Amalgamation of Nucleosides and Amino Acids in Antibiotic Biosynthesis

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds

The feasibility of wastewater recycling that includes residue from dissolved air flotation within a drinking water treatment plant: case study of Midvaal Water Company, South Africa

When purifying water for potable use, wastewater is generated, due to the class of the water treatment plant and the quality of the source water. Midvaal Water Company recycled wastewater that included residue from the dissolved air flotation (DAF), sedimentation and filtration processes in an attempt to save water and reduce costs. The aim of this study was to determine functionality and water quality of such a wastewater recycling system. Samples were collected for analysis, at the sections that contributed to the total wastewater system as well as after various treatment processes. The water quality of these samples was determined, as well as the incidences of water quality failures of the final water, to establish whether the recycle stream that enters the plant together with the source water had any impact on the water quality after the different treatment processes. Data were grouped into periods prior to, during and after recycling to enable comparisons. The water quality of the recycle stream was poorer than that of the source water from the Vaal River with regard to the mean values for total chlorophyll, suspended solids, turbidity and dissolved organic carbon, but the sedimentation process of the wastewater system improved the wastewater quality by drastically reducing total chlorophyll, suspended solids and turbidity. The risk-defined compliance for the final water was excellent (≥95%), despite aluminium, turbidity and total chlorophyll failures of the final water quality during the recycling period. Total chlorophyll was identified as the largest risk during wastewater recycling, especially after the filtration process. It is evident from the data that wastewater recycling, which included wastewater from the DAF, into the main inlet stream of the water treatment plant proved to be effective, based on compliance with national legislation, and had no detrimental impact on overall treatment processes or final water quality.Keywords: dissolved air flotation, sludge balancing dam, total chlorophyll, wastewater recycling, water treatmen

Challenges in the potable water industry due to changes in source water quality: case study of Midvaal Water Company, South Africa

Midvaal Water Company treats hypertrophic water abstracted from the Vaal River to supply bulk wholesome potable water to their consumers in compliance with the South African National Standard (SANS) 241:2015 for drinking water. The facility incorporates conventional and advanced treatment processes. The aims of the study were to identify how the water treatment processes of the plant have changed over time in response to the varying water quality of the Vaal River, and to consider both current and future obstacles as well as possible solutions regarding water quality and treatment. Oxidation steps such as pre-chlorination, potassium permanganate addition, pre-ozonation and intermediate ozonation have either been applied in the past or are still operational. The dissolved air flotation plant accounts for almost 70% of total chlorophyll removal and the significance of this process was confirmed during a brief maintenance shutdown during 2015. Total chlorophyll concentrations of the source water have increased extensively since 1984, while turbidity levels have remained fairly constant but with spikes at times. The facility suffers from severe taste and odour episodes during warm periods due to the presence of methylisoborneol (MIB), released by Cyanophyceae, in the Vaal River. Concentrations of > 300 ng/L MIB have been recorded, whereas the odour threshold concentration for MIB ranges from 4 ng/L to 20 ng/L. The additional application of activated carbon to alleviate taste and odour problems has to be weighed against the cost implications for consumers, the correct type to be purchased for the organic molecules to be adsorbed, the interference of natural organic matter, and the formation of additional sludge mass, as well as the intensity and duration of taste and odour events. Midvaal remains a bulk potable water supplier and therefore has to consider the socio-economic status of their consumers where water pricing is concerned. The study ultimately emphasized the intrinsic value of protecting water resources.Keywords: oxidation processes, dissolved air flotation, chlorophyll a, taste and odou

Construction and evaluation of a scale for creative writing.

Thesis (Ed.M.)--Boston Universit

Intensive care nurse-family engagement from a global perspective: A qualitative multi-site exploration

Background: Critical illness is distressing for families, and often results in negative effects on family health that influence a family\u27s ability to support their critically ill family member. Although recent attention has been directed at improving care and outcomes for families of critically ill patients, the manner in which nurses engage with families is not fully understood. Objectives: To describe nurses’ perceptions and practices of family engagement in adult intensive care units from a global perspective. Design: A qualitative-descriptive multi-site design using content analysis. Settings: The study was conducted in 26 intensive care units of 12 urban, metropolitan, academic medical centers in ten countries, spanning five continents. Participants: A total of 65 registered nurses (77% women, age of M = 39.5, SD = 11.4 years) participated. Most held intensive care certification (72%) and had worked on average 10 (SD = 9.6) years in the ICU. Methods: Semi-structured, individual interviews (M = 38.4 min, SD = 12.0) were held with ICU nurses at the hospital (94%) or their home using an interview guide. Qualitative interview data were analysed using inductive content analysis. Results: We found that nurse-family engagement was an ebb and flow of relational power that needed to be carefully negotiated and balanced, with nurses holding and often exerting more power than families. Constant fluctuations in nurses’ practices of engagement occurred in day-to-day practice from shift-to-shift and from nurse-to-nurse. Family engagement was dependent on individual nurses’ attitudes and perceptions of family, the patient\u27s condition, and workload. Lastly, family engagement was shaped by the ICU context, with team culture, collaborative relationships, unit structures and organizational resources either enabling or limiting nurses’ ability to engage with families. Conclusions: This global study provides an in-depth understanding of the way nurses engage with families in ICU and reflects many different cultures and health systems. We found that nurse-family engagement was marked by a shifting, yet often unequal power distribution in the nurse-family relationship, inconsistent nurse engagement practices, both of which resulted in variable family engagement in intensive care. Our research contributes a detailed description of engagement as practiced in the everyday delivery of health care. A more concentrated team effort, based on a shared culture and defined framework of family care is needed to ensure that families of critically ill persons are fully engaged in all aspects of intensive care

The Biosynthesis of Capuramycin-type Antibiotics: Identification of the A-102395 Biosynthetic Gene Cluster, Mechanism of Self-Resistence, and Formation of Uridine-5\u27-Carboxamide

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5\u27-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5\u27-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5\u27-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures

Preclinical Evaluation of the Novel, Orally Bioavailable Selective Inhibitor of Nuclear Export (SINE) KPT-335 in Spontaneous Canine Cancer: Results of a Phase I Study

The purpose of this study was to evaluate the activity of Selective Inhibitors of Nuclear Export (SINE) compounds that inhibit the function of the nuclear export protein Exportin 1 (XPO1/CRM1) against canine tumor cell lines and perform a Phase I clinical trial of KPT-335 in dogs with spontaneous cancer to provide a preliminary assessment of biologic activity and tolerability.Canine tumor cell lines derived from non-Hodgkin lymphoma (NHL), mast cell tumor, melanoma and osteosarcoma exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE compounds; NHL cells were particularly sensitive with IC50 concentrations ranging from 2-42 nM. A Phase I clinical trial of KPT-335 was performed in 17 dogs with NHL (naive or relapsed), mast cell tumor or osteosarcoma. The maximum tolerated dose was 1.75 mg/kg given orally twice/week (Monday/Thursday) although biologic activity was observed at 1 mg/kg. Clinical benefit (CB) including partial response to therapy (PR, n = 2) and stable disease (SD, n = 7) was observed in 9/14 dogs with NHL with a median time to progression (TTP) for responders of 66 days (range 35-256 days). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg KPT-335 Monday/Wednesday/Friday; CB was observed in 4/6 dogs with a median TTP for responders of 83 days (range 35-354 days). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care, dose modulation and administration of low dose prednisone; hepatotoxicity, anorexia and weight loss were the dose limiting toxicities.This study provides evidence that the novel orally bioavailable XPO1 inhibitor KPT-335 is safe and exhibits activity in a relevant, spontaneous large animal model of cancer. Data from this study provides critical new information that lays the groundwork for evaluation of SINE compounds in human cancer

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen: Focused Compound Screen in Chordoma

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well-characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth-inhibitory effect. Their half-maximal effective concentration (EC50 ) values were determined in chordoma cells and normal fibroblasts. Twenty-seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho-EGFR and its downstream pathways in a dose-dependent manner. Analysis of substituent patterns suggested that EGFR-inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U-CH1 xenograft and a patient-derived xenograft, SF8894). One of the resistant cell lines (U-CH2) was shown to express high levels of phospho-MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p-)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

The European union’s 2010 target: Putting rare species in focus

P. 167-185The European Union has adopted the ambitious target of halting the loss of biodiversity by 2010. Several indicators have been proposed to assess progress towards the 2010 target, two of them addressing directly the issue of species decline. In Europe, the Fauna Europaea database gives an insight into the patterns of distribution of a total dataset of 130,000 terrestrial and freshwater species without taxonomic bias, and provide a unique opportunity to assess the feasibility of the 2010 target. It shows that the vast majority of European species are rare, in the sense that they have a restricted range. Considering this, the paper discusses whether the 2010 target indicators really cover the species most at risk of extinction. The analysis of a list of 62 globally extinct European taxa shows that most contemporary extinctions have affected narrow-range taxa or taxa with strict ecological requirements. Indeed, most European species listed as threatened in the IUCN Red List are narrow-range species. Conversely, there are as many wide-range species as narrow-range endemics in the list of protected species in Europe (Bird and Habitat Directives). The subset of biodiversity captured by the 2010 target indicators should be representative of the whole biodiversity in terms of patterns of distribution and abundance. Indicators should not overlook a core characteristic of biodiversity, i.e. the large number of narrow-range species and their intrinsic vulnerability. With ill-selected indicator species, the extinction of narrowrange endemics would go unnoticedS