Central composite rotatable design for optimization of trihalomethane extraction and detection through gas chromatography: a case study

Central composite rotatable design (CCRD) was employed to optimize initial temperature (ºC), ramp function (ºC/min) and salt addition for trihalomethane extraction/quantification from the drinking water distribution network in Ratta Amral, Rawalpindi., Pakistan. Drinking water samples were collected from the treatment plant, overhead reservoir and consumer’s taps. The USEPA method for trihalomethane detection 551.1 via gas chromatography was applied using liquid–liquid extraction. The experiments with input variables for sample preparation and operational conditions were performed in a randomized order as per design of experiment by central composite rotatable design and responses were evaluated for model development. A significant (p = 0.005) two-factor interaction model was optimized. Initial temperature was observed to be insignificant (p = 0.64), while ramp function (p = 0.0043) and salt addition (p = 0.04) were significant. Product of salt addition and ramp was significant (p = 0.004), while product of initial temperature and salt addition was insignificant (p = 0.008). With a desirability function of 0.97, an initial temperature of 50 ºC, 6 ºC rise/min to 180 ºC and 0.5 g salt were optimized. It was found that development and optimization of the analytical methods for rapid trihalomethane detection would improve optimization of the current treatment practices in the country

Optimization of total trihalomethanes' (TTHMs) and their precursors' removal by granulated activated carbon (GAC) and sand dual media by response surface methodology (RSM)

A response surface methodology (RSM) applying central composite design with rotatable full factorial (14 non-center and six center points) was used to discern the effect of granular activated carbon (GAC), sand and pH on total trihalomethanes (TTHMs) and humic acid (HA) removal from drinking water. Results showed efficient TTHMs and HA removal by GAC while a sand column showed little effect for TTHMs but was significant for total organic carbon (TOC) removal. With GAC and a sand column of 4 cm, a pH increase from 6 to 8 caused an increase in TTHM removal from 79.8 to 83.6% while a decrease in HA removal from 26.6 to 6.6% was observed. An increase in GAC column depth from 10 to 20 cm caused a slight increase in TTHM removal from 99.4 to 99.7%, while TOC removal was increased from an average of 38.85% to 57.4% removal. The developed quadratic model for TTHM removal (p = 0.048) and linear model for TOC removal (p = 0.039) were significant. GAC column depth (p < 0.0117) and column depth2 (p < 0.039) were the most significant factors. A 98% TTHMs, 30%TOC and 51% residual chlorine removal were optimized at 9 cm GAC and 4 cm sand column depth at pH 8 with desirability factor (D) 0.64

Species-specific interaction of trihalomethane (THM) precursors in a scaled-up distribution network using response surface methodology (RSM)

Response surface methodology (RSM) with central composite design (CCD) was used to monitor and optimize species-specific interaction of trihalomethane (THM) precursors in a scaled-up distribution network (DN). Independent variables such as applied chlorine (Cl2), contact time (t), humic acid (HA) and bromide ions (Br−) were analyzed using full factorial CCD. Analysis of variance revealed a good agreement between experimental data and proposed a two-factor interaction model (p = .04, R2 = 0.7983). As a precursor, Cl− and Br− interaction with HA affected THMs’ speciation. These precursor molecules were perceived least significant as discrete elements but HA: Br− and pH product significantly impacted total trihalomethane (TTHM) formation (r = 0.998, p = .007). This mutual interactive fraction was observed pH-dependent and influenced TTHM yield. Dibromochloromethane and bromoform formation was observed pH-dependent provided sufficient Br− in the system. Applied chlorine had significant (p = .01), while time had insignificant (p = .75) effect. Multiple response optimization suggested pH range between 6.0 and 7.6 and HA: Br− ratio between 1.3 and 5.9 were satisfactory for maintaining TTHM below ≤80 μg/L in DN with 0.88 desirability function (D). Their respective concentration may be minimized by changing precursor’s individual concentration and possible combinations

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008435)National Centers for Systems Biology (U.S.) (Grant 1P50GM098792)United States. Defense Threat Reduction Agency (HDTRA1-14-1-0007)Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF13D0001)National Institute of General Medical Sciences (U.S.) (Interdepartmental Biotechnology Training Program 5T32 GM008334)Fonds de la recherche en sante du Quebec (Master's Training Award

Stationary Black Holes: Uniqueness and Beyond

The spectrum of known black-hole solutions to the stationary Einstein equations has been steadily increasing, sometimes in unexpected ways. In particular, it has turned out that not all black-hole-equilibrium configurations are characterized by their mass, angular momentum and global charges. Moreover, the high degree of symmetry displayed by vacuum and electro-vacuum black-hole spacetimes ceases to exist in self-gravitating non-linear field theories. This text aims to review some developments in the subject and to discuss them in light of the uniqueness theorem for the Einstein-Maxwell system.Comment: Major update of the original version by Markus Heusler from 1998. Piotr T. Chru\'sciel and Jo\~ao Lopes Costa succeeded to this review's authorship. Significantly restructured and updated all sections; changes are too numerous to be usefully described here. The number of references increased from 186 to 32

Molecular and Evolutionary Bases of Within-Patient Genotypic and Phenotypic Diversity in Escherichia coli Extraintestinal Infections

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies

A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes

BACKGROUND: Bread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines. RESULTS: A sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies. CONCLUSIONS: Evidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets