evaluated using <it>in vitro </it>standard methods. Spectrophotometry was the basis for the determinations of total phenol, total flavonoids, flavonols, and proanthocyanidins. Tannins, quercetin and catechin equivalents were used for these parameters. The antioxidant activities of the leaves and stem extracts of <it>Buddleja saligna </it>were determined by ABTS, DPPH, and ferrous reducing antioxidant property (FRAP) methods. Laboratory isolates of 10 bacteria species which included five Gram-positive and five Gram-negative strains were used to assay for antibacterial activity of this plant.</p> <p>Results</p> <p>The antioxidant activities of the leaves as determined by the ABTS and DPPH were similar to that of the stem. The flavonoids and the flavonols contents of the leaves were higher than that of the stem but the total phenols, proanthocyanidins and FRAP activities were higher in the methanol extracts of the stem. The extracts did show activity against both Gram-positive and Gram-negative bacteria. For instance, while the methanol extract of the leaves showed good activities on all the organisms except <it>Serratia marcescens </it>and <it>Pseudomonas aeruginosa </it>at MICs of between 2.5 and 5.0 mg/ml, the extract of the stem only showed activities on <it>Bacillus cereus, Streptococcus pyrogens </it>and <it>Pseudomonas aeruginosa </it>at the same concentration.</p> <p>Conclusion</p> <p>The results from this study indicate that the leaves and stem extracts of <it>Buddleja saligna </it>possess antioxidant properties and could serve as free radical inhibitors or scavenger or, acting possibly as primary antioxidants. Although, the antibacterial properties of <it>Buddleja saligna </it>are not as effective as the standard drugs-Chloramphenicol and Streptomycin, they still possess some activity against bacterial strains used in this study. <it>Buddleja saligna </it>may therefore be a good candidate for functional foods as well as pharmaceutical plant-based products.</p

Autism diagnosis differentiates neurophysiological responses to faces in adults with tuberous sclerosis complex

- Background: Autism spectrum disorder (ASD) is a common and highly heritable neurodevelopmental disorder that is likely to be the outcome of complex aetiological mechanisms. One strategy to provide insight is to study ASD within tuberous sclerosis complex (TSC), a rare disorder with a high incidence of ASD, but for which the genetic cause is determined. Individuals with ASD consistently demonstrate face processing impairments, but these have not been examined in adults with TSC using event-related potentials (ERPs) that are able to capture distinct temporal stages of processing. - Methods: For adults with TSC (n = 14), 6 of which had a diagnosis of ASD, and control adults (n = 13) passively viewed upright and inverted human faces with direct or averted gaze, with concurrent EEG recording. Amplitude and latency of the P1 and N170 ERPs were measured. - Results: Individuals with TSC + ASD exhibited longer N170 latencies to faces compared to typical adults. Typical adults and adults with TSC-only exhibited longer N170 latency to inverted versus upright faces, whereas individuals with TSC + ASD did not show latency differences according to face orientation. In addition, individuals with TSC + ASD showed increased N170 latency to averted compared to direct gaze, which was not demonstrated in typical adults. A reduced lateralization was shown for the TSC + ASD groups on P1 and N170 amplitude. - Conclusions: The findings suggest that individuals with TSC + ASD may have similar electrophysiological abnormalities to idiopathic ASD and are suggestive of developmental delay. Identifying brain-based markers of ASD that are similar in TSC and idiopathic cases is likely to help elucidate the risk pathways to ASD

Subliminal Semantic Priming in Speech

Numerous studies have reported subliminal repetition and semantic priming in the visual modality. We transferred this paradigm to the auditory modality. Prime awareness was manipulated by a reduction of sound intensity level. Uncategorized prime words (according to a post-test) were followed by semantically related, unrelated, or repeated target words (presented without intensity reduction) and participants performed a lexical decision task (LDT). Participants with slower reaction times in the LDT showed semantic priming (faster reaction times for semantically related compared to unrelated targets) and negative repetition priming (slower reaction times for repeated compared to semantically related targets). This is the first report of semantic priming in the auditory modality without conscious categorization of the prime

Prognostic value of cortically induced motor evoked activity by TMS in chronic stroke: caveats from a very revealing single clinical case

Background: We report the case of a chronic stroke patient (62 months after injury) showing total absence of motor activity evoked by transcranial magnetic stimulation (TMS) of spared regions of the left motor cortex, but near-to-complete recovery of motor abilities in the affected hand. Case presentation: Multimodal investigations included detailed TMS based motor mapping, motor evoked potentials (MEP), and Cortical Silent period (CSP) as well as functional magnetic resonance imaging (fMRI) of motor activity, MRI based lesion analysis and Diffusion Tensor Imaging (DTI) Tractography of corticospinal tract (CST). Anatomical analysis revealed a left hemisphere subinsular lesion interrupting the descending left CST at the level of the internal capsule. The absence of MEPs after intense TMS pulses to the ipsilesional M1, and the reversible suppression of ongoing electromyographic (EMG) activity (indexed by CSP) demonstrate a weak modulation of subcortical systems by the ipsilesional left frontal cortex, but an inability to induce efficient descending volleys from those cortical locations to right hand and forearm muscles. Functional MRI recordings under grasping and finger tapping patterns involving the affected hand showed slight signs of subcortical recruitment, as compared to the unaffected hand and hemisphere, as well as the expected cortical activations. Conclusions: The potential sources of motor voluntary activity for the affected hand in absence of MEPs are discussed. We conclude that multimodal analysis may contribute to a more accurate prognosis of stroke patients

Eating disorders: the current status of molecular genetic research

Anorexia nervosa (AN) and bulimia nervosa (BN) are complex disorders characterized by disordered eating behavior where the patient’s attitude towards weight and shape, as well as their perception of body shape, are disturbed. Formal genetic studies on twins and families suggested a substantial genetic influence for AN and BN. Candidate gene studies have initially focused on the serotonergic and other central neurotransmitter systems and on genes involved in body weight regulation. Hardly any of the positive findings achieved in these studies were unequivocally confirmed or substantiated in meta-analyses. This might be due to too small sample sizes and thus low power and/or the genes underlying eating disorders have not yet been analyzed. However, some studies that also used subphenotypes (e.g., restricting type of AN) led to more specific results; however, confirmation is as yet mostly lacking. Systematic genome-wide linkage scans based on families with at least two individuals with an eating disorder (AN or BN) revealed initial linkage regions on chromosomes 1, 3 and 4 (AN) and 10p (BN). Analyses on candidate genes in the chromosome 1 linkage region led to the (as yet unconfirmed) identification of certain variants associated with AN. Genome-wide association studies are under way and will presumably help to identify genes and pathways involved in these eating disorders. The elucidation of the molecular mechanisms underlying eating disorders might improve therapeutic approaches

Subliminal Semantic Priming in Speech

Numerous studies have reported subliminal repetition and semantic priming in the visual modality. We transferred this paradigm to the auditory modality. Prime awareness was manipulated by a reduction of sound intensity level. Uncategorized prime words (according to a post-test) were followed by semantically related, unrelated, or repeated target words (presented without intensity reduction) and participants performed a lexical decision task (LDT). Participants with slower reaction times in the LDT showed semantic priming (faster reaction times for semantically related compared to unrelated targets) and negative repetition priming (slower reaction times for repeated compared to semantically related targets). This is the first report of semantic priming in the auditory modality without conscious categorization of the prime

The nasal cavity microbiota of healthy adults

Abstract Background The microbiota of the nares has been widely studied. However, relatively few studies have investigated the microbiota of the nasal cavity posterior to the nares. This distinct environment has the potential to contain a distinct microbiota and play an important role in health. Results We obtained 35,142 high-quality bacterial 16S rRNA-encoding gene sequence reads from the nasal cavity and oral cavity (the dorsum of the tongue and the buccal mucosa) of 12 healthy adult humans and deposited these data in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) (Bioproject: PRJNA248297). In our initial analysis, we compared the bacterial communities of the nasal cavity and the oral cavity from ten of these subjects. The nasal cavity bacterial communities were dominated by Actinobacteria, Firmicutes, and Proteobacteria and were statistically distinct from those on the tongue and buccal mucosa. For example, the same Staphylococcaceae operational taxonomic unit (OTU) was present in all of the nasal cavity samples, comprising up to 55% of the community, but Staphylococcaceae was comparatively uncommon in the oral cavity. Conclusions There are clear differences between nasal cavity microbiota and oral cavity microbiota in healthy adults. This study expands our knowledge of the nasal cavity microbiota and the relationship between the microbiota of the nasal and oral cavities.http://deepblue.lib.umich.edu/bitstream/2027.42/109547/1/40168_2014_Article_56.pd

Remodeling of Purinergic Receptor-Mediated Ca2+ Signaling as a Consequence of EGF-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis