Polyvinyl alcohol-montmorillonite composites for water purification: Analysis of clay mineral cation exchange and composite particle synthesis.

Municipal and residential water purification rely heavily on activated carbon (AC), but regeneration of AC is costly and cannot be performed at the point-of-use. Clay minerals (CMs) comprise a class of naturally abundant materials with known capacities for analyte adsorbance. However, the gel-forming properties of CMs in aqueous suspension pose problems for these materials being used in water-purification. In this study, we have taken three main steps to optimize the use of CMs in these applications. First, we produced several variants of montmorillonite CMs to evaluate the effect of interstitial cation hydrophobicity on the ability of the CM to uptake chargecarrying organic pollutants. These variants include CMs with the following cations: sodium, hexyl(triphenyl) phosphonium, hexyadecyl(triphenyl)phosphonium, and hexyl(tributyl)phosphonium. Second, we synthesized polymer-clay mineral composite films composed of polyvinyl alcohol (PVA), crosslinked in the presence of a CM variant. These films were evaluated for their ability to uptake malachite green (MG). Finally, we developed a one-pot synthetic method for the generation of polymer-clay particles for use in a continuous column process. We synthesized polymer-clay mineral particles using the highest performing CM (based on the film experiments) and evaluated the equilibrium capacity and kinetics of MG uptake from solution.

Mechanistic In Vitro Studies Indicate that the Clinical Drug-Drug Interaction between Telithromycin and Simvastatin Acid Is Driven by Time-Dependent Inhibition of CYP3A4 with Minimal Effect on OATP1B1.

A previous attempt to accurately quantify the increased simvastatin acid exposure due to drug-drug interaction (DDI) with coadministered telithromycin, using a mechanistic static model, substantially underpredicted the magnitude of the area under the plasma concentration-time curve ratio (AUCR) based on reversible inhibition of CYP3A4 and organic anion transporting polypeptide 1B1 (OATP1B1). To reconcile this disconnect between predicted and clinically observed AUCR, telithromycin was evaluated as a time-dependent inhibitor of CYP3A4 in vitro, as well as an inhibitor of OATP1B1. Telithromycin inhibited OATP1B1-mediated [3H]-estradiol 17ß-d-glucuronide (0.02 µM) transport with a mean IC50 of 12.0 ± 1.45 µM and was determined by IC50 shift and kinetic analyses to be a competitive reversible inhibitor of CYP3A4-mediated midazolam1- hydroxylation with a mean absolute inhibition constant (Ki) value of 3.65 ± 0.531 µM. The 2.83-fold shift in IC50 (10.4-3.68 µM) after a 30-minute metabolic preincubation confirmed telithromycin as a time-dependent inhibitor of CYP3A4; the mean inhibitor concentration that causes half-maximal inactivation of enzyme (KI) and maximal rate of inactivation of enzyme (kinact) values determined for inactivation were 1.05 ± 0.226 µM and 0.02772 ± 0.00272 min-1, respectively. After the integration of an enzyme time-dependent inhibition component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above, the newly predicted simvastatin acid AUCR (10.8 or 5.4) resulting from perturbation of its critical disposition pathways matched the clinically observed AUCR (10.8 or 4.3) after coadministration, or staggered administration, with telithromycin, respectively. These results indicate the time-dependent inhibition of CYP3A4 by telithromycin as the primary driver underlying its clinical DDI with simvastatin acid.

Human cytomegalovirus UL97 kinase alters the accumulation of CDK1.

The UL97 protein kinase is a serine/threonine kinase expressed by human cytomegalovirus (CMV) that phosphorylates ganciclovir. An investigation of the subcellular localization of pUL97 in infected cells indicated that, early in infection, pUL97 localized to focal sites in the nucleus that transitioned to subnuclear compartments and eventually throughout the entire nucleus. When UL97 kinase activity was eliminated with a K355M mutation or pharmacologically inhibited with maribavir, the expansion and redistribution of pUL97 foci within the nucleus was delayed, nuclear reorganization did not occur and assembly complexes in the cytoplasm failed to form normally. As UL97 kinase and its homologues appear to be functionally related to CDK1, a known regulator of nuclear structural organization, the effects of the UL97 kinase on CDK1 were investigated. Expression of CDK1 in infected cells appeared to be induced by UL97 kinase activity at the level of transcription and was not tied to other virus life-cycle events, such as viral DNA replication or virion assembly. These results suggest that, in addition to phosphorylating CDK1 targets, the UL97 kinase modifies G2/M cell-cycle checkpoint regulators, specifically CDK1, to promote virus replication.

