It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit.Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT).We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed.We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals. Despite the success in identifying genetic associations with complex disease in recent years, we are still relatively unaware of the proportion of this phenotypic variation that rare variants are responsible for. Study designs over the last decade have largely concerned common variants, and whilst the amount of additive genetic variance explained by these variants is greater than initially expected, the casOne type of approach involves grouping all variants within the same gene together, as they are likely to mark functional effects on the same protein or RNA, followed by analysing the combined effect of these variants using recently developed association tests. However, typically these approaches are underpowered. A majorProtein domains are distinct functional, structural and evolutionary units which can either span sub-sections of a protein or its entire length. The exploration of human disease mutations have found that they tend to cluster together within highly conserved protein positions, 9, whicIn comparison to conventional approaches which aggregate variants according to gene coordinates, we have undertaken a study to evaluate whether collapsing variants across regions based on protein domain coordinates provides a viable alternative. We aggregated variants together using 3 different definitions:Protein domains: Individual regions of the genome which mapped to individual protein domains. Certain protein domains can consist of the entire protein, which would therefore result in an identical analysis to using gene coordinates when analysing variants in exons. The exception to this would be variants at the 3’ and 5’ locations. However, protein domains can also consist of subsections of the protein, which would therefore only map back to a subsection of the corresponding gene region. These individual regions therefore varied in length based on definitions. In these instances we hypothesised that using domain coordinates may aggregate together more functionally relevant variants, thus resulting in a gain of statistical power in the resulting analysis.Domain-Domain Interactions: As two individual protein domains can interact and subsequently lead to the formation of PPIs, it is plausible that the corresponding pair of genomic regions may harbour functionally relevant variants. Whilst a mutation in either domain may be sufficient to affect binding affinity, analysing these regions together may provide stronger evidence of association than analysing each individually as they affect the same interaction. Consequently this definition consists of all variants from within two of the regions analysed using the previous definition, which were predicted to be responsible for a PPI.Protein Families: As previously mentioned, protein families can be defined as multiple protein domains which have a similar sequence and structure. This final definition therefore consists of multiple genomic regions which map to the same type of protein domain. Certain protein families can consist of a large frequency of domains and in these circumstances it seemed unlikely that so many different proteins would be involved along the causal pathway of disease. However, as these protein domains can have similar functionality it was worthwhile investigating whether variants within these regions, after selecting only genes with experimental evidence of interaction, were collectively associated with disease.We hypothesised that, in comparison to conventionally used gene coordinates, aggregating rare variants together across these alternative definitions may result in a greater proportion of variants which have a similar impact along the causal pathway and fewer neutral variants which dilute the observed signal. The potential trade-off to this is that variants with a similar functional effect within the same gene region may end up being collapsed separately. Rare variant analyses using these alternative definitions were conducted using samples from individuals involved in the UK10K project, which consists of participants drawn from the ALSPAC  and TwinsUK cohorts.The UK10K consortium has two main project arms. In this study, we have used data from the cohorts’ arm which was designed to investigate the contribution of genome wide genetic variation to a range of quantitative traits. This arm contains individuals from two intensively studied cohorts of European ancestry, ALSPAC  and TwinsUK:http://www.bristol.ac.uk/alspac).ALSPAC is a population-based cohort study investigating genetic and environmental factors that affect the health and development of children. The study methods are described in detail elsewhere, 13  Committee, South East London, REC 2. Written informed consent was obtained from parents for all measurements made.http://www.twinsuk.ac.uk/).The TwinsUK registry is a cohort of volunteer adult twins from all over the United Kingdom. InitialInformed consent was obtained from participants before they entered the study and ethical approval was granted by the National Research Ethics Service (NRES) Committee, Westminster, London.DNA Samples from 4,030 UK10K study participants  were subjected to low coverage (6-8x average read depth) whole-genome sequencing (WGS). Sequencing was performed at both the Wellcome Trust Sanger Institute (WTSI) and the Beijing Genomics Institute (BGI). DNA (1–3μg) was sheared to 100–1000 bp using a Covaris E210 or LE220 . Sheared DNA was size subjected to Illumina paired-end DNA library preparation. Following size selection (300–500 bp insert size), DNA libraries were sequenced using the Illumina HiSeq platform as paired-end 100 base reads according to manufacturer’s protocol.Data that passed quality control (QC) was aligned to the GRCh37 human reference used in phase 1 of the 1000 Genomes Project. Reads were aligned using BWA (v0.5.9-r16). Of the Missing and low-confidence genotypes in the filtered VCFs were refined out using the imputation procedure in BEAGLE 4 with defALSPAC: Non-fasting blood samples were taken from participants who attended the age 9 clinic . Plasma lipid concentrations , triglycerides (TG) and high density lipoprotein cholesterol (HDLc)) were measured by modification of the standard Lipid Research Clinics Protocol with enzymatic reagents for lipid determination[rmination. Low denrmination:LDLc=TCTwinsUK: Blood samples were taken after at least 6 hours of overnight fasting. The samples were immediately inverted three times and left to rest for 40 minutes at 4°C to obtain complete coagulation. The samples were then centrifuged for 10 min at 2000g and serum was removed. Four aliquots of 1.5 ml were placed into skirted micro centrifuge tubes and then stored in a -45°C freezer until sampling[ sampling. A colorAs a proof of concept, we first undertook analyses using simulated data consisting of 3,000 individuals each with 5,000 rare variants (i.e. MAF ≤ 1%). 4,000 of these were neutral variants and 1,000 were disease causing variants (i.e. variants that were associated with the dichotomous phenotype). The odds ratios for the disease causing variants varied from 1.20 to 1.50, whereas the neutral variants only ever contributed to an odds ratio of 1.0. We randomly allocated variants into 250 ‘synthetic’ genes  of varying number of variants per gene (from 10 to 30 variants). Within these genes we defined subgroups of variants to resemble domains, varying in length between 5–20 variants. 1,000 of these synthetic domains were randomly selected for analysis. We conducted gene-based and domain-based analysis using the sequence kernel association test (SKAT) to examiWe conducted analyses on regions based on three novel definitions of a functional unit. These were:Protein domains: Individual genomic regions which mapped to individual protein domains according to Pfam definitions. These regions comprised of either sections of proteins or in some cases the whole protein. For the latter, analyses would have been identical to analysing variants within the entire exonic region of the gene with the exception of variants at the 3’ and 5’ locations.Domain-Domain Interactions: iPfam was used to identify which pairwise domains were predicted to be responsible for a given protein-protein interaction according to STRINGdb v9.1 [Gdb v9.1 . FirstlyProtein Families: Variants were collapsed together across regions that were located within the same type of domain across the genome . However, only domains within gene regions whose product had experimental evidence of interaction according to STRINGdb (again using a STRING score ≥ 0.8 threshold) were combined. This meant that variants within multiple regions involved in the initial analysis  were analysed together here.Only regions which had at least 2 remaining variants were analysed using SKAT with each lipid trait  in turn. All traits were inverse normal transformed prior to analysis. Further details on trait standardization can be found in the Supplementary Material .To evaluate whether results provided strong evidence of association we used a threshold for multiple comparisons using the Bonferroni correction . All individual protein domains regions were reanalysed using the SKAT-O test  found toQ-Q plots were generated using the distribution of p-values from the results of the individual protein domain analysis with results from a conventional gene-based analysis. This analysis was undertaken with the same dataset but using gene start and end coordinates according to hd19 definitions and analysed as before using SKAT with each lipid trait. The quantiles from the domain-based analyses were therefore interpolated as there were more individual domains analysed compared to the number of genes. Q-Q plo−4 (250 tests)) in comparison to 47 domain-based results ). The tops hits from these analyses can be found in Analysing groups of variants from within simulated domain regions provided stronger evidence of association in comparison to collapsing by gene regions, as 23 gene-based results survived the correction for multiple testing (P<2.0 x 10-6) or were below the VQSLOD score cut-off  that corresponds to the maximum truth sensitivity tranche of 99.5% compared to HapMap3.3. Filtering to only include variants with a CADD C-Score ≥ 15 reduced the final number to 546,334.5,330,943 sites were excluded from further analyses due to showing Sanger/BGI batch effects, failed the test for Hardy-Weinberg equilibrium .−6. No results from this analysis survived this threshold using either the SKAT or SKAT-O tests , the SUPDespite a lack of statistically robust findings in our analyses, there may still be value in examining association signals from rare variants collapsed across highly conserved regions. Moreover, the parameters and resources we have used in this study for the analysis pipeline can be adjusted and this may lead to stronger evidence of association than observed here. For instance, the resources used to define our protein domain regions, the tool used to quantify the predicted deleterious impact of variants (as well as the threshold applied to filter) and the collapsing method used to analyse genotype-phenotype associations are all variables which can be adjusted for in future studies.LDLR, APOB, PCSK9 and LDLRAP1 are known to lead to this condition [In this study, we have focused on lipid traits due to the success reported by other studies in recent years –41. Althondition , althougondition  could bePrevious studies have suggested that larger sample sizes and alternative statistical methodology should help improve findings for collapsing methods. Using a more discrete definition of a functional unit across the genome, such as protein domains and families, provides a feasible alternative to collapsing by gene coordinates, which may yield biologically meaningful inferences and previously unidentified association signals when undertaking rare variant analyses.S1 FileSupplementary Material.(DOCX)Click here for additional data file.


Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628,  Titin, a major component of vertebrate striated muscle sarcomeres, is the largest known polypeptide, with isoforms up to nearly 4 MDa . A59–A60 was uniformly 15N, 13C and 2H-labeled and A60sd was uniformly enriched in 15N and 13C. Cells were grown at 37 °C in D2O-M9 minimal medium containing 15NH4Cl (1 g/L) and either [100 %-D7 and 100 %-13C] glucose (4 g/L) or [100 %-13C] glucose (4 g/L) as the sole nitrogen and carbon sources. For induction of expression, 1 mM IPTG was added when the cells reached an OD600 of 0.7, after which they were left shaking at 37 °C for an additional 15 h. The cells were then disrupted by sonication in a 50 mM TRIS buffer (pH 7.9) containing 6 M guanidinium chloride, 0.5 M NaCl and 15 mM DTT and harvested by centrifugation. Finally the proteins were purified from the supernatant using Ni–NTA resin and eluted with 50 mM EDTA (pH 6.2). Typically 5 mg protein was purified from 1 l of A59–A60 culture. For A60sd yields were much higher, ~15 mg/l of culture. Purity and isotope enrichment were checked by SDS-PAGE and mass spectrometry.The A59–A60 domain tandem and A60sd single domain were cloned into the pET 15b_A170 and PET19b vectors, respectively. They were over-expressed as a N-terminal His6-tagged fusion protein in1H,13C,15N triple resonance probes with z-axis pulsed field gradients; the 750 MHz spectrometer was equipped with a cryogenic probe. Sequence specific backbone assignments were obtained from 2D 15N HSQC-TROSY, 3D HNCA, HN(CO)CA, HN(CA)CB, HN(COCA)CB, HNCO and HN(CA)CO spectra. Data was processed using NMRPipe  were prepared in 0.5 M NaCl, 50 mM MES, 10 mM DTT and 0.01 % azide, pH 6.5. Data were recorded at 293 K on Varian INOVA 600 and 750 MHz spectrometers equipped with 15N–1H HSQC-TROSY spectrum of A59–A60 illustrating the quality of the data and breadth of assignment. The resonances are well dispersed indicating the folded state of the protein. Excluding the resonances of N-terminal His6-tag and 4 Pro amino acids from the very beginning of the domain (labelled −24 to 0), backbone assignment of A59 from A59 to A60 proved nearly complete . In contrast, assignment of A60 in A59–A60 was not as exhaustive: 80.0 % (76/95) for 1HN/15N, 86.4 % (89/103) for 13Cα, 67.7 % (65/96) for 13Cβ, and 73.8 % (76/103) for 13C’. Furthermore, while in A59 most of the unassigned resonances were found in loop regions, in A60 resonances from three spatially separate regions from the first, third and seventh β-strands were totally missing . In order to increase the assignment rate and to help study the interaction between the domains, A60 was expressed alone (A60sd). The completeness of the assignments for A60sd [excluding the N-terminal His6-tag and trailing N terminal residues (25 residues labelled −24 to 0) and the C-terminal trailing residues (residues labelled 199–205)] proved much better than for A59–A60  for 13Cα, 72.9 % (70/96) for 13Cβ, and 93.2 % (96/103) for 13C’. Thus, the source of the missing assignments in the A59–A60 may reasonably be attributed to T2 broadening due to local chemical exchange and the correlation time of the full tandem.Figure Consensus analysis of secondary structure based on CSI (Wishart and Sykes 2 = 0.99) and, excluding changes at the termini where different flanking residues were present, only small shift changes were observed 2]0.5. Aside from the termini, only the loop regions between β-strands A60-β2 and A60-β3, A60-β4 and A60-β5, as well as a number of residues in β- sheets A60-β6 and β7, show a Δ > 0.5 ppm, indicating that their environment has changed significantly. As these regions are localized to the N-terminal face of A60 (assuming a Fn3 fold) this suggests that there are distinct interactions between A59 and A60. Three further strands of evidence support a lack of flexibility between the two domains. Firstly from the observed secondary structure the linker between the β-sheets A59-β6 and A60-β1 is extremely short (residues 95–100). Secondly the observed correlation time of 21 ns for A59–A60 which was measured using a TRACT experiment is consistent with that expected for a rigid double domain construct with small amounts of a higher oligomer present  under accession numbers 19010 and 19011, respectively.Backbone


The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force – the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain – both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2. Once released, the intracellular domain translocates to the nucleus where it can upregulate its target genes. The proteolytic cleavage cascade is initiated when the Notch transmembrane receptor binds to a ligand (Delta/Serrate/Lag-2) in vitroin vitro; removal of the LNRB was also required for S2 cleavage within these experiments Recently, we provided proof of feasibility for the mechanical force model of Notch activation by forcibly unfolding single molecules of the hN2 negative regulatory region. On stretching the hN2 NRR in an atomic force microscope (AFM), cleavage at S2 by the metalloprotease TACE can be directly observed The heterodimerization domain is so-called due to an initial cleavage event carried out by a furin-like convertase at an S1 site located in an exposed loop of the domain. This S1 cleavage occurs during processing and transport of the Notch receptor to the cell membrane and, whilst it does not disrupt the core structure of the HD domain , the domain is effectively divided into two non-covalently associated N- and C-terminal subunits, HD-N and HD-C. The stable core structure of the HD domain comprises a kinked α1-helix and the β4-strand Given the increasing evidence for a role for mechanical force in the Notch signal activation mechanism, we investigate the effect of six mutations, related to those shown previously to destabilise the HD domain within hN1, 2To analyse the effect of the chosen six mutations  on the mechanical stability of the NRR, relevant to a force-induced Notch activation mechanism, a combined experimental  and computational  approach was taken to forcibly unfold the mutated hN2-NRR proteins. The video output representing results from each MD simulation can be found in the 2.1The first key region to be affected by three out of the six mutations investigated is the LNRA:B linker region/LNRB. Force-extension profiles from the MD simulations of mutated constructs compared to the wild-type construct are shown in P < 0.001). This further confirms results observed within the MD simulations indicating a reduction in the force required for unfolding resulting from mutating residues A1647, L1563 and V1623. Furthermore, data suggests this reduction in force is related to the unfolding of the LNR domains, and from the MD simulations can be more specifically located to the removal of the inhibitory plug formed by residues in the LNRA:B linker region and LNRB, key events in removing the auto inhibition and allowing for cleavage at the S2 site. Simulations of the other three mutations  show some reduction in force required for the LNRB unfolding, whilst maintaining the force required for LNRA:B linker removal  from 2800 ps until 4400 ps, where it plateaus at 1.5 nm A, howeveExamination of the AFM data obtained for L1566P shows a reduction in the number of unfolding events requiring a force higher than 500 pN A, in agrAlthough a decrease in the force required for unfolding A1647P relative to WT is observed within the AFM unfolding data A, little3An increasing quantity of evidence supports a force-driven mode of activation of the Notch signalling pathway, whereby exposure of the critical S2 site in the Notch NRR occurs upon mechanical unfolding of the protein during transendocytosis of the Notch extracellular domain into the ligand presenting cell. Having directly examined the mechanical unfolding and cleavage of the hN2-NRR in proof-of-feasibility of this proposed activation mechanism in vitro, which likely corresponds to the reduced forced required for both LNRA:B linker and LNRB removal as observed in silico. Whilst in vitro evidence indicates that the unwrapping of LNRA and LNRB away from the HD domain is necessary and sufficient for S2 site exposure and cleavage by metalloprotease With respect to the LNRA:B linker/LNRB, half of the hN2-NRR mutations made  caused a large and significant reduction in the force required to ‘unplug’ this linker and the LNRB away from the HD domain. In AFM experiments, the force required for NRR unfolding is dramatically reduced compared to WT. In MD simulations, the force barrier attributable to LNRA:B linker removal, identified in previous analyses with WT hN2-NRR as the first significant peak in the force profile, is no longer detectable in the three variants. Furthermore, a significant reduction is observed in the second unfolding peak, corresponding to the removal of the LNRB from its contacts with the HD domain . Our previous simulation study highlighted removal of the LNRA:B linker as the minimal structural change required to allow metalloprotease cleavage at the S2 site In vitro studies looking at the functional effect of the coordinated ions conclude that whilst removal of the calcium and zinc ions with EDTA treatment dramatically affects the mechanical unfolding of the hN2-NRR Two hN2-NRR variants reduce the mechanical stability of LNRC, which is observed to dissociate from the HD domain and unfold in MD simulations . The L1566P mutation is located within the α1-helix adjacent to the Arg1567 residue, shown previously to interact with residue Asp1506 within the LNRC to form a salt bridge Interestingly, A1647P is located within the α3-helix of the HD domain yet affects the LNRC:HD domain interaction, the site of which is some 24 Å away. AFM data of A1647P shows a large reduction in the force observed for the unfolding of the NRR, compared to WT, and the LNRC is observed to move away from the HD domain in simulated unfolding of A1647P . The mutin vivo these particular mutations activate signalling primarily via decreased mechanical stability or a direct increase in the subunit dissociation rate , which can expose S2. However, a comparison of the biochemical data published previously and this structural data highlights the potential for different unfolding mechanisms based on the stress they are subjected to. A number of these mutations, despite showing little change in the ligand-independent activation of hN2 Note that the hN2-NRR proteins in this study have an intact S1-site and are not treated with furin. The importance of furin cleavage still requires full understanding In conclusion, our studies provide insight into key structural elements within the Notch NRR for conferring mechanical stability. The mutations studied all cause destabilisation of one or more of the key regions within hN2-NRR during mechanical unfolding, leading to a more accessible S2 cleavage site. The observations highlight differences between the ligand independent and ligand induced notch signalling pathways and add further weight to a force activation model for Notch signalling. The clear next step is to utilise these single molecule approaches on live cells as recently performed by Gordon et al. 44.13) and C-terminal hexahistidine (His6) tags through expression in T7 Express Escherichia coli cells (NEB), as previously described 2+-His; His-Trap™ HP columns) and size exclusion chromatography (S-200 Superdex) . Purified protein constructs were refolded in a redox buffer  before structural characterisation  was performed to confirm folded protein.Recombinant NRR wild type (WT) and variant constructs were produced as GST-fusion proteins with N-terminal poly-lysine , with a 2 s surface delay to ensure Nickel-His coordination (based on previous experiments). Buffer conditions retained the necessary metal ions (Ca2+ and Zn2+) for protein stability. Controls were performed without protein present to determine the level of features occurring due to surface adhesion contacts and to distinguish these from protein unfolding features observed when protein is present. Attachment controls were also performed using competing ions to disrupt Ni2+-His interactions at the tip. Statistical significance was determined with the Mann Whitney U Test on each data set compared to that of the WT construct.Gold coated AFM tips were functionalised with Ni4.32OO4) using Swiss Model PDB Viewer version 4.0. MD simulations were performed on WT and mutated hN2 NRR constructs using Gromacs Mutated protein constructs were created from the hN2 NRR template  was placed in a 3-dimensional box (dimensions: 10 × 10 × 50 nm) of 100 nM NaCl in simple point charge water JMA designed the research hypothesis; NLS and JMA designed the experiments, and wrote the paper. NLS performed the experiments and analysed the data.


To test the reliability and validity of the Cancer Treatment Satisfaction Questionnaire (CTSQ), to assess its relation with quality of life (QoL), and to assess the interpretability of the domain scores in lung cancer patients receiving intravenous chemotherapy.Patients with stage IIIB and IV non-squamous non-small cell lung carcinoma treated with pemetrexed were enrolled in our study. They completed the 16-item CTSQ and two other  QoL questionnaires. Information about sociodemographic characteristics, cancer stage, and the experience of adverse events was collected. Internal consistency, construct validity, and clinical interpretability were calculated.Fifty-five patients completed the CTSQ. Correlations of the CTSQ items with its domain were all above 0.40. A high correlation between item 8 and the expectations of therapy and satisfaction with therapy domain was observed . The CTSQ domains demonstrated good internal consistency and low to moderate correlations of the CTSQ with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 and World Health Organization Quality of Life-BREF. No significant differences in mean domain scores were observed in relation to the number and severity of different adverse events and chemotherapy-related adverse events.The Dutch version of the CTSQ was found to be a reliable and valid instrument to assess satisfaction and expectations of treatment in lung cancer patients receiving intravenous chemotherapy. Furthermore, the CTSQ proved to be of additional informative value as not all of its domains correlated with the various domains of the existing HRQoL instruments. Anticancer therapies mostly offer modest improvements in survival, making the occurrence of adverse events an important outcome parameter in studies and clinical practice. It is well established that adverse events impair health-related quality of life (HRQoL)  and thatIn 2005, the CTSQ was developed to assess patients’ opinions and feelings concerning their cancer therapy and associated adverse events . A psychGiven these considerations, the objective of our study was focused on three main aspects of the CTSQ: (1) to test the reliability and validity of the Cancer Treatment Satisfaction Questionnaire (CTSQ) in patients with lung cancer receiving intravenous chemotherapy, (2) to assess its relation to health-related quality of life (HRQoL), and (3) to assess the interpretability of the domain scores.This study was approved by the Institutional Review Board of the Erasmus University Medical Center in Rotterdam, the Netherlands. Patients were recruited from a university hospital  and a large teaching hospital  specialized in lung cancer care located in the western part of the Netherlands. Patients were enrolled in our study if they met the following criteria: They provided written informed consent, were aged 18 years or older, and were treated with at least four cycles of pemetrexed monotherapy or in combination with cisplatin or carboplatin as either first or second line. Patients were excluded if they met the following criteria: They were not able to read Dutch or could not complete the questionnaire because of a physical or mental condition (which prohibited participation in the study). A sample size of at least 50 patients was needed in order to perform a validation study .The CTSQ contains three domains covering 16 items: expectations of therapy , feelings about side effects  and satisfaction with therapy . Each item was scored on a scale from one to five with a value of one corresponding with the worst response and a value of five representing the best response. Four items are reverse-coded. Domain score was calculated by the formula: (mean of completed item scores −1) × 25. This results in a domain score ranging from 0 to 100, with a higher score representing a better outcome on each domain.The original CTSQ was translated into Dutch by TransPerfect Translations Inc. according to the forward/backward methodology following international guidelines . QuestioThe European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) is a cancer-specific HRQoL instrument with demonstrated psychometric properties . It consThe World Health Organization Quality of Life-BREF (WHOQOL-BREF) is a shorter version of the original WHOQoL-100 questionnaire. It is a generic QoL instrument and comprises 26 items divided over 4 domains: physical health, psychological health, social relationships, and environment and one facet: overall quality of life and general health. The WHOQOL-BREF domains are scored on a 4–20 scale and the facet on a 2–10 scale with higher scores indicating a better quality of life . The WHOAll questionnaires were completed after patients finished their four-cycle therapy of chemotherapy. In addition to completing the instruments, respondents were asked to provide information about the frequency and severity of adverse events they have experienced (cancer or therapy related). We also collected sociodemographic information  and information about cancer stage, hospitalization (due to cancer or adverse effect of therapy), and the ECOG performance status.Floor and ceiling effects were calculated in our study and were considered to be present if more than 15 % of the respondents achieved the lowest (floor effect) or highest (ceiling effect) possible domain score .Construct validity was evaluated using Pearson’s rank correlation coefficient between questionnaire items and domains. Correlations of 0.40 or higher indicate a good correlation between items and domains .Internal consistency reliability measures to which extent items within a domain correlate with each other to form a (multi-item) domain. Reliability coefficients for the CTSQ domains were estimated using Cronbach’s coefficient alpha where a reliability coefficient of 0.70 or higher was considered to be acceptable .Known-groups validity comparisons were made for the CTSQ domains in relation to the number of different adverse events and its severity. Also the impact of therapy-related adverse events compared to cancer-related adverse events on CTSQ domain score was evaluated. For this analysis, the one-way ANOVA was used to determine whether there are any significant differences between the means of two or more independent groups.The association between the CTSQ domains with domains of the EORTC QLQ-C30 and WHOQOL-BREF was assessed using Spearman’s correlation coefficients.p value below 0.05 was considered to be statistically significant.We assessed interpretability, which is defined as the degree to which one can assign qualitative meaning to quantitative scores. For each CTSQ domain, the MCID was calculated using the approach of 0.5 SD  and 1 stAll analyses were performed using SPSS version 21.0 .Table The mean scores of the ET and FSE domain were 55.6 SD 22.5) and 52.2 (SD 23.8), respectively. The SWT domain had a mean score of 79.7 (SD 13.9), which was much higher compared to the mean scores of the other domains. No patients demonstrated the lowest possible domain score of 0.0. The floor effects for all domains were therefore zero. The FSE domain did not reach the highest possible score of 100, resulting in a negligible ceiling effect for this domain. For the ET and SWT domain, we observed a ceiling effect of 5.5 and 9.1 % respectively, which is below the common accepted limit of 15 %  had a similar correlation with ET domain (0.50) as the SWT domain (0.48). All other comparisons showed good results, as these items correlated better with their own hypothesized domain than with competing domains Table .Table 3CThe internal consistency of the CTSQ domains is given in Table Table The estimates of the MCIDs are given in Table r ≥ 0.40) were observed with global health status, role functioning, emotional functioning and the symptom domains fatigue, nausea and vomiting, and appetite loss. No correlation of 0.40 or higher was observed between the ET domain and the HRQoL domains. The SWT domain only significantly correlated with nausea and vomiting (r = −0.41). The negative correlations between the CTSQ and HRQoL domains indicate that higher scores of the CTSQ domains are associated with worse symptoms.The correlation between the CTSQ domains and domains of the EORTC QLQ-C30 is shown in Table r = 0.52).Results of the association between the CTSQ and WHOQOL-BREF domains are presented in Table Although HRQoL questionnaires inform healthcare professionals about the well-being of their patients, they do not address patients’ expectations and satisfaction with therapy. Brown et al.  demonstrThe previous psychometric validation study demonstrated a positively skewed score distribution of the ET domain with a substantial ceiling effect (20.5) . Even hiAll items correlated better with their own domains than with the other domains, which is in line with the results of the psychometric validation study. However, the correlations between the items and domains were found to be higher in our study compared with the previous study, which might be explained by the homogeneity of the population in our study. We observed that item 8 of the CTSQ (cancer therapy would help you live longer) had strong correlations with the SWT domain and with its own ET domain, which is in line with the results of the previous CTSQ studies , 10. MorIn 2004, a validation study of another patient satisfaction questionnaire (TSQM) was performed and showed significant differences in patient satisfaction and convenience of treatment between different treatment modalities  . As patiAs for the estimates of the MCIDs using the distribution-based methods, we observed similar results for the FSE and SWT domains when we compare our results  with the results of the previous psychometric validation study . However, we found a clear difference of the MCIDs of the ET domain between both studies as in our study a larger change of domain score is needed for it to be considered clinically relevant (MCIDs in our study: 11.25, 9.28; Trask et al. [We observed an increasing number of severe and chemotherapy-related adverse events that corresponded with a decreasing mean FSE domain score. According to Grutters et al. , this maTerwee et al.  suggesteWe were not able to evaluate test–retest reliability since the questionnaire was only given once after the fourth cycle of chemotherapy. If patients fill in the CTSQ a second time after the first completion, it will be hard to define an appropriate interval between those two completions as we included patients who have a relatively poor prognosis. If the interval between these completions is too short, the difficulty may be that they recall their earlier answers upon filling in the CTSQ for a second time. Moreover, when the interval is too long, patients may have progressed in their disease experiencing more adverse events, which may bias our results. However, we do not expect this to be a major problem as this part has already been validated in the psychometric validation study, showing good results .In conclusion, we were able to support the internal consistency reliability and construct validity of the Dutch version of the CTSQ in patients with lung cancer treated with intravenous chemotherapy. Only a few aspects of HRQoL were significantly correlated to items of the CTSQ, indicating the need of using the CTSQ in studies evaluating satisfaction and expectations of patients on cancer chemotherapy. Since patients with disseminated cancer often have a limited prognosis, considering patients’ motivations and needs is of importance to improve HRQoL. We therefore believe that our results may encourage researchers to use the CTSQ to investigate patients’ expectations and satisfaction with therapy in future studies.


The multifunctional signaling adaptor and selective autophagy receptor p62/SQSTM1 is commonly found in dense light-microscopic loci of eukaryotic cells. Recently, Ciuffa et al. demonstrated that p62 is able to form organized polymers of helical symmetry once purified and reconstituted in the test tube . Hence, Structural studies on the protein had been hampered by the fact p62 formed aggregates mediated via the N-terminal PB1 domain. Thus, X-ray crystallographic and NMR structural information on the protein had only been obtained from isolated PB1 domains by preventing it to form large assemblies. While the central part of the protein containing the LC3 interacting region (LIR) is predicted to be disordered, the C-terminal part is composed of the folded UBA domain that recognizes ubiquitinated cargo and had only been structurally characterized in isolation. The recent structural analysis of Ciuffa et al. from a series of successively longer parts of p62 by electron cryo-microscopy (cryo-EM) revealed that the PB1 domain alone is sufficient to form helical polymers with significant flexibility and curvature  .In order to better understand the implications of p62 filament assembly further studies are required to elucidate the mode of interactions with the binding partners. Structural studies on heterooligomeric or heteropolymeric assemblies will be essential to obtain mechanistic insights into the function of the observed p62 filaments. Although it has been established that p62 assembly is critical in autophagy and signaling function, disassembly will be an equally important regulatory mechanism to avoid excessive buildup of large p62 bodies in the cell. Insights into the mechanisms regulating assembly and disassembly of p62 filaments will have important implications for elucidating the roles played by p62 in vital cellular processes as well as in aberrant tumorigenesis.


Here, we report a case of stromal tumor of uncertain malignant potential (STUMP) that was difficult to diagnose. A 53-year-old male was found to have a hard nodule on digital rectal examination; magnetic resonance imaging revealed a large nodule on the left side of the prostate, indicating prostate cancer. However, pathological diagnosis of the biopsy specimen was benign prostatic hyperplasia. Although a papillary tumor in the prostatic urethra was also seen on urethrocystoscopy, the tumor specimen obtained from transurethral resection was not malignant. The tumor in the prostatic urethra recurred only 3 months after transurethral resection, and pathological findings revealed benign hyperplasia not only in the stromal tissue but also in the epithelium; therefore, the prostate tumor was suspected to be STUMP. It took many prostate pathologists a long time to reach the final diagnosis of STUMP. STUMP is a rare benign tumor, difficult to diagnose, and sometimes transforms into stromal sarcoma. Thus, we should consider radical resection in such cases. Stromal tumors of uncertain malignant potential (STUMP) are rare tumors characterized by an atypical, unique stromal proliferation of the prostate. The pathological diagnosis of STUMP is sometimes confused with the stromal proliferations present in BPH, and the distinction may be difficult . Here, wA 53-year-old male was suspected of having prostate cancer because of elevated prostate-specific antigen (PSA) levels at screening. He underwent transrectal biopsy of the prostate at a nearby clinic, which revealed no evidence of malignancy.A year later, he came again to the clinic with the symptom of macroscopic hematuria. The PSA level was 14.7 ng/mL . Digital rectal examination revealed a hard nodule in the left lobe of the prostate, and cystoscopy revealed a papillary tumor in the prostatic urethra. Transrectal biopsy of the prostate and transurethral resection (TUR) of the tumor in the prostatic urethra were performed. The pathological diagnosis was “ductal adenocarcinoma of the prostate” from the specimen obtained from TUR.The left obturator lymph node was slightly enlarged on computed tomography, and the clinical stage was determined to be T3bN1M0. Androgen deprivation therapy (ADT) by bicalutamide was started. Thereafter, he was referred to our institution for radiotherapy.On magnetic resonance imaging, there was a large spread nodule on the left side of the prostate without any signs of seminal vesicle invasion . These cDuring a follow-up visit 6 months later, the patient complained again of macroscopic hematuria. Cystoscopy revealed a papillary tumor in the prostatic urethra with mild inflammatory changes in the mucosa, which was similar to the information obtained from the nearby clinic . We perfFollow-up cystoscopy at only 3 months after TUR revealed recurrence of the large papillary tumor at the same site . We beliSTUMP is a rare tumor characterized by an atypical, unique stromal proliferation of the prostate. Previously, STUMP had been reported in a variety of ways including atypical stromal (smooth muscle) hyperplasia, phyllodes type of atypical stromal hyperplasia, and cystic epithelial-stromal tumors. In 1998, these tumors were called STUMP, when Gaudin et al.  reportedSTUMP shows various features of progression. A retrospective study suggested that the tumor recurred in 46% of cases in patients who did not undergo complete resection . In someThe pathological diagnosis of STUMP is sometimes very difficult, even by pathologists specializing in prostate pathology. STUMP can be histologically misdiagnosed as BPH. It is more difficult to distinguish BPH from STUMP when only small specimen is obtained such as prostate biopsy. However, it is important to recognize that STUMP often recurs and has the potential to change to SS. STUMP can occur at a younger age than BPH, predominantly involve the peripheral zone, and often adhere to the rectum . TherefoIn the present case, it took a long time to establish the final diagnosis because there was proliferation not only in the stromal tissue but also in the epithelium. At first, the pathologist of the other institution  diagnosed ductal adenocarcinoma. We thought that he made a mistake because remarkable epithelial proliferation was seen in the specimen. We corrected the diagnosis to BPH because there were no findings suggesting prostate cancer such as nuclear enlargement and dyskaryosis in the epithelial cells. However, we had to consider that the tumor might have some malignant feature because of the clinical findings such as rapid growing and MRI findings. We suspected STUMP at this time. However, benign epithelial proliferation made it very difficult for us to diagnose STUMP because STUMP is normally recognized as having proliferation of stromal tissue. We had to consult specialists on prostate pathology in Japan and the United States, and we conclude that the diagnosis was “STUMP with a feature of epithelial proliferation” according to their opinion.Before radical prostatectomy, the diagnosis of STUMP was only suspected. However, radical prostatectomy seemed to be a better option than repeat TUR because the tumor in the prostate (suspicious of STUMP) had a tendency of rapid growth and was not amenable to complete resection by TUR, and such a procedure could transform the tumor from STUMP to SS.In summary, we report a case of prostatic STUMP that was difficult to diagnose. STUMP is rare and can be histologically misdiagnosed as BPH. It has the potential to transform into SS. Therefore, STUMP should be managed aggressively with radical resection.


Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products.The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an  Natural products of microorganisms are potential source of bioactives that have been extensively exploited to develop next generation anti-infective drugs proposed by pharmaceutical companies , 2010. BPolyketide synthases (PKSs) are modular proteins involved in the biosynthesis of complex bioactive molecules through sequential catalytic activities. These enzymes mediate biosynthesis of bioactive molecules with diverse structural complexities by combinatorial use of a specific sequential order of catalytic domains. The tailoring of catalytic domains and AA sequence of these domains are drastically changes with natural bioresources and therefore, the nature and chemical structure of end product is varied between/within the species . The mecBacillus and Pseudomonas was well-established. However, biosynthetic pathway of biosurfactants produced by marine actinobacteria, in general remains undisclosed. The biosurfactants invariably showed antibiofilm activity without inhibiting the biomass of pathogens tested. Based on in vitro experiments, it was found that the biosurfactants produced by marine actinobacteria is having antimicrobial and antibiofilm activity. The PCR amplified KS domain from these actinobacteria envisages the biosynthetic pathway of biosurfactants might have mediated through PKS biosynthetic gene clusters. Therefore, in this study, the in vitro findings are integrated with in silico analysis to substantiate the hypothesis that the biosynthesis of biosurfactants produced by marine actinobacteria might have mediated by PKS gene. To date, there are few reports about the interaction between PKS type II gene clusters and biosurfactant production  and D. nigra  collected from southwest cost of India. The 16S rRNA GenBank accession numbers as follows Nocardiopsis alba MSA10: EU563352 (Brevibacterium aureum MSA13: GQ153943 (Brachybacterium paraconglomeratum MSA21: GQ153945 (Streptomyces sp. MAD01: GQ246755 (Streptomyces dendra MS1051: EF417875 (Table 1). The PCR temperature profile used was 95°C for 3 min, and then followed by 30 cycles at 95°C for 30 s, 56°C for 30 s, and 72°C for 60 s and finally an extension step at 72°C for 10 min. The resultant amplified PCR products were purified and cloned using the TOPO TA cloning kit (Invitrogen) for sequencing.The actinobacterial strains used in this study were already been isolated from marine sponges, such as EU563352 , BrevibaGQ153943 , BrachybGQ153945 , StreptoGQ246755 , and StrEF417875 , respectVibrio harveyi. The biofilm was allowed to develop on cover slips and treated with the actinobacterial extracts and incubated for 48 h at 37°C. After incubation the planktonic and spent media were discarded. The cells were washed twice with deionized water air dried and stained with 0.1% acridine orange and examined under confocal laser scanning microscopy (CLSM).The culture supernatant obtained from actinobacterial strains were evaluated for biofilm inhibitory effect against V. harveyi was allowed to develop on 96-well plate and treated with 50 μl culture filtrates of the five actinobacterial strains and incubated for 24 h at 37°C. Untreated wells were set as control. After 24 h the bacterial suspension was collected and then treated with 100 μl of phosphate buffered saline and 50 μl of MTT at concentration of 0.3% were added and then incubated for 2 h at 37°C. The MTT solutions were removed and formazan crystals formed were dissolved in 150 μl of DMSO and 25 μl of 0.1 M glycine buffer of pH 10.2. The absorbance was recorded in a microplate reader at 550 nm.Cell viability of the bacteria in the biofilm was assessed using MTT assay as described by 1 and these deduced AA sequences of type PKS II and ketosynthase were deposited to NCBI-GenBank with the accession numbers of ACS45380–ACS45382 (type II PKS), and ketosynthase bearing following accession numbers ACV31767 and ABP57802. KS domain of type II PKS gene sequences and ketosynthase (Cds) sequences of sponge-associated actinobacteria were retrieved from National center for Biotechnology Information2. GenBank accession numbers of these KS domains and ketosynthase sequences were given as GQ153947 , GQ153948 (B. aureum MSA13), GQ153949 (Brachybacterium sp. MSA21), and GQ246762 (Streptomyces sp. MAD01), EF520724 (Streptomyces dendra MS1051), respectively. The predicted KS domains of all retrieved actinobacterial gene sequences and the PKS type II protein sequences from reference actinobacteria were aligned by CLUSTAL W23 and translated deduced AA sequences were verified using the NCBI-BLAST4 search with expected value set to the default value of 10 was performed using the protein sequences of N. alba, B. aureum, Brachybacterium sp. MSA21, Streptomyces MAD01 and S. dendra, respectively, and the various sequences against 138 complete eubacterial and 20 complete archaebacterial genomes. Phylogenetic tree of the deduced AA sequences of PKS II segments and ketosynthase genes were generated using neighbor-joining method through MEGA programs  actinobacterial KS dataset was analyzed, whether these domains are modular or iterative KS domains. All these three iterative KS domains of PKS type II gene clusters of actinomycetes were modeled using comparative modeling approach. Threading analysis was carried out using a local version of threader package5 to identify the structural templates for modeling of actinobacterial KS domains. The remaining two KS domains (from Streptomyces MAD01 and S. dendra MSI051) have been modeled using fatty acid KAS structure as template (Escherichia coli KAS I), which show only about 40% sequence identity with polyketide KS domains. Even the sequence identity was lesser between the target and template, the two KS proteins structures can be reliable and they adopt similar structure. The secondary structures of type II PKS and ketosynthase domains of 3D models were created using a (PS)2 is an automated homology modeling server  analysis was carried out by HMMER package. The available three (g server . The (PS24) as per Figure 1). The active moieties were identified from GCMS data. The active moieties of MSA10, MSA13, and MSA21 were evidenced as biosurfactant molecules, but the moieties of MAD01 and MSI051 were not related with biosurfactants (Table 2). The antimicrobial moiety of MAD801 was identified as cyclohexane carboxylic acid hexyl ester. It was reported that cyclohexane carboxylic acid is a moiety of the antifungal polyketide ansatrienin A . The KS domain of these gene segments were translated into AAs counts, viz; 191, 212 and 220, respectively. All these KS domains encodes the condensation enzymes (cds), which catalyzes (decarboxylation or non-decarboxylation) Claisen-like condensation reaction, and the KS domains sharing the strong structural similarities are involved in the synthesis and degradation of fatty acids.Sponge-associated actinobacteria: i.e., in silico analysis to identify KS domain counterparts from modular and iterative PKSs and other PKS families. The analyzed domains are separated into distinct clusters in a phylogenetic tree . Based on HMM by the HMMEP package, three actinobacterial KS domain of type II PKS genes were analyzed and the results show that these three isolates contains iterative PKS gene and this outcome provides potential in genome sequencing efforts for the identification of novel PKS genes. Iterative condensation steps play a vital role in biosynthesis by PKS proteins and phylogenetic analysis of iterative KS domains inferred that the clustering of iterative PKS gene sequence is highly correlated with the number of iterations they perform. From this study, we suggest that marine sponge associated actinobacterial community predominantly possesses the iterative KS domain of type II PKS rather than modular type I PKS or NRPS-PKS hybrids. The type II PKS from three different genera is characterized to study and understand their function and diversity. The isolation and identification of PKS with different enzymatic activity in marine actinobacteria has been reported, as well as the occurrence of PKS gene families in a community  type II PKS genes and it can be hypothesized that the antimicrobial biosurfactants synthesis might be mediated by iterative type II PKS genes. Another group of actinobacterial antibiotics producers from the marine sponge D. nigra such as S. dendra MSI051 (Streptomyces sp. MAD01 (Table 1).KS domain of PKS gene is the most conserved catalytic domain and is involved in the tailoring PKS molecule by catalyzing the chain condensation step. We have performed ommunity . This isterature , 2015 eva MSI051  and Strep. MAD01  were incof V. harveyi. Among the extract used the lipopeptide producer MSA10 and MSA 13 inhibit the biofilm formation by 80% compared to the other actinobacterial extracts . The antibiofilm effect may be due to the biosurfactant production mediated by PKS gene.Antibiofilm effect of the culture supernatant was well noticed by CLSM. The culture supernatant inhibits the biofilm formation Figure 4. When compared to the control the extracts from MSA 10 and MSA 13 inhibits the viability of Vibrio cells by more than 80%, followed by MSA 21 by 70%.The viability of the cells were reduced by adding the biosurfactants as shown in In silico analysis of Type II PKS and ketosynthase unveiled an unprecedented organization of various domains encoding discrete ketoacyl synthase (KAS) and thiolase, PKC, CK2, and ACP some are lacking an ACP. Certain polyketides undergoes non-iterative biosynthesis which involves a novel type II PKS that acts directly on acyl CoA substrates. These results demonstrate the capability of nature’s in designing complex bioactive compounds and suggest new methods for PKS design and engineering through synthetic biology approaches to expand the scope and diversity of polyketide library. The structural diversity of PKS would ultimately help in searching for PKS with novel chemistry for combinatorial biosynthesis . KASs are involved in the elongation steps in the pathway of fatty acid biosynthesis. KAS III is involved in the catalysis of the initial condensation and KAS I and II are responsible for elongation steps by Claisen condensation of malonyl-ACP with acyl-ACP. Remaining three protein sequences lack ACP, some non iterative type II PKSs lack ACP, utilize acyl CoAs as substrates for macrotetrolide biosynthesis. It was reported that the PKSs are using ACP to activate the acyl CoA substrate and channel the polyketide intermediates  ACS45380.1 was closely related to those of ACT PKS. Type II PKS  consists of seven structural domains includes Asn Glycosylation, CK2 Phospho site, PKC, Tyr Phosphosite, ACP and KAS (49–161 AA residues) which shares 59% similarity with ACT polyketide putative beta-KAS 2 which contains eight chains, out of which two chains are homologous to our PKS protein which are chain A: beta-KAS/acyl transferase and chain B: ACT polyketide putative beta-KAS 2. The synthesis of aromatic polyketides are mostly begins with the formation of a polyketide chain , 18.32% of Acidic , 15.18% of basic , 3.66% of sulfur , 3.66% of aromatic , and 12.04% of aliphatic hydroxyl .Most of the KS are dimeric with active site at the interface of dimer and type II PKS probably functions by making contact across the twofold axis and the active sites of KS are accessible to ACP . In the de chain . The polB. aureum) ACS45381.1 is sequentially identical to type I ketosynthase (Streptomyces sp. T12-208) ACR61389.1. Structurally it is similar to the human fatty acid synthase (FAS), a modular enzyme involved in the metabolism of fatty acids and a drug target of antineoplastic and anti-obesity agents. Detailed structural study on human FAS has been limited due to its size and flexibility. Large part of human FAS that encompasses the tandem domain of beta-KAS is closely related to the KS domain of B. aureum. The KS domains are appear as the canonical dimer, and its substrate-binding site differs from that of bacterial homologs but is similar to type II PKS of B. aureum.Type II PKS of (Brachybacterium sp. MSA21) ACS45382.1 is identical to type I PKS of Streptomyces sp. and structurally proposed to contain five domains as follows KAS C and KAS N, myristyl site, PKC, and thiolase. The tertiary structure of the protein depicts six alpha helix and six beta sheets and composed of 50.45% of aliphatic , 5% of aromatic , 2.73% of sulfur , 11.36% of basic , 13.18% of acidic , and 13.18% of aliphatic hydroxyl  AAs.According to domain analysis, the PKS is a multi-domain protein consists of 14 domains includes ASN glycosylation, PKC, CK2, beta KAS, ACP synthase III, thiolase C and N terminal, and KAS C terminal domains. The position of KAS domain is 1–151 and 159–212 AA residues. The AA composition of the protein is predicted with 48.58% of aliphatic , 5.19% of aromatic , 2.83% of sulfur , 9.43% of basic , 16.51% of acidic , and 14.15% of aliphatic hydroxyl . Type II PKS , 6.94% of aromatic , 3.47% of sulfur , 13.87% of basic , 19.08% of acidic , and 10.40% of aliphatic hydroxyl . The ketosynthase (Streptomyces dendra) ABP57802.1 found to have seven domains includes CKII phosphorylation site, PKC, KS C terminal and N terminal, phage tail fiber repeat and the AA composition is 42.26% of aliphatic , 4.76% of aromatic , 4.17% of , 13.69% of basic , 18.45% of acidic  and 11.90% of aliphatic hydroxyl . The structural configuration presents six alpha helix and four beta sheets and mimics the structure of chain A. The ACT ketosynthase CLF with the values as follows, E-value: 1.00e – 72, bit-score: 258, aligned-length: 191, and identity to query: 67%. The PSI-BLAST shares 99% of similarity with doxorubicin PKS , 3-oxoacyl-ACP synthase I (Streptomyces avermitilis MA-4680), putative ketosynthase of Streptomyces antibioticus with , and granaticin polyketide putative beta-KAS 1 of Streptomyces hygroscopicus ATCC 53653 . KS domain analysis of type II PKS and ketosynthase was performed using PSI BLAST and MEGA (CLUSTAL W2) to highlight the unique conservative motif of each protein. The strain B. aureum shares specific motif with BAH67362.1 (PKS Streptomyces minoensis) denoted as “VDTACSSSLVALHLAAQALRSG.” Comparative analysis of KS domain of Brachybacterium sp. MSA21 exhibit the presence of unique motif PQQR(H)L in all the reference sequences which are capable of synthesizing cirramycin (BAH67190), minomycin (BAH67362), maridomycin (BAH67036), an anticancer compound (BAH67464), and platiomycin (BAH67144). This is the first report on the possible structural diversity mediated by type II PKS in Brachybacterium sp. MSA21.The position of the KS domain is 1–159 and 167–220 AA residues. In PSI- BLAST, PKS is predicted to have a structure similar to chain A, the ACT ketosynthase chain length factor since 73% identity, the Brachybacterium and Brevibacterium showed a unique clustering, found KS domain of Brevibacterium clustering between two Streptomyces group and each group having two isolates and they possess high similarities among them like 100 and 98, respectively, but having less homology with B. aureum . The Brachybacterium was closely clustering with S. albus J1074 with 100% similarity. N. alba was not clustered with any actinobacterial KS domain since it was having the unique identity with KS domain of Streptomyces MAD01 and S. dendra. KS domain of Streptomyces MAD01 showing 97% of similarities with S. purpeofuces NBRC 14457 and S. dendra was clustering between Streptomyces sp. JS-14 and S. macrosporeus with 98 and 97% similarity, respectively. From the cluster analysis, we observed that two different marine sponge-associated actinobacteria possessing the identical KS domains of iterative PKS.Ketosynthase domain phylogeny was used to infer the phylogeny of type II PKS and ketosynthase. Phylogenetic analysis showed that the sponge associated actinobacterial sequences of PKS II genes and KS fragments were matched to conserved regions of previously characterized functional domains of other PKS I, II, and ketosynthase proteins. The KS domain of Brevibacterium and Brachybacterium represent the KS genes which proves the unexplored resource for natural-product discovery. Conversely, the nearly ubiquitous detection of PKS genes in Streptomyces and Nocardiopsis envisages the possibility similar kind of natural products, but in reality the compounds are expected to be highly complex with diverse bioactivities (Streptomyces MAD01 and S. dendra suggests that they are not associated with the production of iterative type of PKS domains. The mixed clustering of different sponge associated KS domains already been documented here for the first time we are reporting the evolutionary relatedness of KS domains of type II PKS and ketosynthase from D. nigra and F. cavernosa isolates. According to recent literatures, the PKS genes and their products exhibit novel insights in antimicrobial drug discovery (The major aim of this study was to find out the gene diversity of the PKS II and ketosynthase in two marine sponge associated actinobacterial polulation. The KS gene diversity could be useful to understand the evolution pattern of actinobacteria in the marine sponges, mode of interaction between sponge and associated microbes  and chemtivities . To overiscovery . KS domaD. nigra failed to produce biosurfactants but has the capability to synthesis polyketide based antagonistic compounds. There is an existing evidence for the synthesis of lipopeptide biosurfactant in Bacillus subtilis by NRPSs or hybrid PKS/NRPSs (Streptomyces sp. MAD01 possess good biofilm forming capacity as well as antimicrobial activity against test organisms. It also proves that a ketosynthase type II PKS system is responsible for the biosynthesis of the antagonistic compounds in marine actinobacteria. Based on the present findings, the production of biosurfactants might be linked with type II iterative PKS gene cluster and the synthesis of biosurfactant by the sponge-associated actinobacteria might have significant role in the chemical ecology of host sponge (in vitro and in vivo experiments. Based on the functions of biosurfactants including antibacterial/antibiofilm activity, the biosurfactants may play a role in host defense fouling processes (The marine sponge-associated bacteria have been recognized as rich source of biological macromolecules that are of potential interest to various industrial sectors . Study rKS/NRPSs . These mt sponge . Howeverrocesses . TherefoN. alba and B. aureum have the potential of constructing “minimal PKS” and the later species share the specific motif “VDTACSSS” with S. minoensis. Both strains displayed PKS domains structurally similar with encoding ACT. S. dendra is found to have a unique repeat called phage tail fiber repeat which is responsible for altering the host specificity of secondary metabolites through protein–protein interaction. The other three actinobacterial strains Brachybacterium sp., Streptomyces sp., and S. dendra lack ACP results in inactive minimal PKS or may act non-iteratively. This study also provides a new insight on the KS genes of Brevibacterium and Brachybacterium proving that marine resources are still largely unexplored for natural-product discovery. In these regards, in silico gene mining is quite useful for prospecting novel metabolites produced by marine sponge endosymbionts. Further in vitro studies are needed to design novel natural products using a biosynthetic engineering approach.In the current study, the iterative nature of actinobacterial type II PKS was proved by HMM profile. The domain architecture of in silico analysis, ANL performed in vitro assays, NAA and MV performed data analysis and interpretation, GSK, GS, and JS written the manuscript.JS and GSK designed the experiments, GS performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ucides cordatus is an abundant mangrove crab in Brazil constructing burrows of up to 2 m depth. Sediment around burrows may oxidize during low tides. This increase in sediment-air contact area may enhance carbon degradation processes. We hypothesized that 1) the sediment CO2 efflux rate is greater with burrows than without and 2) the reduction potential in radial profiles in the sediment surrounding the burrows decreases gradually, until approximating non-bioturbated conditions. Sampling was conducted during the North Brazilian wet season at neap tides. CO2 efflux rates of inhabited burrows and plain sediment were measured with a CO2/H2O gas analyzer connected to a respiration chamber. Sediment redox potential, pH and temperature were measured in the sediment surrounding the burrows at horizontal distances of 2, 5, 8 and 15 cm at four sediment depths  and rH values were calculated. Sediment cores (50 cm length) were taken to measure the same parameters for plain sediment. CO2 efflux rates of plain sediment and individual crab burrows with entrance diameters of 7 cm were 0.7–1.3 µmol m−2 s−1 and 0.2–0.4 µmol burrows−1 s−1, respectively. CO2 released from a Rhizophora mangle dominated forest with an average of 1.7 U. cordatus burrows−1 m−2 yielded 1.0–1.7 µmol m−2 s−1, depending on the month and burrow entrance diameter. Laboratory experiments revealed that 20–60% of the CO2 released by burrows originated from crab respiration. Temporal changes in the reduction potential in the sediment surrounding the burrows did not influence the CO2 release from burrows. More oxidized conditions of plain sediment over time may explain the increase in CO2 release until the end of the wet season. CO2 released by U. cordatus and their burrows may be a significant pathway of CO2 export from mangrove sediments and should be considered in mangrove carbon budget estimates. Studies investigating the CO2 release by crab burrows or the reduction potential in the sediment surrounding the burrows have only been conducted for the relatively small fiddler crabs 2 release from mangrove sediment approximately 2 to 5 times compared to plain sediment 2 originated from the sediment surrounding the burrows and the remainder from the crab inside the burrow 2 production rates. The rates decreased with increasing distance from the burrow, indicating that the change in reduction potential can influence carbon decomposition processes 2 efflux from the sediments To better evaluate the role of burrowing crabs in carbon cycling, it is important to quantify the amount of carbon stored and released by the crabs and their burrows. Bouillon et al. Ucides cordatus (Ucididae), which, together with much smaller fiddler crabs, influence with their burrows the microtopography of the sediment in Brazilian mangrove forests. Araújo Jr. et al. U. cordatus burrows oxidized the sediment. However, the study did not provide information on the supposed gradient and the horizontal range of the reduction potential in the sediment surrounding the burrows, likewise, no CO2 efflux rates were measured.Araújo Jr. et al. U. cordatus on carbon dynamics and sediment reduction potentials were evaluated by (i) quantifying the CO2 efflux rate of individual burrows compared to plain sediment and (ii) measuring the reduction potential in radial profiles in the sediment around burrows at different depths in a Rhizophora mangle forest, respectively. We hypothesized that (i) sediment with U. cordatus burrows has higher CO2 efflux rates than sediment without burrows and (ii) reduction potentials in radial profiles of the sediment surrounding the burrows down to 50 cm depth decrease gradually from the burrow wall, approximating conditions of non-bioturbated sediment.In the present study the effects of bioturbation by Ucides cordatus is listed as endangered in the Brazilian legislation due to the risk of overfishing. All research activities were allowed by a research permit granted by SISBIO . Field work was performed in the extractive reserve “Reserva Extrativista Marinha de Caeté-Taperaçu”.The artisanally harvested study species Rhizophora mangle (Rhizophoraceae). Other mangrove tree species are also present including Avicennia germinans (Acanthaceae) and Laguncularia racemosa (Combretaceae) The study area is located on the Ajuruteua peninsula, the western bank of the Caeté river estuary in North Brazil . Field wMean annual temperature in 2012 was 26.3±1°C  was then placed on top of the PVC collar. Since the respiration chamber was opaque no CO2 was consumed through photosynthesis during measurements. The CO2 efflux was recorded over 2 min and the measurement repeated five times. Between replicates, the chamber was opened for 25 s to release the accumulated CO2. Most probably, these measurements included the respiration of the crab inside the burrow. The contribution of crab respiration to the measured CO2 efflux rate was estimated by a separate experiment (see below). Sediment temperature was measured outside the collar at 2 cm sediment depth with a thermocouple (OMEGA Engineering).For measuring the CO2 efflux rate of plain sediment without U. cordatus burrows and at the most with very few fiddler crab burrows was performed, following the same measurement protocol as for the sediment with burrows. However, four replicates were taken, because preliminary tests had shown that variance of measured values for plain sediment was smaller than for sediment with U. cordatus burrows.As a control, the CO2 efflux rates were calculated for a linear flux rate U. cordatus burrows, the surface area inside the collar also included the burrow wall surface area. It also had to be considered that the relationship between burrow opening area and surface sediment area inside the collar did not represent the average relationship of burrow opening area to plain sediment area at the study site due to specific selection of single burrows and the small collar area. Under natural conditions, the proportion of burrow to plain area is lower  were obtained. From these values we derived CO2 efflux rate of individual burrows (µmol burrows−1 s−1) with entrance diameters of 5, 6 and 7 cm corresponding to the range of burrow diameters sampled in the field. CO2 efflux rates for mangrove sediment based on the CO2 efflux rate of plain sediment and individual burrows were then obtained with the help of size-specific crab burrow density data. Two burrow densities were chosen, one represents the mean U. cordatus burrow density in a R. mangle dominated forests with 1.7 ind. m−2 for the Furo Grande area −1 m−2 since most U. cordatus burrows have only one opening. The other chosen burrow density was 11.9 burrows−1 m−2 (unpublished data) representing the increased burrow density around dense R. mangle stilt roots.2 efflux rate of a burrow without a live crab was not feasible as removal of the crab would have strongly disturbed the sediment. To nevertheless allow an estimation of the proportion of the CO2 efflux rate contributed by the crabs, their respiration rates were determined in an additional experiment. Adult male U. cordatus were caught nearby the study site. The carapace width (cm) of each animal was measured and the wet mass (g) was calculated by the regression formula for male U. cordatus provided by Diele Measuring the CO2 released by each crab was measured five times for 10 min with breaks of 25 s in-between to open the chamber and release the accumulated CO2. Crabs were not acclimated inside the chamber prior to measurements. Therefore, the five consecutive measurements presented a gradient of crab respiration from a least acclimated state to a more acclimated one. CO2 respiration rates of crabs (µmol CO2 kg−1 s−1) were calculated as described above, but related to wet mass instead of surface area in the calculation.In the laboratory, crabs were placed in a PVC basin  with the respiration chamber mounted on top. Under natural conditions crabs rest in air and water (own observation). Therefore, measurements were performed with different crabs in a dry basin  and in a basin containing seawater . CO2 efflux rate was estimated for individual burrows with a burrow entrance diameter (BED) of 5, 6 and 7 cm. Since the inhabitants of the burrows used for CO2 measurements were not captured, their carapace width was estimated from the BED by a least squared regression formula based on data collected by Piou et al. In the next step the contribution of crab respiration to the COU. cordatus by Diele 2 crab−1 s−1, in air and in seawater) was calculated based on the first and the last measurement of the five consecutive respiration measurements. These two measurements represent a “worst”  and a “best” case scenario (crab with the longest acclimatization time and resting in water), since preliminary tests had shown that crabs resting in air had higher CO2 efflux rates compared to crabs resting in water.The wet mass was then estimated from the calculated carapace width with the formula for male 2 efflux rate measurements in the field were performed, reduction potentials of both burrow walls and of non-bioturbated sediment were measured at the selected sites on the same day. Redox potential (±1.0 mV), pH (±0.1) and temperature (±0.1°C) were measured in the sediment excavated by the crabs outside the burrow and in the upper 2 cm of the burrow water with a Sartorius ORP (redox) combination electrode and a WTW Sentix 41 pH-electrode connected to a WTW portable meter (Multi 340i), respectively. The above mentioned parameters were also measured in the sediment surrounding the burrow  at sediment depths of 1, 10, 30 and 50 cm. To be able to measure the reduction potential in deeper sediment depths, one half of the burrow was carefully opened. When the desired measuring depth inside the burrows was below the water table, the water was manually removed with a small vial before taking the measurement. Measurements beyond 50 cm sediment depth were technically too difficult due to the rapid refilling of the burrow with water from below. At each sediment depth, the electrodes were inserted perpendicularly to the burrow wall, thereby subsequent readings at horizontal distances of 2, 5, 8 and 15 cm from the BWS were obtained  was also measured for comparison. Sediment cores were taken at least 15 cm away from burrows and stilt roots to sample areas where the influence of burrows and roots on the surrounding sediment was most likely low. A peat sampler (Eijkelkamp) of 50 cm length and 6 cm diameter was used to obtain the cores. Redox potential, pH and temperature of the sediment core were measured at depths of 1, 10, 30 and 50 cm.Redox potential, temperature and pH values of each measurement were used to calculate the rH values The statistical analyses were carried out following protocols for data exploration and analysis of Zuur et al. Burrow entrance diameters, depth from the surface sediment to the water surface inside burrows, and rH values of burrow water and excavated sediment were each separately analyzed with a one way ANOVA for differences over time. When the trend over time was not linear, time was considered as a factor and a Tukey post-hoc test was applied to detect differences throughout the wet season.2 efflux rates of burrows (bF) and controls were analyzed with a LME model to test for differences over time. The random intercept of the model allowed for heterogeneity among burrows and between the sampling sites of plain sediment. To find the optimal set of fixed terms, a backwards model selection was used based on the maximum likelihood ratio test (ML) and/or on the Akaike Information Criterion (AIC). The validity of the model was checked by examination of diagnostic plots of model residuals. Final models were checked for homogeneity between residuals versus fitted values and covariates. Independence was examined by plotting residuals versus time. The final model was generated and evaluated with the restricted maximum likelihood estimation (REML) and these models are presented in Gaussian linear mixed-effects (LME) models were used for the following analyses, as they can account for inner variation between sediment cores or burrows The rH data for the BWS were analyzed with a LME model to test for differences among sediment depths, horizontal distances from the burrow wall, time and their interaction terms. The random intercept accounted for heterogeneity among individual burrows. Control rH values were tested with a LME model for differences between sediment depths, time and their interaction term. The random term allowed for heterogeneity between sediment cores. Since general trends over time for the control and burrow rH data had already been analyzed in the two LME models and to keep the following models simple, control and burrow rH data were analyzed separately for each month. Monthly data sets of rH values from burrows and controls were analyzed in four LME models to test for differences among horizontal sampling levels, sediment depths and their interaction terms. BWS values of 2 (BWS2), 5 (BWS5), 8 (BWS8) and 15 cm (BWS15) and the control (>15 cm) represented the five horizontal sampling levels. BWS values and the control value were set as categorical covariates, because the exact horizontal distances of the control cores to the neighboring below ground burrow corridors was unknown. The random term allowed for heterogeneity among sampling locations (sediment core or burrow).2 efflux rates of the plain sediment. Because the CO2 efflux rate of a burrow could be associated to several rH values in the burrow wall sediment  no correlation was performed.A Pearson correlation was applied to measure the relationship between surface rH values and COp = 0.07, p<0.001) and were largest in October  and ranged from 0 (burrow completely filled with water) to 0.1 m2.Burrow entrance diameters did not significantly differ over time  for differences over time showed that the LME model which included the term time was not better than the model without this term  higher than for plain sediment .The model of the control rH data with the interaction term sediment depth×time was significantly better  are probably overestimating the crabs’ respiration under field conditions. Respiration rates for the “best” case scenario (crabs with longest acclimatization time resting in water) on the other hand, probably resemble field conditions more closely. Our data thus suggest that crabs contributed about 20–60% to the CO2 efflux rates of individual burrows, which leaves the burrow CO2 efflux rates without crabs to range between 0.04–0.32 µmol burrows−1 s−1 for burrows of 5 to 7 cm entrance diameter  are 0.19±0.04 µmol CO2 crab−1 s−1 and are similar to rates recorded for a large sesarmid crab  of 0.24 µmol CO2 crab−1 s−1During field measurements, crabs were sometimes seen inside their burrows when the respiration chamber was opened between measurements. The crabs did not appear to be stressed by the chamber, since they moved slowly and did not produce foam, as they do when they are handled or otherwise stressed diameter . Respira2 efflux rates of inhabited U. cordatus burrows were at least 20 times higher (−1 s−1) measured in a Tanzanian mangrove forest 2 efflux rates of U. cordatus burrows are probably caused by the larger body size and higher respiration of the crab and the corresponding larger burrow size (and depth) compared with Uca spp. and their burrows  and 1.38 µmol m−2 s−1 (237±53 burrows−1 m−2) and were similar to ours with 1.0±0.02 until 1.7±0.05 µmol m−2 s−1 based on an average U. cordatus burrow density of 1.7 burrows−1 m−2 . Future investigations should focus on the entire crab community of a mangrove site, involving all burrowing crabs, to obtain more precise estimates of overall CO2 efflux rates from the mangrove sediment.In addition to 2 released by sediment or burrows depends on the prevalence of organic carbon in the sediment and its oxidation pathways by microorganisms U. cordatus burrows (70 cm depth) and were similar to corresponding values of the surface sediment 2 released from burrows may not exclusively originate from carbon oxidation in the BWS, but also from the carbon stock in deeper sediment layers which may be released via the burrows 2 released from the water accumulated inside the burrow is unknown. The water may contain CO2 originating from bacterial, meio- and macrofaunal respiration and from tidal input. How the bicarbonate system of the burrow water is constituted is also an unknown, e.g. how such systems react to increasing/decreasing CO2 concentrations and whether changing water levels inside burrows influence the burrow water CO2 gas exchange. CO2 dissolved in water may also be partly exported by the groundwater during the ebb tide The amount of CO2 is produced by root respiration. Information about root respiration of mangrove tree species is scarce; root respiration rates are reported to be low for R. mangle trees. Kristensen et al. 2 released by pneumatophores of Sonneratia alba and Avicennia marina in a Tanzanian mangrove forest, yielding an equivalent or higher efflux rate than sediment with burrows. Due to technical reasons we had to avoid measuring near aboveground aerial roots. However, underground root respiration may have contributed to the CO2 released by burrows and, equally, by surface sediment without burrows. As U. cordatus prefers areas with R. mangle roots when constructing its burrows 2 produced by root respiration. To test this hypothesis, further work on CO2 release must overcome technical restrictions and focus on areas where crabs and mangrove tree roots occur closely together.Apart from microbial decomposition in the sediment, COReduction potentials of the burrow water and the excavated sediment were similar during the wet season and relatively oxidized compared to the BWS and control rH values in deeper sediment depths. This indicates that burrow water is regularly mixed with more oxidized water or oxygen by, for example, tidal flushing or groundwater flow In contrast to the stable values in the burrow water and the excavated sediment, rH values in the BWS changed over time. In April, BWS had the lowest rH values. At 1 and 10 cm sediment depth rH values were even lower compared to plain sediment. The rising water column inside the burrows  and wateU. cordatus BWS rH values indicated a wider oxidized zone, reaching up to 15 cm away from the burrow, compared to the Uca sp. burrows, where the oxidized zone reached only 1.5–4 cm into the surrounding sediment U. cordatus burrows, leading to improved solute exchange conditions between the BWS and tidal water, groundwater and atmospheric air compared to the smaller fiddler crab burrows. Why rH values for the up to 15 cm horizontal distance from the burrow wall did not approximate control values remains unclear. However, an approximation may have been masked by the high variance of the rH values due to nearby below ground burrow galleries or roots The spatial pattern of the 2 efflux rates of plain surface sediment increased over time. Possibly surface sediment became more oxidized towards the end of the wet season and a change in the microbial decomposition process may thus have resulted in the different CO2 efflux rates 2, which is normally released over the sediment surface, may have been dissolved after rainfalls and stored in rain water filling sediment pore space. This would have increased the sediment CO2 concentrations, while less CO2 would have been released from the sediment, as observed in our study at the beginning of the wet season. Corresponding results have been obtained by studies performed in a temperate pinewood forest 2 efflux rates of plain surface sediment, the CO2 efflux rates of individual burrows did not change over time, despite simultaneously varying rH values. The reasons for this are not clear.COUcides cordatus has been mostly discussed in terms of carbon retention through litter feeding. The results of the present study emphasize the crabs’ importance for carbon export via CO2 release. Respiration by U. cordatus and CO2 release by its burrows increase the CO2 efflux rate compared to plain sediment by 15–71% assuming a conservative average density of 1.7 burrows−1 m−2 for Rhizophora mangle dominated forests at the Caeté estuary in North Brazil. This increase in CO2 efflux rate is particularly high considering the relatively low proportion (0.3–0.7%) for which crab burrow entrances of U. cordatus account for per sediment area. Furthermore, burrows lead to changes in the reduction potential in the sediment and may thereby indirectly influence microbial decomposition pathways and CO2 release. This study further demonstrates that the reduction potential around the burrow walls underlies spatio-temporal variations which need to be considered to improve estimates of CO2 release from mangrove sediments. Future studies should disentangle carbon release and carbon storage by crabs and determine whether these two processes are in balance. Moreover, we underline the importance of including CO2 efflux rates of burrows and respiration rates of crabs into carbon budget estimates of mangrove ecosystems.Until now, the ecological role of Table S12 efflux rate data.Final linear mixed-effects model of burrow CO(PDF)Click here for additional data file.Table S22 efflux rate data.Final linear mixed-effects model of control CO(PDF)Click here for additional data file.Table S3Final linear mixed-effects model of crab respiration data.(PDF)Click here for additional data file.Table S4Final linear mixed-effects model of burrow rH data.(PDF)Click here for additional data file.Table S5Final linear mixed-effects model of control rH data.(PDF)Click here for additional data file.Table S6Final linear mixed-effects model of control-burrow rH data.(PDF)Click here for additional data file.Data S1Complete data set used for statistical analyses.(PDF)Click here for additional data file.


Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H Nature has developed highly efficient ways for signal and substrate processing in living cells. Synthetic biology is inspired by the natural archetype and tries to mimic and optimize biological processes for tailor-made applications  compartmentalization generates an enclosed reaction area that can be defined similar to biological systems ; (III) DNA/RNA building blocks can be utilized for spatial organization of reactive centers; and (IV) protein scaffolding presents a versatile approach in synthetic biology. This last scaffolding principle can be divided further into several approaches: fusion proteins are constructed by linking two or more enzymes into a single protein sequence. Non-covalent protein–protein interactions via the mutual recognition of folded domains or coiled-coil pairs as well as amyloid assemblies can be used to construct scaffolds with defined stoichiometry. A brief overview over all these scaffolding strategies is provided as supplementary information. Another concept in protein-based scaffolding uses protein adaptor domains and peptide ligands for bringing enzymes in spatial proximity, the focus of this review.Multifunctional enzyme systems that mimic natural systems can be artificially constructed via at least four general strategies: (I) Many important physiological protein interactions are mediated by relatively small protein domains, which bind to peptides exhibiting specific sequence motifs Figure A Dinkel. In thisProtein scaffolding based on cognate adaptor domains and peptide motifs requires a careful selection of candidate domains and SLiMs as well as the choice of proper linkers to interconnect these moieties and to attach them to the enzymes of interest Figure B. The prAdaptor domains used in protein-peptide scaffolding need to fulfill two basic requirements. First, they should have a strong affinity toward their peptide ligands to allow for effective coupling. Second, they should provide a distinct specificity for their ligands to allow for defined coupling, when several domain-ligand pairs are used simultaneously. The most often used domains are SH3, SH2, PDZ, and GTPase-binding domain (GBD)  and “Src homology 3” (SH3) domains , which enables them to function as inducible switches.In addition to the key residues necessary for binding, SLiMs also frequently contain variable residues (denoted as “X”) to ensure proper spacing between the binding residues. Due to its lack of a defined structure prior to binding, this peptide–domain interaction differs from the well-known domain–domain interactions in protein complexes. Prominent examples for SLiM sequence patterns include the classical P–x–x–P motif for binding to SH3 domains or a phosphorylated tyrosine with specific sequence neighbors for binding to SH2 domains.The last part necessary for modular protein scaffolding is the linker region connecting the engineered enzymes and the attached peptide ligands Figure B. The imAs a guide for the rational design of artificial linkers, an inspection of natural linkers is helpful. Two independent studies with different data sets gave similar results: while Argos found a preferred mean linker length of 6.5 residues Argos, , George 3 that connected the heavy and light chain domains (VH and VL) of an engineered antibody fragment It should also be noted that linker regions may also have additional benefits. They potentially improve folding and stability  from mouse, and uronate dehydrogenase (Udh) from Pseudomonas syringae were coexpressed in E. coli. A domain-based scaffold for the two enzymes Ino1 and MIOX, which were equipped with the respective peptide ligand sequences, tripled the product titers compared to the original system  by coupling glutamate decarboxylase (GadA/GadB) to the membrane protein glutamate/GABA antiporter (GadC). For that purpose, they attached an SH3 domain to GadA/GadB and three peptide ligand sequences to GadC each separated with flexible linkers. In that way, they could increase the GABA productivity by 2.5-fold.Besides the creation of a scaffold protein containing multiple adaptor domains, domain–ligand interactions can also be exploited in a different fashion as exemplified by the work of Vo et al. . They enA further and different approach used the localization of substrate and enzyme on a self-assembled monolayer for a 30-fold product increase (Li et al., Inspection of Table Recently, Lu et al.  construcSimilarly, Guan et al.  created A new versatile approach for covalent protein linkage is based on CnaB domains from bacteria, which autocatalytically establish isopeptide bonds between the sidechains of a lysine and an asparagine/aspartate residue (Veggiani et al., Synthetic biology is an emerging field with tremendous biotechnological potential. The efforts reviewed above clearly demonstrate that promising steps in this field have been made, though individual system design will require a tailor-made approach for achieving optimization. More complex metabolic pathways or large-scale industrial applications, however, would clearly benefit from an extended and well characterized tool-box of scaffolding components Kwok, .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.