Processes driving freshwater plant production and diversity in upland streams

Upland headwater streams are important sources of freshwater in mountainous temperate to sub-arctic latitude European countries like Scotland. Yet much less is known about the ecology of small, characteristically oligotrophic, mountain streams supporting periphyton and aquatic bryophyte dominated vegetation, and their potential bioindicator capacity of environmental water quality, than lowland rivers impacted by anthropogenic disturbance, in this context. This scarcity of knowledge has significant implications for the success of the recently implemented Water Framework Directive (WFD: 2000/60/EC). The WFD is a major piece of environmental legislation for water policy and sustainable water management in Europe. New contributions are fundamental to environment agencies, such as the Scottish Environment Protection Agency (SEPA), tasked with the responsibility of enforcing WFD statutory requirements and developing effective biomonitoring tools for assessing water quality status in Scotland. A major aim of the WFD is to achieve at least ‘good’ ecological status of inland waterbodies by 2015. Further, in doing so, to ascertain ecological benchmark communities of near-pristine (or minimally-impacted) reference conditions as indicators of high water quality status. The objective is to improve understanding of the environmental processes driving the production and diversity of freshwater plant species-assemblages in upland streams. Such information can be used for assessing perturbations threatening the ecological integrity of rivers impacted by anthropogenic disturbances (human pressure). This enables environment agencies such as SEPA, to respond appropriately by implementing corrective measures and sustainable management strategies. This project monitored a range of near-pristine headwater streams of contrasting underlying geology in the Scottish Highlands. The approach adopted was compatible with current WFD river characterisation and biomonitoring strategies. These were used to investigate the structural and functional response of freshwater plant communities (chiefly diatoms and other algal groups; aquatic bryophyte and vascular submerged macrophyte vegetation) to environmental drivers (e.g. flow, substrate morphology, nutrient inputs, water chemistry, underwater light availability). The work was carried out with the aim of contributing to future development of baseline monitoring tools for assessing upland stream habitat quality in Scotland

A Stakeholder Informed Professional Development Framework to Support Engagement with Learning Analytics

This paper reports on a study aimed at identifying training requirements for both staff and students in higher education to enable more widespread use of learning analytics. Opinions of staff and students were captured through ten focus groups (37 students; 40 staff) and two surveys (1,390 students; 160 staff). Participants were predominantly from two higher education institutions in Ireland. Analysis of the results informed a framework for continuous professional development in learning analytics focusing on aspects of using data, legal and ethical considerations, policy, and workload. The framework presented here differentiates between the training needs of students, academic staff and professional services staff

Biochemical and crystallographic studies of l,d -transpeptidase 2 from Mycobacterium tuberculosis with its natural monomer substrate

The essential l,d-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 3→3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes

Nondestructive quantitative analysis of water potential of tomato leaves using online hyperspectral imaging system

Tomatoes have different water requirements in each growing period. Excessive water use or insufficient water supply will affect the growth and yield of tomato plants. Therefore, precise irrigation control is necessary during cultivation to increase crop productivity. Traditionally, the soil moisture content or leaf water potential has been used as an indicator of plant water status. These methods, however, have limited accuracy and are time-consuming, making it difficult to be put into practice in tomato production. This study developed an online hyperspectral imaging system to measure the leaf water potential of tomato nondestructively. Linear Discriminant Analysis was utilized to automatically and quickly extract the leaf images, with the recognition accuracy of 94.68% was achieved. The mathematical processing of Standard Normal Variate scattering correction was used to remove the spectral variations caused by the defocused leave images. The developed leaf water potential prediction model based on the spectral image information attained using the developed system achieved the standard error of calibration of 0.201, coefficient of determination in calibration set of 0.814 and standard error of cross�validation of 0.230, and one minus the variance ratio of 0.755. The obtained performance indicated the feasibility of apply�ing the developed online hyperspectral imaging system as a real-time non-destructive measurement technique for the leaf water potential of tomato plants

Incidence of Dentinal Defects and Vertical Root Fractures after Endodontic Retreatment and Mechanical Cycling

Introduction: The aim of this study was to evaluate the incidence of dentinal defects and vertical root fractures (VRFs) after endodontic retreatment and mechanical cycling (MC). Methods and Materials: Two hundred mandibular premolars were selected. Forty teeth were left unprepared (control group). The remaining 160 root canals were prepared with ProTaper instruments and filled by using two different techniques [eighty with lateral compaction (LC) and eighty with single-cone (SC)]. Forty canals from each group (LC and SC) received no further treatment. The remaining eighty teeth were divided into two groups (LCR and SCR) (n=40) in order to undergo the removal of the root filling, re-preparation and refilling with lateral compaction and single-cone, respectively. All of the teeth were subjected to MC (1,000,000 cycles, 130 N, 2.2 Hz and 37°C). The roots were sectioned at 3, 6 and 9 mm from the apex and observed under 20× magnification. The defects were classified as: no defect, VRF and other defects. Statistical analysis was performed using the Fisher’s Exact test and the Chi-Squared tests (α=0.05). Results: MC alone did not promote any other defects or VRFs. Experimental groups presented higher dentinal defects than the control group (P=0.021). Retreatment groups did not present a higher amount of dentinal defects than the groups that were subjected to the first treatment (P>0.05). Conclusion: Endodontic treatment and retreatment, regardless of the filling technique and MC, did not influence the occurrence of dentinal defects or VRFs in the human premolars.Keywords: Defects; Endodontics; Retreatment; Root Cana

High-throughput screen with the l,d-transpeptidase LdtMt2 of Mycobacterium tuberculosis reveals novel classes of covalently reacting inhibitors

Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The L,D-transpeptidase LdtMt2, which is responsible for the formation of 3 → 3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of ∼10 000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g., β-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of ∼1 μM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes

Environmental drivers of aquatic macrophyte communities in southern tropical African rivers:Zambia as a case study

The first-ever extensive macrophyte survey of Zambian rivers and associated floodplain waterbodies, conducted during 2006–2012, collected 271 samples from 228 sites, mainly located in five freshwater ecoregions of the world primarily represented in Zambia. The results supported the hypothesis that variation in macrophyte community structure (measured as species composition and diversity) in southern tropical African river systems, using Zambia as a case study area, is driven primarily by geographical variation in water physico-chemical conditions. In total, 335 macrophyte taxa were recorded, and a chronological cumulative species records curve for the dataset showed no sign of asymptoting: clearly many additional macrophyte species remain to be found in Zambian rivers. Emergent macrophytes were predominant (236 taxa), together with 26 floating and 73 submerged taxa. Several species were rare in a regional or international context, including two IUCN Red Data List species: Aponogeton rehmanii and Nymphaea divaricata. Ordination and classification analysis of the data found little evidence for temporal change in vegetation, at repeatedly-sampled sites, but strong evidence for the existence of seven groups of samples from geographically-varied study sites. These supported differing sets of vegetation (with eight species assemblages present in the sample-groups) and showed substantial inter-group differences in both macrophyte alpha-diversity, and geographically-varying physico-chemical parameters. The evidence suggested that the main environmental drivers of macrophyte community composition and diversity were altitude, stream order, shade, pH, alkalinity, NO3-N, and underwater light availability, while PO4-P showed slightly lower, but still significant variation between sample-groups

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [Zr-89]Zr-DFO-PEG(5)-Tz

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-ofconcept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new Zr-89-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t(1/2) = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [Zr-89]Zr-DFO-PEG S -Tz ([Zr-89]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 +/- 0.2 vs 17.1 +/- 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for Zr-89-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.Peer reviewe

Molecular Basis of Class A β-lactamase Inhibition by Relebactam

β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI is in phase-3 clinical trials in combination with imipenem, for treatment of infections by multi-drug resistant Enterobacteriaceae. We show that relebactam inhibits five clinically-important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e. the extended spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM); and the carbapenemases, KPC-2, -3 and -4 (1 - 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared to the clinically approved DBO avibactam, (9 to 120-fold differences in IC50). Minimum inhibitory concentration assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3 and KPC-4 reveal its C2 linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity compared to avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3 and -4. This comprehensive comparison of relebactam binding across five clinically-important class A SBLs will inform the design of future DBOs with the aim of improving clinical efficacy of BLI:β-lactam combinations