Pollution in mediterranean-climate rivers

This review examines information generated over the past decade on the pollution of rivers in regions with a mediterranean-type climate (med-climate). Pollution has clearly increased in the last 100 years and is correlated with the development of industry, agriculture and human population. Important efforts have been made in some med-climate countries in order to characterise the chemical status of rivers. In addition, the number of chemical substances detectable in mediterranean-climate rivers (med-rivers), as well as the limits of detection, have improved from the development of better analytical methods. New substances detected in rivers are gaining attention. We discuss available knowledge regarding real and potential effects of pollutants on the biota and ecosystems in med-rivers, taking into account natural environmental characteristics of these rivers. The extreme seasonal conditions in med-rivers add to the potential risk because these characteristics can enhance pollutant effects. Efforts and policies to prevent or reduce pollution effects on med-rivers are linked to the knowledge about pollution pressures associated with the degree of economic development. Aquatic communities in med-rivers are more sensitive to pollutants because they are exposed to strong natural and human stressors.     

QTL mapping for resistance to Ceratocystis wilt in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Ceratocystis wilt caused by the fungus Ceratocystis fimbriata, is currently one of the major diseases in commercial plantations of Eucalyptus trees in Brazil. Deployment of resistant genotypes has been the main strategy for effective disease management. The present study aimed at identifying genomic regions underlying the genetic control of resistance to Ceratocystis wilt in Eucalyptus by quantitative trait loci (QTL) mapping in an outbred hybrid progeny derived from a cross between (Eucalyptus dunnii × Eucalyptus grandis) × (Eucalyptus urophylla × Eucalyptus globulus). A segregating population of 127 individuals was phenotyped for resistance to Ceratocystis wilt using controlled inoculation under a completely randomized design with five clonal replicates per individual plant. The phenotypic resistance response followed a continuous variation, enabling us to analyze the trait in a quantitative manner. The population was genotyped with 114 microsatellite markers and 110 were mapped with an average interval of 12.3 cM. Using a sib-pair interval-mapping approach five QTLs were identified for disease resistance, located on linkage groups 1, 3, 5, 8, and 10, and their estimated individual heritability ranged from 0.096 to 0.342. The QTL on linkage group 3 overlaps with other fungal disease-resistance QTLs mapped earlier and is consistent with the annotation of several disease-resistance genes on this chromosome in the E. grandis genome. This is the first study to identify and attempt to quantify the effects of QTLs associated with resistance to Ceratocystis wilt in Eucalyptus.     

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.     

Ontogenetic integration between force production and force reception: a case study in Ctenomys (Rodentia: Caviomorpha)

During ontogeny, complex adaptations undergo changes that sometimes entail different functional capabilities. This fact constrains the behaviour of organisms at each developmental stage. Rodents have ever‐growing incisors for gnawing, and a powerful jaw musculature. The incisors are long enough, relative to their diameter, to be affected by bending stresses. This is particularly true in the subterranean Ctenomys that uses its incisors for digging. We measured bite force (BF) in individuals of different ages using a force transducer. We estimated incisor section modulus Z, a geometrical parameter proportional to bending strength. A relative strength indicator was calculated as S = Z/BF incisor length. We found that ontogenetic BF scales to body mass with positive allometry. However, an anova showed non‐significant differences in S, neither between sexes nor among age classes. This result implies that during growth, incisors might have a rather similar ability to withstand bending stresses from increasing masticatory forces, what may be considered evidence of ontogenetic integration of force production (by muscles) and force reception (by the incisors). This fact well correlates with the observation that pups and juveniles of C. talarum incorporate solid foods shortly after birth, and they are able to dig burrows early in life.     

Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd

In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC₅₀?=?260?±?0.112?µg/ml) and antiplasmodial (IC₅₀?=?8.5?±?1.6?µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31?µg/ml?≥?IC₅₀?≥?5.6?µg/ml; antiplasmodial: 14?µg/ml?≥?IC₅₀?≥?2?µg/ml) with very minimal toxicity.     

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.     

Vibrational and electronic circular dichroism study of the interactions of cationic porphyrins with (dG-dC)10 and (dA-dT)10

The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.     

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed approximately 200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn.