Crystal structure and biophysical characterization of IspD from Burkholderia thailandensis and Mycobacterium paratuberculosis.

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13-19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate Tm values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.

Undifferentiated Hepatic Sarcoma: A Rare Case.

Primary hepatic undifferentiated pleomorphic sarcoma (UPS) is a rare malignant mesenchymal tumor with a nonspecific clinical and radiologic presentation. Primary hepatic UPS is often a diagnosis of exclusion made by multiple immunohistological testing that rules out hepatic, hematologic, neural, and epithelial origin. Stains for mesenchymal origin are usually the only positive stain and do not demonstrate evidence of specific mesenchymal cell differentiation. We report a case of a 56-year-old male with no significant past medical history that presented with complaint of epigastric abdominal pain of six months duration. A computed tomography (CT) scan of the abdomen and pelvis exhibited numerous hepatic masses involving right and left hepatic lobe. A CT-guided core needle biopsy discovered undifferentiated/pleomorphic sarcoma. Histomorphology showed spindle cell neoplasm without recognizable hepatic tissue. Immunohistochemistry (IHC) stains were positive for smooth muscle actin (SMA) but failed to establish a more specific histogenesis. Furthermore, IHC stains revealed spindle neoplastic cells with focal and patchy positive h-caldesmon (approximately 10-15% of neoplastic cells), and negative for desmin. Given these results, the diagnosis of undifferentiated/pleomorphic sarcoma was established. It is imperative to consider UPS in the differential diagnosis of large liver lesions without evidence of differentiation. Early identification of this rare tumor can prevent the possibility of distant metastasis and improve survival among patients.

The role of emptying services in provision of safely managed sanitation: A classification and quantification of the needs of LMICs.

Classifications for onsite sanitation in terms of facility type (septic tanks, pit latrines) exist, but connecting these facilities to the wider sanitation value chain via improved containment, emptying, and collection has not been well explored. Using existing Joint Monitoring Programme facility classifications and secondary data on piped water access, a Service Typology was developed to classify and quantify the primary emptying service needs of household level onsite sanitation facilities. Facilities in six Sustainable Development Goal (SDG) regions were classified as Emptiable (faecal sludge can be removed either via Mechanized or Non-Mechanized means) or Unemptiable. Of the 722 million household level sanitation facilities assessed in these regions, 32% were found to be emptiable via Mechanized means, 50% via Non-Mechanized means and 18% were found to be Unemptiable pits. The volume (by number of facilities) and density (as a proportion of the full population) of each service type were estimated by SDG region and by country. Results from this study provide background data on the role of emptying sanitation facilities in achieving SDG6, and can be incorporated into investment priorities, policy framing, technology development, infrastructure development, and targeted behaviour change strategies.

Changes in DNA methylation profiles of myalgic encephalomyelitis/chronic fatigue syndrome patients reflect systemic dysfunctions.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a lifelong debilitating disease with a complex pathology not yet clearly defined. Susceptibility to ME/CFS involves genetic predisposition and exposure to environmental factors, suggesting an epigenetic association. Epigenetic studies with other ME/CFS cohorts have used array-based technology to identify differentially methylated individual sites. Changes in RNA quantities and protein abundance have been documented in our previous investigations with the same ME/CFS cohort used for this study. RESULTS: DNA from a well-characterised New Zealand cohort of 10 ME/CFS patients and 10 age-/sex-matched healthy controls was isolated from peripheral blood mononuclear (PBMC) cells, and used to generate reduced genome-scale DNA methylation maps using reduced representation bisulphite sequencing (RRBS). The sequencing data were analysed utilising the DMAP analysis pipeline to identify differentially methylated fragments, and the MethylKit pipeline was used to quantify methylation differences at individual CpG sites. DMAP identified 76 differentially methylated fragments and Methylkit identified 394 differentially methylated cytosines that included both hyper- and hypo-methylation. Four clusters were identified where differentially methylated DNA fragments overlapped with or were within close proximity to multiple differentially methylated individual cytosines. These clusters identified regulatory regions for 17 protein encoding genes related to metabolic and immune activity. Analysis of differentially methylated gene bodies (exons/introns) identified 122 unique genes. Comparison with other studies on PBMCs from ME/CFS patients and controls with array technology showed 59% of the genes identified in this study were also found in one or more of these studies. Functional pathway enrichment analysis identified 30 associated pathways. These included immune, metabolic and neurological-related functions differentially regulated in ME/CFS patients compared to the matched healthy controls. CONCLUSIONS: Major differences were identified in the DNA methylation patterns of ME/CFS patients that clearly distinguished them from the healthy controls. Over half found in gene bodies with RRBS in this study had been identified in other ME/CFS studies using the same cells but with array technology. Within the enriched functional immune, metabolic and neurological pathways, a number of enriched neurotransmitter and neuropeptide reactome pathways highlighted a disturbed neurological pathophysiology within the patient group.

