Such metrics have not been well characterized for recent advances in BOLD acquisition.Application of BOLD data from the HCP 500 Subjects Release was performed with FIX ICA and with WM, CSF and motion parameter regression. Analysis with ROIs covering the gray matter at 5 mm resolution was performed to assess functional connectivity. ROIs in key areas were used to demonstrate statistical differences between specific connections. Reproducibility of group‐mean functional connectivity and for single connections for individuals was evaluated for both resting state and task acquisitions.Analysis of multiband Systematic differences in group‐mean connectivity were demonstrated during task and rest and during different tasks, although individual differences in connectivity were maintained. Reproducibility of a single connection for a subject and across subjects for resting and task acquisition was demonstrated to be a linear function of the square root of imaging time. Randomly removing up to 50% of time points had little effect on reliability, while truncating an acquisition was associated with decreased reliability. Reliability was highest within the cortex, and lowest for deep gray nuclei, gray‐white junction, and near large sulci.ROIs. Longer scans may be facilitated by acquisition during task paradigms, which will systematically affect functional connectivity but may preserve individual differences in connectivity on top of task modulations.This study found systematic differences in group‐mean connectivity acquired during task and rest acquitisions and preserved individual differences in connectivity due to intrinsic differences in an individual's brain activity and structural brain architecture. We also show that longer scan times are needed to acquire data on single subjects for information on connections between specific  Functional connectivity MRI is an evolving, diversely utilized technology for identifying brain network organization  is the largest publicly available functional connectivity datasets with such higher spatial and temporal resolutions , which may allow dramatic improvements in single subject characterization. The HCP (Human Connectome Project) datasets  allowed for analysis of functional connectivity in precise circuits, and a coarser parcellation consisting of 264 regions in strategic locations within canonical brain networks allowing assessment of clinically relevant connections . The multiband BOLD resting state data released includes both minimally processed as well as FIX ICA cleaned .Subjects ranged from 22 to 35 years in age, with 280 female and 196 male participants. These subjects comprised all those for whom four 15‐min resting BOLD sequences were available. Minimally processed task‐based fMRI data were also used for analysis for each of the seven task sequences for each subject for whom two acquisitions of the task were available   between 0.001 and 0.1 Hz, and each time series was subjected to a linear detrend operation in conjunction with the WM, CSF, and motion regression (each time series and covariate was detrended and bandpass filtered prior to regression).A bandpass filter was applied  was used to analyze the functional connectivity networks at higher spatial resolution. Second, analysis of a second paracellation of 264 ROIs, a subset of the first, in strategic locations allowed for analyses of statistical differences involving computationally more intensive comparisons.x = 42, y = 12, z = 10); Seed 2, left posterior cingulate ; Seed 3, right occipital pole ; Seed 4, right precentral gyrus ; Seed 5, left frontal eye field . Connectivity of the other 6922 ROIs to each of the five seed ROIs was used to obtain snapshots of core canonical resting state networks with different preprocessing strategies, in resting state acquisition data, and in task acquisition data for each sequence analyzed.The Fisher‐transformed correlation coefficients were obtained for each pair of 6923 × 6923 ROIs. These extensive correlation coefficients represent the functional connectivity and were analyzed for the spatial resolution of the functional networks. In order to compare canonical network patterns, five of the 6923 ROIs were selected a priori to represent distinct functional networks: Seed 1, right anterior insula  were used to compare reliability of functional connectivity of each of the 6923 ROIs to each of the 264 ROI subset for each subject in the four 15‐min acquisitions. For each of the 6923 ROIs, this allowed calculation of a mean ICC for the connections across the strategic 264 ROI subset and across subjects. Display of the mean ICC for each of the 6923 ROIs allows display of a 5 mm resolution map of functional connectivity reliability. We also calculated ICC for connections between each of the 14 subcortical ROIs and the same 264 ROI subset to evaluate for differences in ICC associated with segmentation of larger ROIs defined by subject‐specific structural gray matter boundaries rather than MNI coordinates.Intraclass correlation coefficients for each ROI in each subject was also calculated after systematically varying the amount of data present using two separate strategies. First, a “simulated scrubbing” was performed where randomly selected datapoints were removed from the time series at each ROI and the remaining time points concatenated, analagous to a scrubbing procedure to delete high motion timepoints  and for each task (average of two acquisitions for 476 subjects).For each individual connection, the root‐mean‐square difference between two acquisitions of the task or resting state data in Fisher‐transformed correlation was used as an estimate of the reproducibility. When averaged across all connections this yielded an estimate of the likely uncertainty in Fisher‐transformed correlation for an individual functional connectivity measurement between two 5‐mm diameter ROIs.To evaluate the reproducibility of subject‐specific differences in functional connectivity, correlation coefficient across subjects was calculated for each individual connection acquired during two different task or resting acquisitions. This correlation coefficient was averaged across connections after Fisher transformation. If the same subjects showed relatively higher connectivity for a particular connection in both task and rest scans, this would be evidence that individual differences in connectivity persist during rest and task acquisitions. Furthermore, this would also show that, in the same task conditions, differences in connectivity for that individual compared to other subjects may be expected to be similar regardless of the task condition for which the data were acquired.The WM, CSF, motion‐regressed method resulted in slightly higher Fisher‐transformed correlations of group mean connectivity for each pair of 6923 × 6923 ROIs for each of the four resting state sequences as compared to the correlation coefficients for the same ROIs for each resting state acquisition FIX ICA‐cleaned BOLD resting state data. However, the overall spatial pattern of seed‐based functional connectivity between the diffferent preprocessing strategies was similar . The FIX ICA method preprocessing strategy yielded systematically reduced connectivity but preserved spatial distribution across five distinct functional networks: salience, default mode, visual, sensorimotor, and dorsal attention in resting state and in task acquisition .q < 0.05 was applied to determine which connections differed between task and rest. A difference in connectivity of ±0.12 in Fisher‐transformed correlation units corresponded to a significant difference between task and rest acquisitions.To look at differences in group mean functional connectivity between resting state and task acquisitions, a parcellation of 6923 regions of interest covering the gray matter was used, with measurements of connectivity between each pair of ROIs. This resulted in 23.9 million connections between ROIs. Differences in resting state compared to task‐driven functional connectivity were measured by subtracting Fisher‐transformed correlation for rest versus task acquisitions for each connection, and a two‐tailed t‐test was performed across 476 subjects to test whether the difference was significantly different from 0. To account for multiple comparisons, a false discovery rate of Functional connectivity for five seeds corresponding to different functional networks to the rest of the brain had a similar spatial distribution for corresponding seeds in task and rest conditions Fig. . IncreasBut there are exceptions where a task may not result in higher connectivity within a related network. For example, during the motor task, regions of the sensorimotor network were often anticorrelated due to the temporal structure of the task. The motor task involves alternating motor activity in ipsilateral and contralateral body regions, so that when one part of the motor cortex is active, other regions of the motor cortex may not be. With unilateral seed placement in the motor cortex, the signal is the sum of the resting fMRI signal and the forced timing of the task. In addition to connectivity changes reflecting the temporal structure of the task, there are also network‐level changes where a network that participates in a given task may show higher connectivity, for example, the dorsal attention network during a working memory task.Reproducibility of group mean functional connectivity for both resting state and task acquisitions were measured by evaluating differences between multiple acquisitions of a sequence. Each task acquisition was performed twice in each subject, and each 15‐min resting state acquisition was performed four times for each subject. We compared group mean functional connectivity acquired during one task to functional connectivity acquired during the second acquisition of the same task versus during a different task. Correlation of the group mean connectivity across 23.9 million connections was used as a similarity metric, shown in Figure During resting state and during task acquisitions, repeat measurements using the same task paradigm tended toward a mean value that lies along the main diagonal when considering a scatter plot of connectivity acquired during two analogous acquisitions. When comparing data acquired during resting state versus task acquisitions, connectivity was much less similar across connections. That is, the repeat measurements using the same task paradigm were more highly correlated than the correlations between different tasks. The systematic differences attributable to different tasks may be related to brain regions artificially synchronized by the timing of task stimuli or cognitive events required by the task. These systematic differences in functional connectivity may be due to the networks engaged in the tasks and the positive or negative correlations of networks as suggested by Figure The range for correlations of group mean results for repeated runs of the same task was 0.79–0.91 (except for the outlier emotion task at 0.51), with repeated runs of the resting task showing r = 0.95. Comparing group mean results for different tasks ranged from 0.55 to 0.89 (mean 0.71 ± 0.088 SD). The emotion task was the only task for which repeated runs of the same task were less correlated than for comparisons between tasks.3) in the ROI  represent the Fisher‐transformed resting state functional connectivity for a given subject m between ROI i and ROI j and task acquisition condition k. This task acquisition condition could represent functional connectivity calculated from data acquired during either a task or resting state acquisition. Then the mean functional connectivity for connection ij and task acquisition condition k across subjects may be represented as rsfc¯ and the standard deviation for functional connectivity across subjects can be represented by Sij,k Then let rij represent the Pearson correlation coefficient between ROI i and ROI j across subjects for task acquisition conditions k1 and k2: For This correlation coefficient was Fisher‐transformed and averaged across all connections ij to give the mean correlation between any two task acquisition conditions (comparing either 2 runs of the same task or 2 runs of different tasks) shown in Figure The correlation across subjects improved with the square root of imaging time of the shortest sequence. Figure Intraclass correlation coefficient was calculated for each ROI in each subject with “simulated scrubbing” where randomly selected data volumes were removed showed almost no effect on reliability. However, truncation of the time series by a similar number of volumes had dramatic effect on reliability, shown in Figure We report an analysis of data from the Human Connectome Project 500 Subject Release that reliability and reproducibility of multiband functional connectivity is determined by scan duration of acquisition. More specifically, reliability and reproducibility can be estimated by 1 over the square root of imaging time. This relationship was true for data obtained acquired during resting state as well as for data acquired during each of seven task paradigms. Functional connectivity results acquired during tasks were significantly modulated by the timing of the task paradigm, likely representing artificial synchrony of brain regions coactivated by the task. Nevertheless, individual differences in connectivity between subjects were similar regardless of the particular task performed, and these differences also become more reliable as a function of imaging time. Scan duration, not necessarily more volumes due to faster imaging sequences, was the critical factor in achieving higher reliability.One direction to improve reliability and reproducibility of functional connectivity measures is standardization of data processing. The HCP MRI data  was evaluated, a similar spatial distribution between task and rest acquisitions was seen. Although the group mean functional connectivity showed modulation with the task, connectivity differences appeared to be preserved across individuals. Furthermore, the different tasks, including the timing and structure, changed the pattern of connectivity in a systematic way. The higher correlation for a specific connection between rest and task across subjects demonstrated in our study is further evidence that individual differences in connectivity persist during rest and task acquisitions. Individual differences in connectivity may be expected to be similar regardless of the task condition for which the data were acquired. The differences may be due to intrinsic differences in individual's brain activity, whether that is due to structural white matter connections and/or greater gray matter volume.The results from this study highlight that longer scan times are needed to acquire data on single subject networks and information on the connectivity from single brain regions. Specifically, the reliability and reproducibility of functional connectivity a single connection as well as across subjects for resting and task acquisition were demonstrated to be a linear function of the square root of imaging time  versus truncation of scans corroborate that the primary driver of reliability is the length of scan. Random scrubbing up to 50% of the datapoints had very little effect on reliability while truncating the data to a similar degree had a much larger effect. Since functional connectivity measures relatively slow (<0.08 Hz) changes, removal of random time points did not appear to apreciably effect the results, but shorter scan durations allowing less time for the brain to sample different states had a much larger effect on reliability. Resting state connectivity metrics are ultimately a measure of synchrony and are very sensitive to specific mental processes that are taking place during the scan. Changes in mood, content of thought, and arousal state are relatively slow processes, and sampling a short duration of brain connectivity is likely to be skewed toward a given state. This improves with data collated from multiple scan sessions or longer imaging times.Longer imaging times for measuring functional connectivity may provide increased reliability and reproducibility, but the limiting factor is the subject's ability to tolerate prolonged scanning. There are the realistic issues of maintaining wakefulness and stillness, discomfort in the prolonged prone position and pathology‐related hindrances. Acquiring functional connectivity data during stimulation or task paradigms will enable longer scan durations with better characterization of brain states during scanning. Task acquisition may facilitate holding a subject's attention with less risk of falling asleep during the scan and, as shown by our results, there are preserved individual differences in functional connectivity at task and rest acquisitions.Clinical application of functional connectivity research to individual patients and to the development of disease classifications requires test–retest reliability and reproducibility. Our study found systematic differences in group‐mean connectivity acquired during task and rest and preserved individual differences in connectivity during rest and task acquitisions due to intrinsic differences in an individual's brain activity. Reproducibility in task and rest acquisitions is similar, although the group mean functional connectivity will show modulation with the task. We also showed that longer scan times are needed to acquire data on single subject networks and information on the connectivity between focused brain regions. Our results illustrated that the primary driver of reliability and reproducibility is the length of the scan, with longer scan times allowing the brain to sample a broader range of cognitive states with improved reliability and reproducibility.None declared.Figure S1. Effect of preprocessing strategy on group mean connectivity for 6923 × 6923 ROI pairs.Click here for additional data file.Figure S2. Systematic changes in connectivity associated with FIX ICA consist of reduced connectivity but similar spatial distribution across five different seeds.Click here for additional data file.Appendix S1. Effect of preprocessing methods on fMRI data analysis.Click here for additional data file.


DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions. Traditional ensemble approaches analyse the entire population of molecules averaging their characteristics, which does not take this variability into account. DNA combing stretches single DNA molecules uniformly at high density on silane coated glass surfaces134DNA replication initiates at origins of replication located at specific sites along chromosomes and cells utilise a subset of available origins to complete genome duplication144101617Schizosaccharomyces pombe where we can analyse molecules whose length average is 2 Mb with some molecules occasionally reaching 5.6 Mb, allowing the entire length of all three fission yeast chromosomes to be investigated. We have extended our new combing method to human U2OS cells and have analysed single DNA molecules on average 8 Mb in length and occasionally up to 12 Mb. Changing the scale of analysis of replicating single DNA molecules by more than an order of magnitude reveals a more extended structure of origin firing. Replication origins fire stochastically and form clusters of closely spaced origins separated by regions of sparsely firing origins. Our findings suggest that the spatial organisation of fired origins is similar in different eukaryotes.Here we report a modified DNA combing protocol that allows the analysis of DNA replication on single DNA molecules up to 12 Mb in length. We have developed this methodology in the fission yeast in vivo with thymidine analogues such as BrdU or EdU in a synchronous cell population  molecules such as the ionic strength and pH of the combing buffer, the temperature, the base composition and the pulling velocities exerted on the DNA molecule2526272325We used the new DNA combing method to investigate the pattern of replication origin firing on multi-Mb long single DNA molecules in fission yeast. We first synchronised cells for entry into S-phase and pulse labelled newly synthesised DNA with BrdU . Genomicin silico manipulation. We therefore developed an algorithm that enables representation of long, partially replicated DNA molecules as “barcode” diagrams, where stretches of replicated and un-replicated DNA are shown as coloured segments. These diagrams were built from the measurements of successive replicated and un-replicated DNA segments, together with a reference position on the chromosome determined by FISH. Due to the considerable number of measured segments, we used a machine-independent C routine that takes Excel data files as input to generate the bar code diagrams in Postscript vectorial format (see Comparing replication patterns on long DNA molecules imaged across consecutive microscopic fields of view requires rmat see . Such bain silico our longer molecules into fragments of 500 kb and comparing their inter replication tracks distances (IRTD) with those analysed in the earlier study. 15One 4 Mb partially replicated single DNA molecule is shown in in silico into 44 consecutive fragments) and the synthetic image of the whole DNA molecule is shown below. Visual examination showed that replication origins fire in clusters of closely spaced origins separated by long regions with sparsely fired origins. Clusters of closely spaced origins have been reported previously for Xenopus, mouse and human cell lines3334We next extended our improved DNA combing protocol to organisms with larger chromosomes to determine the maximum length of DNA molecules that could be analysed. We investigated the distribution of origin firing in human U2OS cells, synchronising the cells by first blocking cell cycle progression at S-phase onset using thymidine followed by release and a second block in mitosis using nocodazole . At the 11To analyse the distribution of origin firing along human chromosomes we analysed 7 additional single DNA molecules averaging 8 Mb in length and replicated up to 50% . The aveNext, we characterised cluster size by setting the maximal IODs within clusters to be 40 kb, twice the average 20 kb IOD, and estimated the minimal number of origins per cluster by varying the number of origins defining a cluster from two to 15, and plotting it against the number of clusters per genome. We also measured replication fork velocity in U2OS cells by chasing BrdU with EdU for 20 minutes at mid S-phase . To deri36We have modified DNA combing protocols to allow imaging of single DNA molecules up to 12Mb in length compared with previous limits of around 1 Mb. Our protocol allows analysis of origin firing and fork velocities on single DNA molecules genome-wide for organisms with small genomes and across large sub-chromosomal fragments in human cells. We showed that in human cells origin selection is a stochastic process forming clusters of closely spaced fired origins.Previously, analysis of DNA replication on short DNA molecules resulted in different values for experimentally measured IODs and replication fork velocities for the same cell lineEach cell must complete genome duplication in a timely fashion before the onset of mitosis. Failure to replicate even a small region of chromosomes would lead to genomic instability. Understanding the mechanisms underlying the timely completion of genome duplication will benefit from the analysis of models using experimentally measured parameters of the process of DNA replication. In organisms with small genomes and few chromosomes such as fission yeast, these parameters can be measured using DNA combing because a single DNA molecule at the scale of a full-length chromosome represents a significant part of the genome and can be used as a surrogate for the extent of DNA synthesis within the cellDNA combing of multi-Mb-scale single DNA molecules allows the characterisation of the distribution of fired origins in clusters along human chromosomes. The absolute position of these clusters can be determined using FISH, allowing origins and clusters to be mapped to specific chromosomal sequences. In addition mutants and small molecules that perturb cluster characteristics can be screened to gain insights into their formation. Understanding the mechanisms responsible for cluster formation will bring insight to the higher order organisation of interphase chromosome in human cells. Finally, epigenetic modification such as DNA methylation can be detected on combed single DNA molecules using specific antibodiescdc25-22 strains were grown in minimal media (EMM) at 25 °C to 1–2 × 106 cells/ml, shifted to 36 °C for 4 hours to block cells in late G2, and then released at 25 °C. 2 μM BrdU was added to a synchronised culture at 34 minutes after release (corresponding to G1-phase) allowing cells to enter S-phase in the presence of BrdU. Cell cycle progression was stopped at 75 inutes after release  and DNA was prepared for DNA combing Images for DNA molecules were collected in Metamorph  using an epifluorescence microscope  equipped with a Zeiss Plan-FLUAR 63x/1.40 lense  and CoolSNAP HQ camera (Roper Scientific).How to cite this article: Kaykov, A. et al. Molecular combing of single DNA molecules on the 10 megabase scale. Sci. Rep.6, 19636; doi: 10.1038/srep19636 (2016).


Influence of laser treatment on mechanical properties, wear resistance, and Vickers hardness of aluminum alloy was studied. The specimens were treated by using Nd:YaG laser of energy 780 mj, wavelength 512 nm, and duration time 8 ns. The wear behavior of the specimens was studied for all specimens before and after treatment by Nd:YaG laser and the dry wear experiments were carried out by sing pinon-disc technique. The specimens were machined as a disk with diameter of 25 mm and circular groove in depth of 3 mm. All specimens were conducted by scanning electron microscopy (SEM), energy-dispersive X-ray florescence analysis (EDS), optical microscopy, and Vickers hardness. The results showed that the dry wear rate was decreased after laser hardening and increased Vickers hardness values by ratio of 2.4 : 1. The results showed that the values of wear rate for samples having circular grooves are less than samples without grooves after laser treatment. The solid surface hardening by laser treatment represents the structural transformations of the material; this can be established by irradiating the surface with a laser pulse. Surface heat treatment with laser beam uses the characteristics of self-quenching that cools rapidly into materials without cooling water unlike general surface heat treatment , 2. If sAluminum alloy samples were prepared and divided into two groups: the first group operated in the form of a cylinder diameter of 25 mm and a thickness of 4 mm and a second group is also operated in the same shape and dimensions but were drilled three circular grooves on the samples surface in depth of 2 mm to reduce the contact area. All samples were cleaned and polished and then washed with HCl and NaOH with four molarities. Finally the samples immersing in distilled water and drying carefully.2. These samples immersed in distilled water with depth of 3 cm and all samples are dried before starting the experiments of wear.Laser treatment of samples' surface was performed for two types of samples (with grooves and without grooves) by using Nd:YAG pulses laser type Rofin DY044 (as shown in The X-ray fluorescence (XRF) model (TSMJ) made in Germany has been analyzed for aluminum alloy samples to determine the elemental composition of samples.W1 and W2 is weight of specimen before and after the test (gm):r is radius from the center of the specimen to the center of the disc (cm), n is nimber of rotating disc (r.p.m), and t is time of sliding (min).Pin on disk was used to conduct the dry wear experiments and the wear rate was measured by using the weighing method, and each specimen was weighed before and after wear testing by using sensitive digital balance of accuracy 1 mg. Wear rate values were calculated by using the following equations:F is applied load (kg f) and d is the main diagonal of indentation (mm).Hardness type Vickers was conducted for all samples before and after laser surface treatment by using (Hensddt Wetzlar), with applied load 500 gm. Vickers hardness values were calculated according to the following equation:The elemental composition of aluminum alloy samples used in this work was made by using X-ray fluorescence (XRF) analysis technique as shown in Vickers hardness results of aluminum alloy samples before and after laser surface treatment increased from 98 HV to 235 HV. This enhancement of surface hardness after laser treatment can be represented by the ratio of 235%. Vickers hardness measurements included three readings and then taken average.Figures The plasma causes a shock wave by its expansion; this shock increased by distilled water pressure that covers sample during shooting laser pulse. For this reason the hardening was increased and the grain size decreased. The surface morphology of aluminum alloy samples was conducted by using SEM technique as shown in Figures Based on the obtained results, we can conclude that laser surface treatment allows decreasing the wear rate by 50% as an approximate ratio when we used 500 pulses and by when we used 250 pulses and grooves. Also, the Vickers microhardness of laser treated aluminum alloy samples is higher than that of the microhardness of the untreated aluminum alloy samples.


Chalcogenide semiconductor quantum dots are emerging as promising nanomaterials due to their size tunable optoelectronic properties. The commercial synthesis and their subsequent integration for practical uses have, however, been contorted largely due to the toxicity and cost issues associated with the present chemical synthesis protocols. Accordingly, there is an immediate need to develop alternative environment‐friendly synthesis procedures. Microbial factories hold immense potential to achieve this objective. Over the past few years, bacteria, fungi and yeasts have been experimented with as eco‐friendly and cost‐effective tools for the biosynthesis of semiconductor quantum dots. This review provides a detailed overview about the production of chalcogen‐based semiconductor quantum particles using the inherent microbial machinery. Of particular relevance is the quantum particles composed of groups II–VI or III–V elements having unique electronic and optical properties resulting from quantum confinement effects  solvent, when the methyl radicals formed during the reaction combine with the oxygen in TOPO, ethers may form Collins, , which set al., et al., et al., The development of eco‐friendly and green approaches for synthesizing these nanoclusters is thus required , where the binding energy within the exciton cannot be overcome through the absorption photon energy. Unlike in the bulk semiconductors, enhanced Coulomb interaction in QDs results in much more tightly bound excitons that force them to interact with each other, resulting in shifts in the energy spectrum 3 times larger than that of the bulk exciton  is often the nanoparticle semiconductor of first choice because of the ease of its fabrication: there is no high‐temperature, anaerobic chemistry involved and no molten solvents  QD production has generally been reported as a subset of CdSe preparation  QDs and zinc selenide (ZnSe) QDs have each been synthesized by numerous groups around the world. Current research focuses on producing larger quantities, doping basic ZnS with manganese, lead (II) or cadmium, and varying the capping structures of the QDs Yoffe, . On the Numerous biological resources have been exploited for bioproduction of Nanoparticles (NPs), including bacteria, fungi, algae, viruses, plants and plant extracts. Bacterial structures, such as the S‐layer, flagella and spores, are regarded as potent green nano‐factories for the production of NPs with exquisite sizes and shapes. Viruses are also fascinating nano‐biotemplates that can form NP arrays based on their inherent nanostructures, e.g. capsids (Narayanan and Sakthivel, et al., et al., et al., et al., Because of their biotransformation and metal bioaccumulation ability, fungi and bacteria are receiving special focus for the biological generation of metallic nanoparticles (Sastry Candida glabrata and Schizosaccharomyces pombe, cultured in the presence of cadmium salts (Dameron et al., et al., Biosynthesis of semiconductor NCs was pioneered as early as 1989 in the yeasts S. pombe (Kowshik et al., Torulopsis (Kowshik et al., 2+ ions by glutathione‐related peptides and a consequent production of CdS within the yeast cells (Dameron et al., Later, the intracellular biogenesis of CdS and PbS nanoparticles was initiated in et al., Escherichia coli (Mi et al., E. coli cells were lysed using a hyper‐acoustic cell grinder, the lysed cell suspension was centrifuged, the re‐suspended cells were further freeze‐thawed at −70°C, and the QDs were purified using anion exchange resin columns. Pandian and colleagues (Brevibacterium casei cells prior to their characterization. Similar recovery and purification steps have been reported by other researchers for the intracellular biosynthesis of chalcogenide QPs (Mousavi et al., A shortcoming of the above‐discussed studies was that the metallic nanoparticles were synthesized ‘intracellularly’. Indeed, when the site of nanoparticle synthesis is intracellular, downstream processing becomes difficult and often defeats the purpose of developing a simple and cheap process (Kowshik lleagues  also repFusarium oxysporum in a facile enzymatic process has been reported (Ahmad et al., 2− and the Cd2+ ions in the reaction mixture when incubated with the yeast cells induce a specific defence mechanism generating proteins to coordinate with Cd and Te ions in an effort to detoxify the metal ions. In this biochemical process (Fig. 2+ and Te2− ions can eventually become incorporated as caps on the surface of the QDs forming a coating layer (Bao et al., To overcome the downstream processing steps of intracellular means of QD biosynthesis, one‐step scalable extracellular biogenesis of semiconductor nanocrystallites in microorganisms has been developed. In this regard, the extracellular biosynthesis of CdS NPs using the fungus et al., et al., et al., et al., According to the authors (Bao et al., et al., The protein cap confinement also leads to a uniform size and good degree of dispersion without any aggregation. According to Bao and colleagues , the CdTSaccharomyces cerevisiae was initiated by Prasad and Jha (Candida glabrata cells exposed to cadmium ions in the form of excretion of the enzyme phytochelatin synthase and a protein HMT‐1, which effectively aborts the CdS NCs from cytosol. Once entered into the cytosol, the CdS might have triggered the family of oxygenases harboured in the endoplasmic reticulum, chiefly meant for cellular‐level detoxification through oxidation/oxygenation. The authors have also drawn a comparison of the efficiency of bacteria and fungi as candidate systems for biosynthesis. Based on their findings, the authors concluded better size quantization effects in yeast in comparison to bacterial‐mediated synthesis for the production of CdS nanoparticles (Prasad and Jha, Another important research effort for the extracellular biosynthesis of CdS nanoparticles in  and Jha . It is ret al., Klebsiella pneumonia was proposed (Malarkodi et al., et al., et al., et al., The involvement of 5′‐adenylylsulfate reductases in sulfate‐assimilating bacteria that could efficiently detoxify cadmium ions by the formation of extracellular CdS particles has also been detailed (Bick Capsicum annuum that could effectively reduce selenite to selenide ions through an enzymatic reaction was also reported (Li et al., 2SeO3 and subsequently with CdCl2 resulted in the intracellular synthesis of CdSe QD and an increase in the glucan content of their cell walls, resulting in their enhanced mechanical strength (Luo et al., E. coli strains harbouring plasmids containing phytochelatin synthase from S. pombe and γ‐glutamylcysteine synthetases are effective biofactories for the synthesis of CdS QDs (Kang et al., E. coli cell lysates was also demonstrated (Liu et al., Similar observations explaining the involvement of a 30 kDa protein in the extract of et al., et al., Regardless of the site of QP synthesis, it is noteworthy that the microbial sources for biosynthesis are largely unexplored and that a more comprehensive understanding regarding the mechanisms that initiate biosynthesis holds significance. According to Ramezani and colleagues , the metAlthough the constraints in the cultivation of microorganisms and the shape and size control of the microbiologically generated NPs have been reported as significant drawbacks of biomediated processes (Gericke and Pinches, Aspergillus terreus.Table S. pombe by maximizing the biomass yield under conditions where glucose repression is absent. To achieve this, they developed a feedback controlled fed‐batch system for the increased yield of biomass available for continuous CdS production. The degree of S. pombe glucose repression caused by the addition of cadmium was reduced so as to induce CdS QDs production. The study concluded that the glucose feed rate reduction followed by minimal glucose repression and ethanol production maximized the biomass yield and hence the QD yield. Further, the yeasts S. pombe and Candida glabrata were successfully cultivated in a fed‐batch process at cadmium levels up to 100 mg l−1 (Williams et al., Schizosaccharomyces pombe incorporated higher amounts of cadmium per gram of dry biomass compared with C. glabrata. The higher Cd uptake from S. pombe cells was correlated with the elevated glucose concentrations during the cultivation (Williams et al., Williams and colleagues  reportedChalcogen‐based semiconductor QDs have initiated an application‐oriented research in nanotechnology. These NCs with excellent quantum confinement effects are mostly prepared by chemical means, which raise toxicity and cost‐related issues. Thus, there is a critical need in the field of nanotechnology to develop reliable and eco‐friendly protocols for the synthesis of QDs. The microbial community is relatively unexplored for their biosynthesis machinery for the production of chalcogenide QDs. Biological systems have the capacity to initiate the synthesis of these nanoclusters using the enzymes in their metabolic pathways at ambient temperature, pressure and neutral pH. Quantum dots can be synthesized both in bacteria and fungi, with fungal‐based systems having an advantage of easy handling and extracellular synthesis. Although bacterial synthesis has an advantage of involving directed evolution or genetic manipulation, fungi have a niche with respect to the cost factor on scale‐up and large‐scale synthesis. This area of nanotechnology offers future scope for exploring newer biosynthetic sources and the optimization of production conditions for the commercialization of QD to apply them in solar power harnessing and biosensing.None declared.


The plant holobiont comprises the plant and its associated microbiota, which interact with each other and determine holobiont functioning and plant performance. We have started to understand the complexity of the involved microorganisms and their interactions, however, we need more research on plant–microbiome interactions to understand holobiont functioning. By 2020 we expect that our knowledge on these interactions will have considerably increased facilitating crop management practices based on the interactions of the plant holobiont. Already in 1994, Richard Jefferson introduced in a symposium lecture his idea that the evolutionary selection unit is not a single organism but a macro‐organism  and all its associated microorganisms that act in consortia  phytofirmans PsJN in potato  production of the fungal endophyte, although the bacterium does not show IAA production when grown alone (Hoffman and Arnold, Rhizopus microsporus, the causal agent of rice seedling blight, and its interaction with the endofungal bacterium Burkholderia endofungorum, which is responsible for toxin production (Partida‐Martinez and Hertweck, Dichanthelium lanuginosum as well as its fungal endophyte Curvularia protuberata can tolerate temperatures of 40°C when grown separately, but in symbiosis, the combination of the host plant and the fungal endophyte infected with a virus can tolerate soil temperatures as high as 65°C (Márquez et al., et al., Numerous studies report on the complexity of plant microbiomes comprising multiple fungal and bacterial members (reviewed by Vorholt, et al., These examples demonstrate how important it will be to shed more light on the interplay between microbiome members with regard to interacting with the plant, and a multitude of yet unknown interactions impacting plant growth and health are still to be expected. A better understanding on the type of interactions, the mechanisms and signalling cascades involved will be applicable in different directions. Defined microbial consortia comprising bacteria as well as fungi may be designed to provide a specific set of functions. Understanding the interactions between pathogens or pests with other plant‐associated microbiota may lead to novel avenues in plant disease prevention (Massart et al., et al., By 2020, we should have acquired better knowledge on the ecology and functioning of plant–microbiome interactions in different plant compartments. Of main interest are the root environment and reproductive organs such as seeds Fig. . Our undOur understanding of higher organisms is changing fundamentally. Plants are no longer understood as monogenetic individuals but as polygenetic entities, in which the microbiota play a central role in fitness, adaption and diversification of the holobiont (Bordenstein and Theis, Authors have no conflict of interest to declare.


The acquisition of procedural skills is an essential component of learning for medical trainees. The objective of this study was to assess which teaching method of performing urinary catheterisation is associated with most efficient procedural skill acquisition and retention. We evaluated factors affecting acquisition and retention of skills when using simulators as adjuncts to medical training.Forty-two second year medical students were taught urinary catheter insertion using different teaching methods. The interactive group (n = 19) were taught using a lecture format presentation and a high fidelity human urinary catheter simulator. They were provided with the use of simulators prior to examination. The observer group (n = 12) were taught using the same method but without with simulator use prior to examination. The didactic group (n = 11) were taught using the presentation alone. Student characteristics such as hand dexterity and IQ were measured to assess for intrinsic differences. All students were examined at four weeks to measure skill retention.Catheter scores were significantly higher in the interactive group (p < 0.005). Confidence scores with catheter insertion were similar at index exam however were significantly lower in the didactic group at the retention testing (p < 0.05). Retention scores were higher in the interactive group (p < 0.001). A significant positive correlation was observed between laparoscopy scores and time to completion with overall catheter score (p < 0.05). Teaching method, spatial awareness and time to completion of laparoscopy were significantly associated with higher catheter scores at index exam (p = 0.001). Retention scores at 4 weeks were significantly associated with teaching method and original catheter score (p = 0.001).The importance of simulators in teaching a complex procedural skill has been highlighted. Didactic teaching method was associated with a significantly higher rate of learning decay at retention testing.The online version of this article (doi:10.1186/s12909-014-0264-3) contains supplementary material, which is available to authorized users. With the introduction of the European working time directive and the regulation of the profession and training bodies, there is an increased emphasis surrounding the acquisition, assessment and retention of procedural skills within medical training facilities -3. With Prior to the introduction of the Halsted’s apprenticeship model of training at the beginning of the 20th century, trainees were immersed in the hospital setting and expected to learn the relevant skills in an unstructured manner. With the advent of more complex procedures such as laparoscopy and robotics, increasing efforts have been deployed to teach fundamental skills in surgical teaching laboratories . PioneerSimulation based training is cost effective and benefits the health sector budget with faster procedural times, fewer complications encountered and overall greater efficiency within the hospital -14. ExteThe aims of this study was to assess which teaching method in performing urinary catheter insertion is associated with most efficient skill acquisition and longer learner retention in undergraduate medical students. Furthermore we aimed to understand human characteristics affecting acquisition and retention of skills when using simulators as adjuncts to medical training and to examine factors which are associated with “learning decay” at retention testing.Forty-two second year medical students at NUI Galway were randomly allocated into three groups to attend teaching sessions on urinary catheter insertion. The first 21 males and 21 females that voluntarily agreed to participate in the study were included. Consent was obtained from all participants taking part in the study. All teaching sessions and lecture format presentations were carried out by an experienced urologist with more than 5 years teaching experience. The lecture presentation was a 15 slide tutorial lasting 90 minutes outlining the indications, consent, complications and diagrammatical instructions for urinary catheter insertion in patients. The Interactive group (n = 19) were taught using the lecture presentation and a high fidelity human urinary catheter simulator All students within this group were provided with simulators to facilitate practice prior to examination for 30 minutes. The Observer group (n = 12) were taught using the same presentation and observed the urologist carrying out catheter insertion on the simulator for 30 minutes, however they were not provided with hands on experience prior to examination. The Didactic group (n = 11) were taught using lecture format presentation alone and had no access to the simulator. Exclusion criteria included previous experience with urethral catheterisation. All students were asked to complete questionnaires measuring their confidence with catheterisation at different stages of the teaching process along with individual characteristics such as hobbies, examination results, type of learning style as outlined by Honey and Mumford and career motivation which may account for differences in skill acquisition and retention . StudentWithin 2 hours of each teaching session the students underwent an Observed Structured Clinical Examination (OSCE). Students were examined using 4 stations at which examinations ran for 6 minutes each. They were graded by two observers using a standardised marking sheet to prevent bias  derived from the standardised MENSA question bank assessing spatial awareness, memory testing, concentration, and subgroup classification .Hand dexterity was measured using a beginner’s virtual reality laparoscopy exercise on the ProMis Laparoscopy trainer® Figure . The tasOutcomes measured at the individual stations were:All students were re-examined at 4 weeks to measure retention of catheterisation skills. Students were requested not to up-skill in the interim.Ethical approval was obtained from the Galway University Hospitals ethics committee, following submission of the standard ethics form and chairperson’s review.p-value of < 0.05 was considered significant.Data was collected and analysed using Minitab version 16. Analysis of Variance (ANOVA) was conducted to examine the difference between group means. Regression analysis was carried out to determine the relationship between variables. A This study comprised of 42 second year medical students from National University of Ireland Galway. The average age was 20.95 years with 21 males and 21 female participants, respectively. Twenty nine students were interested in pursuing a surgical career.p = 0.59, Figure p < 0.05). Student confidence levels in performing catheterisation were found to be significantly higher for all teaching groups after they had undergone the examination process  respectively.All students were asked to document their level of confidence with catheterisation at different stages of the teaching and examination process. Prior to teaching there was no significant difference in the confidence of students in performing urinary catheter insertion (p > 0.05) in estimated and actual confidence level with catheter insertion for both the interactive group and observed group .Index examination scores for urinary catheter insertion were measured within 1 hour of each student undergoing a teaching session. The interactive group (n = 19) had significantly higher scores at index examination compared to the observed (n = 12) and didactic (n = 11) teaching groups .Retention scores for urinary catheter insertion were measured at 4 weeks post initial teaching. Students taught using the interactive simulation method scored significantly higher at their retention test than the other teaching methods  respectively  respectively.A multivariate analysis was carried out to assess the relationship between factors associated with scores for catheter insertion at index examination and at the retention test four weeks after teaching. Teaching method, time to completion of laparoscopic task and spatial awareness measured at IQ testing were significantly associated with catheter scores . There was also a trend towards significance noted for those students who played music (p = 0.058).A multivariate analysis was carried out to identify the factors associated with higher catheter scores at the retention test. Teaching method and catheter score at index examination were found to be significantly associated with catheter scores at retention testing (The role of simulation is to recreate a clinical scenario that is representative of a true life situation. Multiple studies have shown that skills learned at the bench using simulators are translated into the operating room . This alIn this study the degree of readiness or confidence levels of individual students with catheter insertion was measured during different time points. There was no significant difference in confidence levels for each group prior to teaching. Confidence levels were found to be significantly lower in the didactic group after their initial teaching session. Interestingly however, confidence levels were significantly highest for all students after undergoing an OSCE format examination for catheter insertion. This outlines the importance of the examination process in assuring students of their ability to perform a procedural skill competently. A limitation to this study is that although all students underwent the OSCE examination process, it is difficult to assess whether this increase in confidence may simply be due to more hands on experience rather than the examination process itself. Assessments have been shown to promote reflective practice . This heIn the current study, significant differences in performance were also observed at the four week retention test. The interactive group significantly outperformed the observed and didactic groups. The retention of skills in medical practice is critical. A limitation acknowledged in this current study was the relatively short duration from index exam to retention testing, small study group and lack of randomisation. Although well documented in the literature that proficiency based progression training has been qualitatively shown to the optimal approach to skill acquisition, due to time constraints it was no possible to analyse this within our cohort . RetentiSkill degradation is a serious issue in medical education and is associated with increased procedural times, costs and complications -32. FactWe found that students who scored higher in spatial awareness and hand dexterity tasks such as time to laparoscopic task completion, economy of movement and instrument pathway scored higher in catheter insertion at index exam. Moreover there was a significant correlation between scores achieved by individual students at initial testing and those at four weeks highlighting that some students possess innate skills. This is easy to understand as it has been long recognised that some individuals master a certain skill set such as laparoscopy in a relatively short period while others struggle to learn, take longer and may never reach competency. Interestingly, computer gaming and playing sports did not impact on performance in this study. Previous studies have shown significant association with certain pre-learned skills such as computer gaming, music and sport with easier acquisition of new procedural skills -40. It iThe importance of simulators in teaching a complex procedural skill has been highlighted. Teaching and allowing students to practice their skills on simulators is associated with higher index and retention examination scores. This study also highlights the importance of the examination process during teaching in assuring students of their ability to carry out procedural skills. Simulator training has been shown to suit all types of individuals regardless of learning style. Students with increased manual dexterity and spatial awareness score significantly higher with the use of simulators in teaching. Skill degradation or “decay” was significantly higher in the didactic teaching group. In order to optimise training in procedural skills for the future there needs to be an increase in the provision of simulation based training. This will allow for a more efficient and smoother translation of procedural skills from the non clinical environment of the skills laboratory to the hospital wards and operating room.


A major goal of evolutionary biology and ecology is to understand why species richness varies among clades. Previous studies have suggested that variation in richness among clades might be related to variation in rates of morphological evolution among clades . Other studies have suggested that richness patterns might be related to variation in rates of climatic‐niche evolution. However, few studies, if any, have tested the relative importance of these variables in explaining patterns of richness among clades. Here, we test their relative importance among major clades of Plethodontidae, the most species‐rich family of salamanders. Earlier studies have suggested that climatic‐niche evolution explains patterns of diversification among plethodontid clades, whereas rates of morphological evolution do not. A subsequent study stated that rates of morphological evolution instead explained patterns of species richness among plethodontid clades . However, they did not consider climatic‐niche evolution. Using phylogenetic multiple regression, we show that rates of climatic‐niche evolution explain most variation in richness among plethodontid clades, whereas rates of morphological evolution do not. We find little evidence that ecological limits explain patterns of richness among plethodontid clades. We also test whether rates of morphological and climatic‐niche evolution are correlated, and find that they are not. Overall, our results help explain richness patterns in a major amphibian group and provide possibly the first test of the relative importance of climatic niches and morphological evolution in explaining diversity patterns. Explaining patterns of species richness among clades is a major goal of evolutionary biology and ecology. For example, why do some clades have a single species and others have over a million ? An important approach for answering this question is to test whether particular ecological, morphological, behavioral, or genetic traits are correlated with patterns of diversification  and species richness. For example, recent studies have found significant relationships between increased rates of species diversification and occurrence in tropical regions . Similarly, all organisms have morphology (a characteristic shape and size). Second, theory and empirical studies suggest that climatic‐niche divergence may be an important driver of speciation  of the study by Rabosky and Adams  is that Here, we explore the relationships among species richness and rates of niche and morphological evolution among the major clades of plethodontid salamanders. We also test whether patterns of diversification over time within clades may be associated with ecological limits to clade diversity. We show that patterns of species richness among plethodontid clades are explained primarily by rates of climatic‐niche evolution. Specifically, using phylogenetic multiple‐regression models, we show that morphological variables explain relatively little variation in richness that is not already explained by rates of climatic‐niche evolution. Moreover, we find that rates of morphological evolution are unrelated to rates of climatic‐niche evolution. Finally, we show that apparent slowdowns in diversification within plethodontid clades are unlikely to be explained by saturation or “filling” of morphological or ecological space. Together, these results may offer the first direct comparison of the relative importance of morphological evolution and climatic‐niche evolution in driving patterns of species richness within and among clades, and one of the relatively few tests of the ecological limits hypothesis to directly incorporate ecologically relevant variables.We tested the hypothesis that species richness of plethodontid clades is significantly and positively related to the rate of climatic‐niche evolution and compared this relationship with that between species richness and rates of size evolution and shape evolution. We then tested whether the size and shape rates still significantly explain species richness in a multiple‐regression model that included the rate of climatic‐niche evolution. We also tested whether these rates of morphological and climatic change are related to each other.Furthermore, we tested whether declining rates of diversification within plethodontid clades  for PC2–PC7 combined. Estimation of σ2 followed O'Meara et al. , and obtaining data for 19 climatic variables for each locality. They obtained the mean value for each variable across the localities for each species. Then, as with the morphological data, they performed PCA and reduced the 19 climatic variables to three PCs that explained 83% of the variation among species. They then estimated σ2 for each of these three PCs within each clade and combined all three PCs to obtain a single multivariate estimate of climatic‐niche rate. Sample sizes of species within each clade are given in Table For analyses of climatic‐niche rates, we used the multivariate estimates from Kozak and Wiens . Howevers et al.  or Raboss et al. . The nics et al.  estimateσ2 of PCs) were used to estimate rates for each type of variable in each clade. Furthermore, the sample sizes for each variable type within each clade were not always identical, but were strongly associated . Similarly, sample sizes within each clade were strongly related to the total number of species within each clade . We also note that Kozak and Wiens  climatic‐niche rates alone; (2) size rate alone; (3) shape rate alone; (4) size and shape rates together; (5) size rate and climatic‐niche rate; (6) shape rate and climatic‐niche rate; and (7) size, shape, and climatic‐niche rate. We calculated the AIC of each model, and evaluated whether models including one or both morphological rates had substantially better fit than models including only climatic‐niche rate. An AIC difference of four or greater between competing models was considered to show substantially better fit of one over another , whereas size and shape rate each explain closer to one‐third . Most importantly, even though a model including all three variables had the best fit (AIC = 15.04822), this model was not substantially better than one based on climatic‐niche rate alone . There is no significant relationship between size or shape rate and rates of climatic‐niche evolution  is somewhat uncoupled from their ecology . However, even though clades with greater species richness should have more climatic‐niche divergence among species based on sampling alone, this would not explain higher rates of change per unit time, as found here. Furthermore, analyses of rates of niche divergence and rates of net species diversification also support our inference that the number of species alone does not explain climatic‐niche divergence  remains to be seen. More broadly, our results illustrate the potential shortcomings of considering only a single predictor variable when trying to understand patterns of richness and diversification. Similarly, our results here contribute to many other lines of evidence arguing against ecological limits on species richness in plethodontids related to morphological evolution. These latter results underscore the need for objective, detailed ecological analyses to test hypotheses of ecological limits, rather than merely testing relationships between clade age and richness.None declared.


COPD subjects. The blood eosinophil count is a biomarker of sputum eosinophilia, but whether it is associated with bronchial submucosal eosinophils is unclear. In 20 COPD subjects and 21 controls we assessed the number of bronchial submucosal eosinophils and reticular basement membrane thickening and found these were positively correlated with the blood eosinophil percentage. In COPD, blood eosinophils are a good biomarker of bronchial eosinophilia and remodelling.A sputum eosinophilia is observed in 10–40% of  COPDchronic obstructive pulmonary diseaseIL5Interleukin‐5MBPmajor basic proteinRBMreticular basement membrane and lamina reticularisThere is an increasing need to find non‐invasive reliable biomarkers in asthma and chronic obstructive pulmonary disease (COPD) to identify different phenotypes and direct treatment. A sputum eosinophilia >3%) is present in 10–40% of COPD subjects.% is presWe hypothesized that the bronchial submucosal (lamina propria) eosinophil count and thickening of the reticular basement membrane and lamina reticularis (RBM) is related to the peripheral blood eosinophil percent in COPD. To test our hypothesis, we analysed large airway bronchial tissue samples from COPD subjects and controls to determine the association between bronchial and blood eosinophil counts.n = 20) were all ex or current smokers with spirometric evidence of airflow obstruction according to the Global Obstructive Lung Disease criteria for COPD. Subjects with a history of asthma were excluded. Control subjects (n = 21) included those with and without a smoking history without airflow obstruction. All subjects gave written informed consent. The study was approved by the Leicestershire, Northamptonshire and Rutland local ethics committee.We undertook a single‐centre observational study at Glenfield Hospital, Leicester, UK. Subjects with or without COPD undergoing surgical lung resection for cancer or suspected cancer were recruited. COPD subjects (2 submucosa (lamina propria between the RBM and airway smooth muscle bundle), and the RBM was measured as the mean of 50 measurements at 20 μm intervals. All measurements were undertaken by an observer blinded to the clinical characteristics.Bronchial tissue was dissected from the lung resection material and embedded in glycol‐methacrylate and stored at −80°C. Two micrometre sections were stained with monoclonal antibodies directed to major basic protein , tryptase  for eosinophils and mast cells respectively and corresponding isotype controls (Dako). The number of eosinophils and mast cells were enumerated per mmigh and eosinophillow determined by the group median peripheral blood eosinophil percentage as well as using the peripheral blood percentage cut‐off of 2% and an absolute peripheral blood eosinophil count of 0.3 × 109/L.t‐tests and analysis of variance or Mann–Whitney and Kruskal–Wallis for parametric and non‐parametric analyses as appropriate and correlations by Pearson and Spearman rank tests. A P value of <0.05 was considered statistically significant.Statistical analysis was performed using Prism version 6 . In addition to comparisons between COPD and controls, the subjects were stratified into eosionophilhSupplementary Table S1. There were no differences in gender between those with COPD and controls. COPD subjects were older and had a greater pack year smoking history and poorer lung function. The use of corticosteroids was similar between the eosinophillow (3/10) and eosinophilhigh (4/10) COPD groups (Fisher's Exact Test P = 1.0). There were no significant differences in the blood or submucosal eosinophil counts between COPD and controls nor in submucosal mast cell counts, although the RBM thickness was increased in COPD (9.7 (0.6)μm versus 7.1 (0.4)μm; P < 0.001) . In the COPD subjects, there was a strong correlation between the peripheral blood eosinophil percent and the submucosal eosinophil count  and RBM thickness  ‐5 monoclonal antibody mepolizumab.There were few potential drawbacks in our study. Baseline differences in age and smoking history between COPD and control groups might affect the differences observed in inflammation and remodelling. Additionally, we did not formally study whether subjects had atopy, and although we excluded subjects with asthma, we did not assess for asthma–COPD overlap syndrome. The study was retrospective, and contemporaneous sputum analysis was not performed. The number of subjects included was relatively small especially when they were divided into four groups based on blood eosinophil levels, although the correlations in the whole group were strong suggesting the findings were robust. However, further large‐scale prospective studies are warranted in order to validate and replicate our findings.In conclusion, in COPD, peripheral blood eosinophils are associated with submucosal eosinophils and airway remodelling. Therefore, consistent with previous reports of the peripheral blood eosinophil count as a biomarker of a sputum eosinophilia, it is also likely to be related to airway tissue eosinophilia. These data further support the peripheral blood eosinophil count as an important biomarker of an airway eosinophilia and is likely to be important in targeting current and future anti‐eosinophil therapies for COPD.The authors sincerely thank all the research volunteers who participated in the study and wish to express their gratitude to the following people for their valuable assistance in the study: Rachid Berair, Aarti Parmar, Fiona Symon, Jane Middelton and Mitesh Pancholi.This work was part funded by Medical Research Council and Wellcome Trust Senior Fellowship (CEB) and a sponsored studentship by the Libyan embassy in London on the behalf of Benghazi University in Libya. This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.Supplementary Table S1 Clinical characteristics of the COPD and control subjects stratified by the median peripheral blood eosinophil count percentage.Click here for additional data file.


In the next decade, advanced biocatalytic systems will increasingly be used for the production of chemicals that cannot be made by current processes and/or where the use of enzyme catalysts is more resource efficient with a much reduced environmental impact. We expect that in the future, manufacture of chemicals and materials will utilize both biocatalytic and chemical synthesis synergistically. The realization of such advanced biomanufacturing processes currently faces a number of major challenges. Ready‐to‐deploy portfolios of biocatalysts for design to production must be created from biological diverse sources and through protein engineering. Robust and efficient multi‐step enzymatic reaction cascades must be developed that can operate simultaneously in one‐pot. For this to happen, bio‐orthogonal strategies for spatial and temporal control of biocatalyst activities must be developed. Promising approaches and technologies are emerging that will eventually lead to the design of  In the coming decade, industrial biology is expected to accelerate and facilitate the production of high‐value chemicals that cannot be made by current methods or of high‐volume chemicals where biological processes are more economical and resource efficient with a much reduced environmental impact. Consequently, manufacturing of a range of chemicals and materials will increasingly utilize both enzymatic and chemical synthesis synergistically. Biomanufacturing of chemicals using cell‐based or in vitro synthesis of highly pure compounds from precursor molecules. While microbial production with metabolically engineered strains holds great promise for the production of commodity chemicals, synthesis of many other industrially relevant complex molecules such as fine chemicals and chemical building blocks currently is best achieved using cell‐free biocatalytic systems. A major advantage of in vitro systems is the ability to assemble with relative ease non‐natural biocatalytic cascade reactions by mixing and matching enzymes with the desired activities isolated from different sources  must be reduced considerably, process efficiency increased and catalytic processes optimized for scalable downstream processing  for a given reaction and/or substrate binding , many challenges still remain. The above examples illustrate that both enzyme choice and reaction engineering are critical for achieving robust cascade reactions that operate concurrently and at maximal performance. Therefore, in addition to developing a functionally diverse portfolio of robust, ready‐to‐use biocatalysts, engineering of optimal reaction environments for the operation of multi‐enzymatic cascades – much like the operation of metabolic pathways in cellular systems – will be critically important for realizing advanced biocatalytic processes suitable for the chemical industry.Although a range of promising bioactalytic cascade reactions have been developed, demonstrating the feasibility of carrying out complex one‐pot reaction schemes at preparative scale  using natural or synthetic protein scaffolds and nucleic acid scaffolds  in bacteria. Encapsulation peptides target metabolic enzymes into the compartments during their self‐assembly enzymatic cascades that execute synthetic tasks akin to the metabolic networks of living cells.


Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine‐tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger‐scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics. Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. The biosynthetic pathway of patellamides consists of a seven‐gene cluster  make up a wide group of natural compounds with various biological activities.100 human leukemic cell line,Cyanobactins, including patellamides, have diverse and valuable biological activities.mac (macrocyclisation domain of PatG) tolerates significant diversity in the amino acids in the core peptide,cis‐proline) and is flanked by a C‐terminal macrocyclisation signature AYD.mac as well as the selective derivatisation of the subsequent cyclic peptides. We also report the synthesis of a patellamide‐like cyclic peptide containing both a heterocycle and an unnatural amino acid in a one‐pot procedure.Biochemical studies have shown that PatG3) or a dehydroalanine (Dha) reactive group. The azide moiety is a well known bio‐orthogonal group that reacts with alkynes both ex and in vivo.3) and Dha, were introduced at different positions  in their respective core peptides to explore whether their incorporation presented any challenges for PatGmac processing. For simplicity, we made the first two compounds from peptides that lacked any cysteine residues, and therefore heterocycles. Having established a suitable approach, we advanced to adding the azide to a cysteine‐containing peptide. We were able to enzymatically heterocyclise the cysteine to a thiazoline within the sequence and then macrocyclise the resulting product in a one‐pot process, to make a genuine patellamide analogue.The introduction of bio‐orthogonal or specific reacting groups on the side chains of linear/cyclic peptide residues would be highly desirable, as they would allow regiospecific and facile derivatisation. This approach has been previously used to study binding and/or activity,1 (ITAA(N3)ITAPAYD/G) and 3  were incubated in the presence of PatGmac. The corresponding cyclic peptides cyclo(‐ITAA(N3)ITAP‐) (2) and cyclo(‐VDhaAGIGFP‐) (4) were obtained in 45 and 63 % purified yield, respectively . Furthermore, the Dha group was found to be highly stable under the macrocyclisation reaction conditions .The synthetic precursor peptides 2 was then reacted with cyclooctyne 5 in a copper‐free, strain‐promoted azide–alkyne cycloaddition , which contains the fluorescent cyanine tag  and emission (λ=671 nm) properties of conjugate 8 were in good agreement with those of the parent Cy5 molecule . As these types of compounds could potentially be used for target identification by fluorescence microscopy, conjugate 8 (dark blue colour) was tested to ensure there was no unexpected behaviour (such as quenching or precipitation) in cells. When incubated with permeabilised HeLa cells, a diffuse staining pattern of 8 (red colour) throughout the cytoplasm and nucleus was visualised by fluorescent microscopy .Cyclic peptide mac processes substrates with unnatural amino acids at similar rates to other sequences,1 was replaced with a cysteine (peptide 12) that could be enzymatically heterocyclised. Like PatGmac, the heterocyclase enzymes of the cyanobactin pathways (known as the D enzymes)As PatG12 overnight with the engineered heterocyclase LynDfusion (from the aestuaramide pathway (Lyngbya sp.)) in the presence of ATP and MgCl2. The fully heterocyclysed product 13 was detected by MS but not isolated ITAhetC‐) in 58 % yield.We incubated peptide Milligram quantities of cyanobactin derivatives with fluorescent components will greatly facilitate the target identification of many of these natural biologically active products.We have demonstrated that both the heterocyclases and macrocyclases from the patellamide (and a related) pathway can be used in vitro with entirely synthetic substrates that contain such chemically reactive unnatural amino acids. Moreover, we have shown that the resulting macrocycles can be derivatised with high efficiency. The ability to combine the diversity of chemical synthesis with the exquisite catalysis of enzymes is well known and recognised to be powerful in developing natural products into therapeutics.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


In this review, we discuss the application of mouse models to the identification and pre‐clinical validation of novel therapeutic targets in colorectal cancer, and to the search for early disease biomarkers. Large‐scale genomic, transcriptomic and epigenomic profiling of colorectal carcinomas has led to the identification of many candidate genes whose direct contribution to tumourigenesis is yet to be defined; we discuss the utility of cross‐species comparative ‘omics‐based approaches to this problem. We highlight recent progress in modelling late‐stage disease using mice, and discuss ways in which mouse models could better recapitulate the complexity of human cancers to tackle the problem of therapeutic resistance and recurrence after surgical resection. CRCcolorectal cancerEGFepidermal growth factorGEMMgenetically engineered mouse modelsHNPCChereditary non‐polyposis CRCSNPssingle‐nucleotide polymorphismsTfhT follicular helperTGFβtransforming growth factor βAPC gene (>80% CRCs) AXIN2 (Axis inhibition protein 2) and AMER1 (APC membrane recruitment 1) are found in tumours that also harbour APC mutations Colorectal cancer (CRC) accounts for 700,000 deaths and 1.4 million newly diagnosed cases globally per annum, making it the number one cause of non‐smoking related cancer deaths PC gene >0% CRCs ASMAD gene family members or TGFβ receptors, promote the development of advanced adenomas or invasive adenocarcinomas TP53 (Tumour protein p53) or IGF2R (Insulin‐like growth factor 2 receptor) occurs with greater frequency in established carcinomas that invade submucosal layers than in adenomas Activation of the Ras‐MAPK  pathway, usually via point mutations that constitutively activate KRAS or BRAF , and inactivation of the TGFβ (transforming growth factor β) pathway through inactivation of Endoscopic or surgical resection is routinely used for patients with early premalignant adenomas, as well as for the treatment of most early stage carcinomas and selected patients with late stage or advanced metastatic disease, for whom chemotherapy  is also a key treatment modality KRAS, an intracellular signalling pathway that converges with the EGF receptor pathway. Finally, the drug discovery pipeline attrition rate for oncology investigational compounds is extremely high. Only 5% of compounds that show promise in pre‐clinical studies are eventually approved for clinical use as the majority fail in Phase I/II trials Aside from our lack of understanding of the molecular events that drive metastases, there are several other plausible explanations for our limited success in treating CRC. Firstly, there are currently few biomarkers that are predictive of early disease, the likelihood of favourable treatment response, recurrence or the development of metastatic disease Xenografting involves the injection of cells or the surgical transplantation of primary tissue into immune‐deficient mice such as the nude mouse, which is athymic and does not produce T‐lymphocytes, or the severe combined immune‐deficient (SCID) mouse, which has altered adaptive immunity but normal innate immunity, to prevent rejection. It was thought that the large number of commercial colorectal cell lines available would represent the inter‐tumour heterogeneity of the human disease, but in reality, few cell lines lead to reliable primary tumour growth, and fewer still to naturally metastatic CRC Microenvironmental differences likely explain why the morphology of subcutaneous CRC cell line xenografts, especially the stromal elements, are often markedly different from the human disease and may explain why they have a low metastatic potential Patient derived xenografts (or ‘PDXs’) avoid the natural selection of dominant clones and epigenetic and genetic alterations that occur during long‐term cell culture as well as temporarily preserving the original, species‐specific, tumour‐stromal cell interactions. The suturing of small fragments of fresh surgical specimens of CRC liver metastases to the caecum of immune‐deficient mice has been reproducible in generating models of metastatic CRC Smad4 in T‐cells excessively secrete proinflammatory cytokines and develop gastrointestinal tumours The two major limitations of all xenograft mouse models of human CRC cell lines are the species mismatch between tumour and stroma, which likely affects cell communication, and the use of immune‐deficient hosts. The role of the immune system appears to be particularly important for the development of CRC; T cell immune infiltrate is an important predictive criterion for patient survival Genetically engineered mouse models (GEMM) offer several advantages over cancer cell lines as research tools in cancer research. Despite their evolutionary distance, the gene content of the mouse and human genomes has been largely conserved through evolution 2. Furthermore, there is a lack of inter‐tumour heterogeneity of GEMM tumours because laboratory mice are inbred and therefore lack the genetic heterogeneity present in the outbred human population. Moreover, GEMM tumours are often initiated and maintained by the same genetic mutation of a strong cancer driver gene, which may limit the number of pathways to tumourigenesis. While the difference in complexity of tumours is advantageous when applying a cross‐species oncogenomics approach to cancer gene validation Sequencing of DNA from several tumour types that have developed in mouse or man has recently revealed that human tumours are more heterogeneous than their GEMM‐derived counterparts Min/+Apc mouse was subsequently recognised as a paralog for human familial adenomatous polyposis (FAP) syndrome and provided confirmation of the causal gene mutation in sporadic CRCs with 5q21 deletions APC is observed in >80% of sporadic CRCs Table MLH1, PMS1, PMS2, MSH2 or MSH6,  that encode the enzymatic machinery of DNA mismatch repair. As with sporadic cancers, intestinal cancers from HNPCC patients have a high frequency of mutations in APC, as well as KRAS, TP53 and TGFBR2MLH1 promoter −/−Msh2 deficient mouse Cre‐recombinase driven by a tissue‐specific promoter. Cre‐recombinases catalyse recombination between two loxP sites that are situated in the introns surrounding a critical exon(s) and result in excision of the DNA between the sites. Using a conditional strategy that results in expression of Cre recombinase from the Villin promoter  a LoxPVillin‐Cre/Msh2 mouse was generated that resulted in intestinal adenomas and adenocarcinomas in the absence of lymphoma Hereditary non‐polyposis CRC (HNPCC) or Lynch Syndrome, is the most common inherited, autosomal dominant CRC syndrome and accounts for around 3% of all CRCs. HNPCC is most frequently caused by mutations in Min/+Apc mouse limits the utility of the model and it was thought that mice with fewer tumours would live longer, and that if tumours were restricted to the colon, they might progress to carcinoma and metastasise Apc mutant mice with germline or inducible cell‐type specific conditional alleles of Apc were developed  mice had developed metastases to distant organs such as the liver Apc may be disrupted by introduction of conditional alleles, which may elevate Wnt signalling levels in tissues other than the intestine, and which appears to drive the formation of life‐limiting hepatocellular tumourigenesis before the onset of intestinal tumourigenesis. For example Apcfl/fl and 580S/580SApc mice show reduced expression of Apc protein throughout their tissues , and develop hepatocellular carcinomas between 9 and 15 months  such as ulcerative colitis predispose patients to developing colorectal cancer as a result of chronic inflammation. It is becoming apparent that these tumours may develop through alternative TCGA has identified genes that are frequently mutated in many different types of malignancy (frequently referred to as ‘pan‐cancer’ genes) suggesting that they are likely to accelerate the process Villin‐Cre recombinase system APC, FBXW7 (F‐box and WD repeat domain containing 7), PTEN (phosphatase and tensin homolog), and SMAD4, thus validating the approach. The remainder, including RSPO2 (R‐spondin 2), had not been previously implicated in the disease. R Spondin is a ligand for LGR4‐6 receptors , which stimulate the canonical Wnt signalling pathway. We now know that RSPO2 fusion genes occur in a subset of colorectal carcinomas and are mutually exclusive with APC mutations One of the first insertional mutagenesis studies to identify novel genetic drivers of intestinal tumourigenesis involved restricting transposition to epithelial cells of the gastrointestinal tract using a Apc and hyperactivation of the Wnt pathway, two independent teams of researchers mobilised the Sleeping Beauty transposon in Min/+Apc mice or conditional fl/+Apc mice Apc, consistent with the requirement for loss of heterozygosity of Apc for intestinal tumour induction. Interestingly, all three mutagenesis screens identified a high number of insertions in the WW domain‐containing adaptor with coiled‐coil gene (Wac), which is also mutated in human cancers Apc mutant mice identified common insertions in the voltage‐gated potassium channel subunit Kcnq1Min/+Kcnq1−/−Apc double mutant mice developed more intestinal adenomas than Min/+Apc control mice, and progression to aggressive adenocarcinomas was observed. The authors went on to show that reduced KCNQ1 expression correlated with poor survival in patients; however, the exact mechanism of tumour suppression remains unknown To identify mutations that cooperate with inactivation of Apc mutations, which model the initiation of intestinal tumourigenesis, to screen for early disease biomarkers. Clusterin was a ‘proof‐of‐principle’ for the cross‐species approach to biomarker discovery Min/+Apc mice and human tumours, and was recently shown to be highly‐expressed in intestinal stem cells Most screening strategies aimed at the early detection of adenomas and CRC involve endoscopy, and thus have limited applicability. Early detection of CRC is critical for disease management, and so the identification of reliable, early disease biomarkers would improve the outcome for many patients. Mouse models that are predisposed to colorectal tumour formation can be an efficient tool for early disease biomarker discovery Fig. . Since e580S/ΔApc mice was screened by mass spectrometry, and cathepsin B and D were found to be elevated compared with control mice 15lox/+ Fabpl‐CreApc mice resulted in the identification of 192 proteins that were more highly excreted by tumours relative to control, including Chitinase 3‐like 1 (Chi3l1) Recent improvements in proteomic profiling methods, based primarily on mass spectrometry, have allowed the detection and identification of peptides in the blood and biological fluids of cancer patients Fig. . The plaMin/+Apc mice has also been useful in the identification of patient relevant, early disease biomarkers  that includes human SNP rs6983267 are resistant to intestinal tumours induced by the ApcMin mutation Apc loss, it has not yet been possible to pharmacologically regulate Myc. Instead, a selective small‐molecule BET bromodomain inhibitor, JQ1, inhibits proteins that are regulatory factors for Myc The transcription factor Myc proto‐oncogene protein (Myc) is thought to be a target of the β‐catenin–Tcf4 transcription complex in CRC cells in vitro, in normal crypts in vivo and in intestinal epithelial cells acutely transformed on in vivo deletion of the TGFB1 cells were conferred a survival advantage by suppressing apoptotic stimuli encountered during organ colonization. Moreover, inhibition of TGFBR1 has been shown to inhibit metastasis formation in multiple xenograft models CKO/CKO/KrasG12DApc colon cancer model A large proportion of CRCs display mutational inactivation of the TGFβ pathway. However, paradoxically, they are characterised by elevated TGFβ production. In other cancer types, such as breast or prostate, which retain a functional TGFβ signalling pathway, TGFβ induces a variety of prometastatic programmes that range from induction of epithelial‐to‐mesenchymal‐like transition (or ‘EMT’) to expression of genes that allow colonisation of foreign organs −/Cxcr5− mice (CXCR5 is the receptor for CXCL13) compared to control, and rejection of tumour cells when recombinant CXCL13 was injected into the colonic submucosa of wild‐type mice before transplantation In a recent study, Bindea and colleagues examined the spatio‐temporal dynamics of 28 different immune cell types (the ‘immunome’) that were infiltrating colorectal tumours were analysed by whole‐genome expression, PCR arrays and multicolored immunohistochemistry In combination with studies of CRC cell lines and organoid models, mouse models of late‐stage CRC will help us to build a comprehensive picture of the molecular alterations that drive tumour formation and disease progression, through cross‐species analysis of the genomes, epigenomes, transcriptomes, proteomes and even immunomes of cancers of mice and men. Mouse models will be critical for reductionist approaches that aim to validate the cancer drivers from mutations identified by the TCGA.Human CRC is the result of interactions between genes, including those involved in host immunity, and the environment, including diet and the microbiome


RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N‐terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC‐helix and charged residues upstream of the NtA motif. Additionally, we show that the R‐spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure‐based mechanism for the auto‐transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization‐related drug resistance. RAF kinases connect the Ras GTPase to activation of the MEK‐ERK pathway. This pathway regulates many fundamental cellular functions, including cell proliferation, and is dysregulated in approximately 50 % of human cancers.Based on recently available crystallographic data for RAF . Remarkably, the C‐terminal end of the αC‐helix is neutral in most kinases, whereas in RAFs it carries a +3 charge. Moreover, mutations of these charged residues impair kinase activityOur simulations also reveal that the phosphorylated NtA motif is connected to the active site via the R‐spinePrevious experiments showed that mutation of a conserved Trp residue  impedes transactivation and impairs the dimerization of BRAF.Importantly, our results indicate phosphorylation‐induced large‐scale structural changes in RAF dimers, whereas the unphosphorylated dimers remain structurally similar to their starting structures, in agreement with the crystallographic structures. Only when all phosphorylated residues, including the two activation‐loop residues near V600, are present in the simulated system, we observe large changes between the universally conserved HRD and DFG motifs Figure . These cIn summary, our results show that phosphorylation of the NtA motif promotes dimerization through several interprotomer salt bridges formed between the NtA motif of one of the protomers and the positively charged C‐terminal end of the αC‐helix of the other protomer. They further reveal the importance of the conserved tryptophan residue located at the N‐terminal end of the kinase domain (W450 for BRAF), which plays a crucial role by connecting the R‐spines of the two protomers. This suggests a cooperative interprotomer interaction that is mediated by salt bridges involving the phosphorylated NtA motif. The direct interaction of the R‐spines via W450 explains why mutation of W450 abolishes the transactivation of RAF dimers.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.