Citizen science through a recreational underwater diving project supports the collection of large-scale marine litter data: The Oceania case study.

In recent years, a growing body of literature on seafloor macro-litter has been produced worldwide. However, the spatial coverage of these studies is still limited and highly unbalanced, resulting in considerable knowledge gaps in some regions. To address this lack of information in Oceania, we extracted data from the Citizen Science project Dive Against Debris® to characterize marine debris collected by volunteer scuba divers on the coastal seafloor. Overall, the average litter density was 58.22 items/100m2, with plastics accounting for approximately 50 % of the total abundance and Single Use Plastics accounting for nearly 17 %. Notably, 36 % of the total litter abundance consisted of lost Fishing Gear including fishing lines, sinkers, baits and hooks as the most abundant debris items. To reduce lost fishing gear, clean-up initiatives by divers along with management actions such as education programs for fishermen, gear restrictions and the identification of designated fishing sites are recommended.

Chester treadmill police tests as alternatives to 15-m shuttle running.

BACKGROUND: Police officers require a specific level of aerobic fitness to allow them to complete personal safety training and specialist roles. Officers' aerobic fitness is assessed using the 15-m multi-stage fitness test (MSFT); however, due to the agility required and risk of injury, two alternative treadmill tests have been designed to predict four of the key minimum VO2 criteria of 35, 41, 46 and 51 ml·kg-1·min-1. AIMS: To investigate the validity and reliability of Chester Treadmill Police Walk Test (CTPWT) and Chester Treadmill Police Run Test (CTPRT). METHODS: Seventy-eight UK police officers (18 females) completed the CTPWT (n = 53) or CTPRT (n = 35), or both, generating a total of 88 data sets. To assess reliability, 43 participants returned for a second visit (T2), to repeat the treadmill test. RESULTS: Mean differences between predicted and actual VO2 at 35, 41, 46 and 51 ml·kg-1·min-1 were as follows -1.1, -2.1, -0.1 and -1.2 ml·kg-1·min-1. Despite a significant under prediction (p = 0.001), a minimum of 92% of participants were within 10% of target VO2 at all levels. There was no significant difference between actual and predicted VO2 in the CTPRT, at 46 ml·kg-1·min-1 (T1 46.0 ± 1.4 or T2 45.1 ± 1.3 ml·kg-1·min-1). Similarly, there was no significant difference at 51 ml·kg-1·min-1 (T2 50.5 ± 1.4 ml·kg-1·min-1). We observed no differences for gender or trial. Ninety-five per cent limits of agreement were at worst -0.25 ± 4.0 ml.kg-1.min-1 between trial 1 and 2. CONCLUSIONS: The CTPWT and the CTPRT provide a valid and reliable alternative to the 15-m MSFT.

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.

Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution.

Anaerobic synthesis of vitamin B12: characterization of the early steps in the pathway.

The anaerobic biosynthesis of vitamin B12 is slowly being unravelled. Recent work has shown that the first committed step along the anaerobic route involves the sirohydrochlorin (chelation of cobalt into factor II). The following enzyme in the pathway, CbiL, methylates cobalt-factor II to give cobalt-factor III. Recent progress on the molecular characterization of this enzyme has given a greater insight into its mode of action and specificity. Structural studies are being used to provide insights into how aspects of this highly complex biosynthetic pathway may have evolved. Between cobalt-factor III and cobyrinic acid, only one further intermediate has been identified. A combination of molecular genetics, recombinant DNA technology and bioorganic chemistry has led to some recent advances in assigning functions to the enzymes of the anaerobic pathway.

Aerobic synthesis of vitamin B12: ring contraction and cobalt chelation.

The aerobic biosynthetic pathway for vitamin B12 (cobalamin) biosynthesis is reviewed. Particular attention is focused on the ring contraction process, whereby an integral carbon atom of the tetrapyrrole-derived macrocycle is removed. Previous work had established that this chemically demanding step is facilitated by the action of a mono-oxygenase called CobG, which generates a hydroxy lactone intermediate. This mono-oxygenase contains both a non-haem iron and an Fe-S centre, but little information is known about its mechanism. Recent work has established that in bacteria such as Rhodobacter capsulatus, CobG is substituted by an isofunctional protein called CobZ. This protein has been shown to contain flavin, haem and Fe-S centres. A mechanism is proposed to explain the function of CobZ. Another interesting aspect of the aerobic cobalamin biosynthetic pathway is cobalt insertion, which displays some similarity to the process of magnesium chelation in chlorophyll synthesis. The genetic requirements of cobalt chelation and the subsequent reduction of the metal ion are discussed.

Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX).

One of the four operons required for cobalamin biosynthesis in Bacillus megaterium is also associated with sirohaem synthesis, and contains three genes, sirA, sirB and sirC. By undertaking functional complementation experiments and in vitro assays using recombinantly produced enzymes, we have been able to demonstrate that (1) SirA acts as a uroporphyrinogen III methyltransferase, transforming uroporphyrinogen III into precorrin-2, (2) SirC acts as an NAD(+) dehydrogenase, responsible for the oxidation of precorrin-2 into sirohydrochlorin, and (3) SirB acts as a ferrochelatase, responsible for the insertion of a ferrous ion into sirohydrochlorin to give sirohaem. Comparative sequence analysis reveals that the primary structure of SirB is highly similar to that of the cobalt chelatase involved in cobalamin biosynthesis in Bacillus megaterium, CbiX, with the exception that CbiX contains a C-terminal histidine-rich motif. Surprisingly, CbiX has been shown (using EPR) to contain a 4Fe-4S centre, a redox centre that is absent from SirB.

Cobalamin (vitamin B(12)) biosynthesis in Rhodobacter capsulatus.

In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically. However, a comparison of the main cobalamin biosynthetic operon found within R. capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected. Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon. When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein. Some of this protein is cleaved within E. coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein. When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound. Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein. We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R. capsulatus.

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy.

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy.

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Functional characterization of missense mutations at codon 838 in retinal guanylate cyclase correlates with disease severity in patients with autosomal dominant cone-rod dystrophy.

Three different mutations in codon 838 of GUCY2D, the gene for retinal guanylate cyclase 1, have been linked to autosomal dominant cone-rod dystrophy at the CORD6 locus. To examine the relationship between enzyme activity and disease severity, the three disease-causing substitutions (R838C, R838H and R838S) and four artificial mutations (R838A, R838E, R838L and R838K) were generated. Assay of GCAP1-stimulated cyclase activity in vitro shows that, compared with wild-type, R838E, R838L and R838K possess only low activity, whereas R838A, R838C, R838H and R838S have activity equal or superior to wild-type at low Ca(2+) concentrations. These four latter mutants showed a higher apparent affinity for GCAP1 than did wild-type. The Ca(2+) sensitivity of the GCAP1 activation was also altered with marked residual activity at high Ca(2+), the effect increasing: wild-type < R838C < R838H << R838A < R838S. Within the photoreceptor, this would result in a failure to inactivate cyclase activity at high physiological Ca(2+ )concentrations. Amongst the three disease-associated mutations, the effect correlates directly with disease severity. The wild-type and R838H mutant displayed a difference in pH sensitivity, with the mutant showing a higher specific activity with pH > 6.0. Site 838 is in the dimerization domain that forms a coiled-coil in the active protein. A computer-aided structure prediction of this region indicates that R838 in the wild-type breaks the structure at four helical turns, and there is an increasing tendency for the structure to continue for further turns in the order R838C < R838H,S,K << R838E < R838A < R838L.