A granular activated carbon/electrochemical hybrid system for onsite treatment and reuse of blackwater.

Over 1/3 of the global population lacks access to improved sanitation, leading to disease, death, and impaired economic development. Our group is working to develop rapidly deployable, cost-effective, and sustainable solutions to this global problem that do not require significant investments in infrastructure. Previously, we demonstrated the feasibility of a toilet system that recycles blackwater for onsite reuse as flush water, in which the blackwater is electrochemically treated to remove pathogens due to fecal contamination. However, this process requires considerable energy (48-93 kJ/L) to achieve complete disinfection of the process liquid, and the disinfected liquid retains color and chemical oxygen demand (COD) in excess of local discharge standards, negatively impacting user acceptability. Granular activated carbon (GAC) efficiently reduces COD in concentrated wastewaters. We hypothesized that reduction of COD with GAC prior to electrochemical treatment would both improve disinfection energy efficiency and user acceptability of the treated liquid. Here we describe the development and testing of a hybrid system that combines these technologies and demonstrate its ability to achieve full disinfection with improved energy efficiency and liquid quality more suitable for onsite reuse and/or discharge.

Improving energy efficiency of electrochemical blackwater disinfection through sequential reduction of suspended solids and chemical oxygen demand.

Onsite reuse of blackwater requires removal of considerable amounts of suspended solids and organic material in addition to inactivation of pathogens. Previously, we showed that electrochemical treatment could be used for effective pathogen inactivation in blackwater, but was inadequate to remove solids and organics to emerging industry standards. Further, we found that as solids and organics accumulate with repeated recycling, electrochemical treatment becomes less energetically sustainable. Here, we describe a pilot study in which concentrated blackwater is pretreated with ultrafiltration and granular activated carbon prior to electrochemical disinfection, and show that this combination of treatments removes 75-99% of chemical oxygen demand, 92-100% of total suspended solids, and improves the energy efficiency of electrochemical blackwater treatment by an order of magnitude.

Early postpartum pup preference is altered by gestational cocaine treatment: associations with infant cues and oxytocin expression in the MPOA.

Cross-fostering studies suggest cocaine-induced deficits in maternal behavior could be associated with altered behavior of offspring following prenatal cocaine-exposure. Neonatal vocalizations are an important offspring cue facilitating early interactions between dam and rodent pup offspring and have been shown to be altered following prenatal cocaine-exposure. It is unclear how variations in acoustic parameters of USVs impact maternal behavior and the mechanism(s) underlying these processes. The present study examined differences in cocaine-exposed and control rodent dam maternal preference of cocaine-exposed or untreated pups in a dual choice apparatus. Relationship of preference-like behavior with pup USVs and dam oxytocin expression was explored. Gestational cocaine-exposure interfered with preference-like behavior of dams on postpartum day 1 with cocaine-exposure associated with decreased time spent on the cocaine-exposed pup side compared to the control pup side, and decreases in preference-like behavior associated in part with decreased number of USVs being emitted by cocaine-exposed pups. On postpartum day 5, decreased oxytocin expression in the medial preoptic area was associated with altered preference-like behavior in cocaine-exposed dams, including frequency and latency to touch/sniff pups. Results indicate cocaine's effects on the mother-infant relationship is likely synergistic, in that cocaine influences mother and offspring both independently and concertedly and that variations within pup vocalizations and the oxytocin system may be potential mechanism(s) underlying this synergistic relationship during the postpartum period.

Evaluation of water quality threats to the endangered Okaloosa darter (Etheostoma okaloosae) in East Turkey Creek on Eglin Air Force Base.

The threatened Okaloosa darter (Etheostoma okaloosae) is found almost exclusively on the Eglin Air Force Base in the Choctawhatchee Bay watershed of Florida. Portions of this limited habitat are threatened with soil erosion, altered hydrology, and impaired water quality. In the present study, general water quality parameters (i.e., dissolved oxygen, specific conductance, pH, temperature, relative turbidity, and primary productivity) were characterized in East Turkey Creek, which is a body of water potentially impacted by treated wastewater sprayfields, and Long Creek, an adjacent reference stream that does not border the sprayfields. Water quality was assessed during a 30-day exposure using passive samplers for both non-polar and polar effluent parameters. Because the Okaloosa darter was listed as endangered at the time of sampling we chose a closely related species from the same creeks, the sailfin shiner (Pteronotropis hypseleotris) in which to measure metal body burdens. Additionally, fathead minnows (Pimephales promelas) were used for microarray analysis on gonad and liver tissues after 48 h exposures to water collected from the two creeks and brought into the laboratory. Waters from all sites, including reference sites, affected the expression of genes related to various biological processes including transcription and translation, cell cycle control, metabolism, and signaling pathways, suggesting that the sum of anthropogenic compounds in the site waters may cause a generalized stress response in both liver and testis, an effect that could be related to the generally low populations of the Okaloosa darter. Furthermore, effects of site waters on fish gene expression may be related to the impact of human activities other than the wastewater sprayfields, as nearby areas are closed to the public for military testing, training, and administrative activities and due to ordnance contamination.