The finished DNA sequence of human chromosome 12.

Human chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.

The DNA sequence, annotation and analysis of human chromosome 3.

After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

Human cytomegalovirus UL97 kinase prevents the deposition of mutant protein aggregates in cellular models of Huntington's disease and ataxia.

The presence of aggregates of abnormally expanded polyglutamine (polyQ)-containing proteins are a pathological hallmark of a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia-3 (SCA3). Previous studies in cellular, Drosophila, and mouse models of HD and SCA have shown that neurodegeneration can be prevented by manipulations that inhibit polyQ aggregation. We have shown that the UL97 kinase of the human cytomegalovirus (HCMV) prevents aggregation of the pp71 and pp65 viral tegument proteins. To explore whether UL97 may act as a general antiaggregation factor, we examined whether UL97 prevents aggregation of cellular non-polyQ and polyQ proteins. We report that UL97 prevents the deposition of aggregates of two non-polyQ proteins: a protein chimera (GFP170*) composed of the green fluorescent protein and a fragment of the Golgi Complex protein (GCP-170) and a chimera composed of the red fluorescent protein (RFP) fused to the Werner syndrome protein (WRN), a RecQ helicase and exonuclease involved in DNA repair. Furthermore, we show that UL97 inhibits aggregate deposition in cellular models of HD and SCA3. UL97 prevents the deposition of aggregates of the mutant huntingtin exon 1 containing 82 glutamine repeats (HttExon1-Q82) or full length ataxin-3 containing a 72 polyQ track (AT3-72Q). The kinase activity of UL97 appears critical, as the kinase-dead UL97 mutant (K335M) fails to prevent aggregate formation. We further show that UL97 disrupts nuclear PML bodies and decreases p53-mediated transcription. The universality of the antiaggregation effect of UL97 suggests that UL97 targets a key cellular factor that regulates cellular aggregation mechanisms. Our results identify UL97 as a novel means to modulate polyQ aggregation and suggest that UL97 can serve as a novel tool to probe the cellular mechanisms that contribute to the formation of aggregates in polyglutamine disorders.

Genome sequence of the Brown Norway rat yields insights into mammalian evolution.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Human cytomegalovirus UL97 kinase activity is required for the hyperphosphorylation of retinoblastoma protein and inhibits the formation of nuclear aggresomes.

Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinase-dependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding motif or the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.

Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir.

The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb) and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.

Comparison of vaccination with rhesus CMV (RhCMV) soluble gB with a RhCMV replication-defective virus deleted for MHC class I immune evasion genes in a RhCMV challenge model.

A human cytomegalovirus (HCMV) vaccine to prevent infection and/or reduce disease associated with congenital infection or visceral disease in transplant recipients is a high priority, but has remained elusive. We created a disabled infectious single cycle rhesus CMV (RhCMV) deleted for glycoprotein L (gL) and the MHC class I immune evasion genes Rh178 and Rh182-189, and restored its epithelial cell tropism by inserting the Rh128-131A genes. The resulting virus, RhCMVRΔgL/178/182-189, was used to vaccinate rhesus monkeys intramuscularly and was compared with vaccination of animals with soluble RhCMV glycoprotein B (gB) in alum/monophosphoryl lipid A or with PBS as a control. At 4 weeks after the second vaccination, an increased frequency of RhCMV-specific CD8 T cells was detected in animals vaccinated with the RhCMVRΔgL/178/182-189 vaccine compared to animals vaccinated with soluble gB. In contrast, monkeys vaccinated with soluble gB had 20-fold higher gB antibody titers than animals vaccinated with RhCMVRΔgL/178/182-189. Titers of neutralizing antibody to RhCMV infection of fibroblasts were higher in animals vaccinated with gB compared with RhCMVRΔgL/178/182-189. Following vaccination, monkeys were challenged subcutaneously with RhCMV UCD59, a low passage virus propagated in monkey kidney epithelial cells. All animals became infected after challenge; however, the frequency of RhCMV detection in the blood was reduced in monkeys vaccinated with soluble gB compared with those vaccinated with RhCMVRΔgL/178/182-189. The frequency of challenge virus shedding in the urine and saliva and the RhCMV copy number shed at these sites was not different in animals vaccinated with RhCMVRΔgL/178/182-189 or soluble gB compared with those that received PBS before challenge. Although the RhCMVRΔgL/178/182-189 vaccine was superior in inducing cellular immunity to RhCMV, it induced lower titers of neutralizing antibody and antibody to gB than the soluble gB vaccine; after challenge, animals vaccinated with soluble gB had a lower frequency of virus detection in the blood than those vaccinated with RhCMVRΔgL/178/182-189.

Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human In Vitro Systems.

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5-10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

A novel inhibitor of Mycobacterium tuberculosis pantothenate synthetase.

Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential adenosine triphosphate (ATP)-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast, and plants; pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). Because the enzyme is absent in mammals and both CoA and ACP are essential cofactors for bacterial growth, PS is an attractive chemotherapeutic target. An automated high-throughput screen was developed to identify drugs that inhibit Mycobacterium tuberculosis PS. The activity of PS was measured spectrophotometrically through an enzymatic cascade involving myokinase, pyruvate kinase, and lactate dehydrogenase. The rate of PS ATP utilization was quantitated by the reduction of absorbance due to the oxidation of NADH to NAD+ by lactate dehydrogenase, which allowed for an internal control to detect interference from compounds that absorb at 340 nm. This coupled enzymatic reaction was used to screen 4080 compounds in a 96-well format. This discussion describes a novel inhibitor of PS that exhibits potential as an antimicrobial agent.

Why do inguinal hernia patients have pain? Histology points to compression neuropathy.

PURPOSE: The purpose of this study is to describe the known soft tissue neuro-histology factors associated with compression neuropathy in relation to the incidence of preoperative pain in primary inguinal hernia. Enlargement of the ilioinguinal nerve occurs in 63% of patients with primary inguinal hernia; compression neuropathy has similar gross features. METHODS: Patients completed pain questionnaires pertaining to preoperative pain and the quality of pain experienced. During routine inguinal hernia repair, nerve segments were sampled for histologic evaluation. RESULTS: Twenty-two thickened nerve segments (63% of total) with proximal and distal specimens were resected for examination and comparison. We quantified various histologic indicators including nerve diameter, fascicle count, myxoid content within the epineurium, perineurium and endoneurium. Increased preoperative patient pain scores correlate with increased nerve diameter, increased fascicle count and increased myxoid material both within the perineurium and endoneurium. CONCLUSION: These findings support the concept that preoperative hernia pain is associated with compression neuropathy.

The Availability of Advanced Airway Equipment and Experience with Videolaryngoscopy in the UK: Two UK Surveys.

Fibreoptic intubation, high frequency jet ventilation, and videolaryngoscopy form part of the Royal College of Anaesthetists compulsory higher airway training module. Curriculum delivery requires equipment availability and competent trainers. We sought to establish (1) availability of advanced airway equipment in UK hospitals (Survey I) and (2) if those interested in airway management (Difficult Airway Society (DAS) members) had access to videolaryngoscopes, their basic skill levels and teaching competence with these devices and if they believed that videolaryngoscopy was replacing conventional or fibreoptic laryngoscopy (Survey II). Data was obtained from 212 hospitals (73.1%) and 554 DAS members (27.6%). Most hospitals (202, 99%) owned a fiberscope, 119 (57.5%) had a videolaryngoscope, yet only 62 (29.5%) had high frequency jet ventilators. DAS members had variable access to videolaryngoscopes with Airtraq 319 (59.6%) and Glidescope 176 (32.9%) being the most common. More DAS members were happy to teach or use videolaryngoscopes in a difficult airway than those who had used them more than ten times. The majority rated Macintosh laryngoscopy as the most important airway skill. Members rated fibreoptic intubation and videolaryngoscopy skills equally. Our surveys demonstrate widespread availability of fibreoptic scopes, limited availability of videolaryngoscopes, and limited numbers of experienced videolaryngoscope tutors.

A seroprevalence study of primate workers for asymptomatic rhesus cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) infection can cause severe disease in neonates and immunocompromised persons, and infectious mononucleosis in healthy adults. While, rhesus CMV (RhCMV) infects human cells in culture, it is unknown whether the virus can infect humans. OBJECTIVES: We sought to determine whether primate workers, including those with injuries from animals, might be infected asymptomatically with RhCMV. STUDY DESIGN: We developed serologic assays that distinguish RhCMV from HCMV antibodies. We tested two groups of primate workers: those with documented injuries or mucosal splashes associated with rhesus macaques, and those with no documented exposure who worked with these animals. RESULTS: None of over 200 primate workers, including 119 with injuries or mucosal splashes associated with exposures to macaques, were seropositive for RhCMV. CONCLUSIONS: The frequency of asymptomatic RhCMV infection in persons who work with rhesus macaques was <0.5% (<1/200 primate workers).

Don't Count Your Chicken Livers: an Outbreak of Campylobacter sp. Not Associated with Chicken Liver Parfait, England, November 2013.

In England, several recent campylobacter outbreaks have been associated with poultry liver consumption. Following a lunch event in a hotel in Surrey in November 2013 where chicken liver parfait was served, guests reported having gastrointestinal symptoms. A retrospective cohort study showed 46 of 138 guests became unwell, with a median incubation period of two days and for 11 cases campylobacter infection was laboratory confirmed. Food item analysis identified an association between illness and consumption of roast turkey (aOR=3.02 p=0.041) or jus (aOR=3.55 p=0.045), but not with chicken liver parfait (OR=0.39 p=0.405). The environmental risk assessment did not identify non-compliance with standard food practice guidelines. This study presents a point-source outbreak of campylobacter with a high attack rate and epidemiological analysis results show that the jus or roast turkey was the likely source of infection although this could not be confirmed by the environmental assessment. Consuming the chicken liver dish was not a risk factor for developing symptoms as was initially hypothesised. Prior knowledge on the association between poultry liver food items and campylobacter outbreaks should not overly influence an outbreak investigation to ensure the true aetiology is identified and on-going public health risk is minimised.

Coding potential of UL/b' from the initial source of rhesus cytomegalovirus Strain 68-1.

Rhesus cytomegalovirus (RhCMV) 68-1 is the prototypic strain of RhCMV that has been used for pathogenesis and vaccine development. We determined the complete sequence of the RhCMV 68-1 UL/b' region directly from the original urine from which RhCMV 68-1 was isolated in 1968, and compared it to other RhCMVs. The laboratory passaged RhCMV 68-1 has inversions, deletions, and stop codons in UL/b' that are absent in the original isolate and other low passage RhCMV isolates. Fourteen of the 17 open reading frames (ORFs) in 68-1 UL/b' in the original isolate share >95% amino acid identity with low passage RhCMV. The original isolate retains 6 ORFs that encode α-chemokine-like proteins, including RhUL146 and RhUL146b that share only 92% and 81% amino acid identity, respectively, with a contemporary low passage RhCMV isolate. Identification of the original RhCMV 68-1 UL/b' sequence is important for using RhCMV 68-1 in pathogenesis and vaccine studies.

Rationally tuned micropores within enantiopure metal-organic frameworks for highly selective separation of acetylene and ethylene.

Separation of acetylene and ethylene is an important industrial process because both compounds are essential reagents for a range of chemical products and materials. Current separation approaches include the partial hydrogenation of acetylene into ethylene over a supported Pd catalyst, and the extraction of cracked olefins using an organic solvent; both routes are costly and energy consuming. Adsorption technologies may allow separation, but microporous materials exhibiting highly selective adsorption of C(2)H(2)/C(2)H(4) have not been realized to date. Here, we report the development of tunable microporous enantiopure mixed-metal-organic framework (M'MOF) materials for highly selective separation of C(2)H(2) and C(2)H(4). The high selectivities achieved suggest the potential application of microporous M'MOFs for practical adsorption-based separation of C(2)H(2)/C(2)H(4).