Evolution of virulence in Fusarium oxysporum f. sp. vasinfectum using serial passage assays through susceptible cotton.

Fifty strains of Fusarium oxysporum, recovered from rhizosphere soil around native Gossypium species and found to be mildly virulent on cotton (Gossypium hirsutum), were used to assay the propensity for evolution of virulence using serial passage assays through cotton. Only one lineage A strain, 2613, successfully completed 10 successive passages, while all others lost the ability to cause foliar disease symptoms at various stages during this process. Based on 46 amplified fragment length polymorphism (AFLP) markers generated with four EcoRI x MseI primer combinations, mutants were identified in offspring isolates from strain 2613 regardless of whether serial passages occurred in cotton or on water agar, suggesting the occurrence of spontaneous mutations. Significantly increased virulence was observed in the offspring isolates generated on cotton, while no increasing virulence was found in those obtained on water agar, suggesting that the evolution of virulence in F. oxysporum f. sp. vasinfectum is associated with the presence of cotton. No clear correlation was observed between the AFLP mutations and increased virulence in this study.

Long-term regulation of N-methyl-D-aspartate receptor subunits and associated synaptic proteins following hippocampal synaptic plasticity.

Synaptic plasticity in the dentate gyrus is dependent on activation of the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors. In this study, we show that synaptic plasticity in turn regulates NMDA receptors, since subunits of the NMDA receptor complex are bidirectionally and independently regulated in the dentate gyrus following activation of perforant synapses in awake animals. Low-frequency stimulation that produced a mild synaptic depression resulted in a decrease in the NMDA receptor subunits NR1 and NR2B 48 h following stimulation. High-frequency stimulation that produced long-term potentiation resulted in an increase in NR1 and NR2B at the same time point. Further investigations revealed that in contrast to NR2B, NR1 levels increased gradually after long-term potentiation induction, reaching a peak level at 48 h, and were insensitive to the competitive NMDA receptor antagonist 3-3(2-carboxypiperazin-4-yl) propyl-1-phosphate. The increased levels of NR1 and NR2B at 48 h were found associated with synaptic membranes and with increased NMDA receptor-associated proteins, postsynaptic density protein 95, neuronal nitric oxide synthase and Ca(2+)/calmodulin-dependent protein kinase II, alpha subunit. These data suggest that the persistence of long-term potentiation is associated with an increase in the number of NMDA receptor complexes, which may be indicative of an increase in synaptic contact area.

A dynamic competition between release factor 2 and the tRNA(Sec) decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H.

Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.

Heterosynaptic metaplasticity in the hippocampus in vivo: a BCM-like modifiable threshold for LTP.

The homeostatic maintenance of the "modification threshold" for inducing long-term potentiation (LTP) is a fundamental feature of the Bienenstock, Cooper, and Munro (BCM) model of synaptic plasticity. In the present study, two key features of the modification threshold, its heterosynaptic expression and its regulation by postsynaptic neural activity, were tested experimentally in the dentate gyrus of awake, freely moving rats. Conditioning stimulation ranging from 10 to 1,440 brief 400-Hz trains, when applied to medial perforant path afferents, raised the threshold for LTP induction heterosynaptically in the neighboring lateral perforant path synapses. This effect recovered slowly over a 7- to 35-day period. The same conditioning paradigms, however, did not affect the reversal of long-term depression. The inhibition of LTP by medial-path conditioning stimulation was N-methyl-D-aspartate (NMDA) receptor-dependent, but antidromic stimulation of the granule cells could also inhibit lateral path LTP induction, independently of NMDA receptor activation. Increased calcium buffering is a potential mechanism underlying the altered LTP threshold, but the levels of two important calcium-binding proteins did not increase after conditioning stimulation, nor was de novo protein synthesis required for generating the threshold shift. These data confirm, in an in vivo model, two key postulates of the BCM model regarding the LTP threshold. They also provide further evidence for the broad sensitivity of synaptic plasticity mechanisms to the history of prior activity, i.e., metaplasticity.

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli.

The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.

Release factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis.

The decoding of stop signals in mRNA requires protein release factors. Two classes of factor are found in both prokaryotes and eukaryotes, a decoding factor and a stimulatory recycling factor. These factors form complexes at the active centre of the ribosome and mimic in overall shape the complexes found at other stages of protein synthesis. The decoding release factor is shaped like a tRNA and has a domain for codon recognition at the decoding site of the ribosome, and a domain for peptidyl-tRNA hydrolysis that is inferred to be near the peptidyltransferase centre. Initial interaction of the decoding factor with the ribosome is a low fidelity event involving multiple contacts with the ribosomal components. A subsequent discrimination step, at present poorly defined, ensures high fidelity of codon recognition.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation.

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-1 DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP.

Functional characterization of yeast mitochondrial release factor 1.

The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.

Metabotropic glutamate receptors trigger homosynaptic protein synthesis to prolong long-term potentiation.

We investigated the mechanisms by which previous "priming" activation of group I metabotropic glutamate receptors (mGluRs) facilitates the persistence of long-term potentiation (LTP) in area CA1 of rat hippocampal slices. Priming of LTP was elicited by either pharmacological or synaptic activation of mGluRs before a weak tetanic stimulus that normally produced only a rapidly decaying phase of LTP that did not involve protein synthesis or mGluRs. Pharmacological priming of LTP persistence by a selective group I mGluR agonist was blocked by an inhibitor of group I mGluRs and by inhibitors of translation, but not by a transcriptional inhibitor. The same mGluR agonist increased (35)S-methionine incorporation into slice proteins. LTP could also be facilitated using a synaptic stimulation priming protocol, and this effect was similarly blocked by group I mGluR and protein synthesis inhibitors. Furthermore, using a two-pathway protocol, the synaptic priming of LTP was found to be input-specific. To test for the contribution of group I mGluRs and protein synthesis to LTP in nonprimed slices, a longer duration control tetanization protocol was used to elicit a more slowly decaying form of LTP than did the weak tetanus used in the previous experiments. The persistence of the LTP induced by this stronger tetanus was dependent on mGluR activation and protein synthesis but not on transcription. Together, these results suggest that mGluRs couple to nearby protein synthesis machinery to homosynaptically regulate an intermediate phase of LTP dependent on new proteins made from pre-existing mRNA.

The ribosomal binding and peptidyl-tRNA hydrolysis functions of Escherichia coli release factor 2 are linked through residue 246.

Replacing a cassette of 31 residues from Escherichia coli release factor 1 with the equivalent residues in release factor 2 gave a protein active in codon-specific binding to the ribosome but inactive in peptidyl-tRNA hydrolysis. Such a phenotype is also found unexpectedly with release factor 2 when expressed at high concentration in bacteria. Substituting threonine with the release factor 1 equivalent serine at position 246 within the cassette restored the impaired activity of the chimeric protein, and also that of inactive recombinant release factor 2, both in vitro and in vivo. The differences in activity are not due to posttranslational modifications or a lack of it at this residue. Random mutagenesis of codon 246 suggests that this position is pivotal for the function of the release factor, being able to affect differentially both its binding to the ribosome and its peptide release activities. We propose that amino acid 246 is close to a sharp turn (GGQ motif at position 250), and is essential for transmitting the signal from cognate codon recognition by correctly positioning the peptidyl-tRNA hydrolysis domain of the release factor into the peptidyltransferase center.

Effect of finishing and polishing procedures on the surface roughness of packable composites.

This study examined the average surface roughness (Ra, micron) of three packable composites and one hybrid composite cured against mylar, before and after treatment with a fine finishing diamond bur, a resin finisher followed by fine and extrafine polishing paste, two silicone-based finishing and polishing systems, fine and super-fine aluminum-oxide polishing disks, a silicon carbide-impregnated polishing brush and a surface-penetrating composite sealant. Additionally, the Ra was examined for one of the packable composites before and after treatment with a finishing carbide, prior to the finishing and polishing procedures detailed above. The finishing diamond significantly increased the Ra for all composites (ALERT, SureFil, Solitaire and Z-100). The finishing carbide used with SureFil (SureFil + C) also increased the Ra; however, it also produced surfaces up to 3.5x smoother when compared to SureFil surfaces finished with the diamond. Overall, Sof-Lex Contouring and Polishing Discs were able to produce the smoothest surfaces, followed by the Jiffy Composite Polishing Cups, the Enhance Composite Finishing & Polishing System/Prisma-Gloss Composite Polishing Paste, the Diacomp Intra-Oral Composite Polishers and the Jiffy Composite Polishing Brushes, respectively. The smoothest surfaces were produced using Z-100, followed by SureFil + C (carbide finishing bur), Solitaire, SureFil and ALERT, respectively. In general, Protect-It Composite Surface Sealant had little effect on the Ra, except with ALERT, where a slight increase in Ra was observed.