The acquisition and mastery of speech-motor control requires years of practice spanning the course of development. People who stutter often perform poorly on speech-motor tasks thereby calling into question their ability to establish the stable neural motor programs required for masterful speech-motor control. There is evidence to support the assertion that these neural motor programs are represented in the posterior part of Broca’s area, specifically the left pars opercularis. Consequently, various theories of stuttering causation posit that the disorder is related to a breakdown in the formation of the neural motor programs for speech early in development and that this breakdown is maintained throughout life. To date, no study has examined the potential neurodevelopmental signatures of the disorder across pediatric and adult populations. The current study aimed to fill this gap in our knowledge. We hypothesized that the developmental trajectory of cortical thickness in people who stutter would differ across the lifespan in the left pars opercularis relative to a group of control participants. We collected structural magnetic resonance images from 116 males (55 people who stutter) ranging in age from 6 to 48 years old. Differences in cortical thickness across ages and between patients and controls were investigated in 30 brain regions previously implicated in speech-motor control. An interaction between age and group was found for the left pars opercularis only. In people who stutter, the pars opercularis did not demonstrate the typical maturational pattern of gradual gray matter thinning with age across the lifespan that we observed in control participants. In contrast, the developmental trajectory of gray matter thickness in other regions of interest within the neural network for speech-motor control was similar for both groups. Our findings indicate that the developmental trajectory of gray matter in left pars opercularis is abnormal in people who stutter. Stuttering is a complex developmental communication disorder affecting 1% of the population  44 and 45. BA44 and 45 are relatively synonymous with the pars opercularis and pars triangularis of the left hemisphere which are labeled via the cortical folding nomenclature  = 0.37 p = 0.71). The Stuttering Severity Index—3rd Edition was used to measure stuttering severity in the patient group. Stuttering severity ranged from very mild (5) to very severe (49) . One patient’s stuttering severity score was not available due to technical difficulty.Participants were 116 right-handed males with English as their primary language and a normal developmental and medical history. The 55 boys and men with persistent developmental stuttering ranged in age from 7 to 47 years  and the 61 matched control participants from 6 to 48 years . The two groups did not differ in age (T1 images for 51 participants (23 people who stutter) were collected on a 1.5-T MRI system  using a standard quadrature head coil at the Toronto Western Hospital in Toronto, Ontario, Canada. A T1-weighted three-dimensional inversion recovery-prepared fast spoiled gradient echo (FSPGR) sequence  = 5.2 ms, repetition time (TR) = 12 ms, preparation time = 300 ms) was used to generate 124 1.5 mm-thick sagittal slices (256 × 256 matrix). T1 images for 47 participants (23 people who stutter) were collected on a 1.5-T MRI system at the Hospital for Sick Children in Toronto, Ontario, Canada. A 1.5-T Signa Excite HD 12.0 MRI System  with an 8-channel head coil was used to obtain neuroantomic images. A T1-weighted 3D FSPGR sequence  was used to generate 114 1.5 mm-thick axial slices . Pixels were squared post processing ultimately resulting in a 256 × 256 viewing matrix. The images for the remaining 18 participants (9 people who stutter) were collected on a 3-T MRI system  using an 8-channel head coil at the Magnetic Resonance Research Center at Yale University in New Haven, Connecticut, USA. A T1-weighted three-dimensional MPRAGE sequence  was used to generate 176 1 mm-thick sagittal slices (256 × 256 matrix).T1 images were registered to the ICBM 152 template with a 12-parameter linear transformation , separately for each hemisphere, based on our hypothesis. The ROIs are depicted in Figure F = 10.63, p = 0.001, q = 0.04). Gray matter in this region was thinner in the younger people who stutter relative to their same aged peers but this association inversed with age such that gray matter was thicker in older people who stutter relative to their same age peers. By contrast, the developmental trajectory of thickness for the other ROIs was similar for both groups and no statistically significant interactions were identified.The results of the ROI statistical analyses are shown in Table Our cross-sectional study is the first to examine the maturational time course of cortical gray matter thickness associated with persistent developmental stuttering across the lifespan in a large cohort of patients. Our data indicate that the developmental trajectory of gray matter thickness in Broca’s area, specifically the left pars opercularis, is abnormal in people who stutter. Within the group of people who stutter, the gray matter in the left pars opercularis did not demonstrate the typical linear maturational pattern of gradual life-long thinning associated with the controls but rather was unchanging across the lifespan. No other regional group differences were identified. Our findings suggest that the neuroanatomic microstructure of the left pars opercularis is crucially important for the development of fluent speech-motor control and that deviations in the development of this region are associated with the onset and persistence of stuttered speech.The acquisition of speech-motor control spans the course of infant, child and adolescent development (Green et al., Our observed abnormal developmental pattern may contribute to the chronic nature of stuttering, as adult-like efficiency of neural processing in the pars opercularis is not attained with maturation. There is an emerging literature of behavioral evidence to support this assertion. Children who stutter as young as 4-years-old are known to have abnormal speech-planning and execution skills as evidenced by higher variability in oral-motor coordination relative to typically developing peers (Smith et al., Importantly, there is a precedent for associating changes in cortical gray matter development with speech-motor and language related behaviors. The pars opercularis and posterior superior temporal gyrus show protracted development relative to other brain regions over the school-age years suggesting the importance of continued cortical maturation in these areas for speech-motor and language development during this period (Sowell et al., Our control data demonstrate the typical pattern of gray matter development defined by thicker gray matter cortex in pre-adolescent followed by a steady and consistent post-adolescent decrease across the majority of neural regions for the remainder of life Figure , 2004. EThe identification of a developmental pathology within the left pars opercularis in people who stutter is consistent with the existing structural and functional neuroimaging literature and modern hypotheses of stuttering onset and persistence. At this time it is impossible to determine if the deficient development of gray matter within the left pars opercularis is causal or the result of the repeatedly documented limited white matter connectivity that this region has with critical nodes within the neural network for speech production in both children and adults who persistent phenotypes of the disorder (Sommer et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila brain have typically been considered from either a developmental or functional perspective without reference to how the circuits might have been inherited from ancestral forms. For example, two candidate pathways for ON- and OFF-edge motion detection in the visual system act via circuits that use respectively either T4 or T5, two cell types of the fourth neuropil, or lobula plate (LOP), that exhibit narrow-field direction-selective responses and provide input to wide-field tangential neurons. T4 or T5 both have four subtypes that terminate one each in the four strata of the LOP. Representatives are reported in a wide range of Diptera, and both cell types exhibit various similarities in: (1) the morphology of their dendritic arbors; (2) their four morphological and functional subtypes; (3) their cholinergic profile in Drosophila; (4) their input from the pathways of L3 cells in the first neuropil, or lamina (LA), and by one of a pair of LA cells, L1 (to the T4 pathway) and L2 (to the T5 pathway); and (5) their innervation by a single, wide-field contralateral tangential neuron from the central brain. Progenitors of both also express the gene atonal early in their proliferation from the inner anlage of the developing optic lobe, being alone among many other cell type progeny to do so. Yet T4 receives input in the second neuropil, or medulla (ME), and T5 in the third neuropil or lobula (LO). Here we suggest that these two cell types were originally one, that their ancestral cell population duplicated and split to innervate separate ME and LO neuropils, and that a fiber crossing—the internal chiasma—arose between the two neuropils. The split most plausibly occurred, we suggest, with the formation of the LO as a new neuropil that formed when it separated from its ancestral neuropil to leave the ME, suggesting additionally that ME input neurons to T4 and T5 may also have had a common origin.Synaptic circuits for identified behaviors in the  The problem of how a complex brain with highly organized pathways and centers could have arisen from simpler forms lacking clear structural compartments and with less differentiated cellular components, has generally attracted discussion chiefly in seeking a transition from very basal forms to those in which brain structure assumes some canonical stage —and the outer part of the second neuropil, or medulla (ME), neuropil splitting is said to have occurred among neuroblast proliferation centers, or anlagen, and to be accompanied by the formation of a chiasma of axons connecting between the two new descendant neuropils  form a chiasma, those from neighboring transmedulla (Tm) cells inverting their retinotopic order within alternating fiber sheets, first depicted by Braitenberg  as sheetThe morphologies of T4 and T5 cells are highly characteristic, and each has unmistakable similarities to the other Figures  especialIn a further final similarity, T4 and T5 cells are both also innervated by a complex tangential cell (CT1), a newly discovered giant contralateral tangential neuron from the central brain. This cell has laterally directed neurites that split and innervate terminals in both the ME and LO, one per column in all columns of the M10 and Lo1 strata , and that receive reciprocal input from the corresponding T4 (ME) and T5 (LO) cells  reconstruction of synaptic circuits , but a lack of immunoreactivity to the vesicular GABA transporter VGAT  how the LO arose, and whether, as we are about to suggest, this could have been from an ancestral neuropil fused with what then became the modern proximal medulla (PM); (2) the topological requirements for this transition, especially those of axon trajectories within the internal chiasma  the eyeless sine oculis mutant lacks both LA and outer ME, whereas the inner ME, LO and LOP are all still present  and the new LO, share some significant anatomical features in common. These include a population of visual projection neurons (VPNs) conveying visual inputs to the central brain, and the nearly-symmetrical innervation of both neuropils by LOP Y cells  in the central brain Figure . We firsThe only significant cell population with a major innervation site in the LO is in fact that of the VPNs, which are actually not optic lobe neurons at all, but develop from neuroblasts in the central brain  circuit, comparing this against an input from a unicolumnar partner, Mi1  diverged further from each other than Tm4 did from Tm3. Two broad options for a unicolumnar input exist: (1) Either there was a single input terminal in each column, one that terminated in ME strata M9, M10, and possibly Lo1, and this became Mi1 for T4, with its own input in turn from the L1 pathway. For input to a new T5 cell population from the L2 pathway, an Mi1-like ancestor would then have had either to extend deeper to LO stratum Lo1 or to retract selectively from M9 and M10 Figures , and thiThe options for the “VPN-duplication” model Figure  are muchDid L1 and L2 arise by duplication in the LA to initiate the segregation between T4 and T5? The circuits driving modern T4 and T5 differ in receiving respective inputs from the L1 and L2 pathways. How might these have arisen as separate pathways? Evidence is mostly only suggestive, but both L-cells are similar in two important ways. First, their dendrites cooperate closely in occupying two of the four postsynaptic sites at all photoreceptor tetrads ; both thus have a cholinergic phenotype. This might be held to support their common ancestral origin, but the weakness in this argument is that most elements of the L2 pathway are also cholinergic as well. L2 and Tm2 (Takemura et al., cha transcripts and ChAT immunoreactivity, and Tm1 is likewise ChAT positive (Shinomiya et al., cha transcripts and ChAT immunoreactivity (Shinomiya et al., Finally, what can be learned from the neurotransmitter phenotype of these cells? From a survey of molluskan species, Sakharov  viewed tDrosophila. It remains to be determined whether during the course of evolution the dichotomy arose first with L1 and L2 in the LA or, deeper in the optic lobe, with the origin of T4 and T5 as evolutionary siblings. Looking yet further afield, it seems plausible that an evolutionary driving force toward ON- and OFF-coding pathways arose more generally, because this dichotomy appears very widely in sensory systems of many stripes. Other benefits may result from an ON/OFF split in sensory pathways, in particular that such a split may simplify downstream computations (Gjorgjieva et al., The existence of dichotomously antagonistic ON- and OFF- pathways has of course long been known in the vertebrate RE. There, an ON-OFF split in the visual pathways already occurs at the first synapse (Schiller, For all that we hope it to be closely reasoned, most of what we propose is entirely speculative and unlikely to receive detailed support from comparative anatomy or the fossil record. On the other hand, we can anticipate that future comparative transcriptome analyses among identified neurons may bear with greater force on our proposals, and help address the question of how neural circuits might have arisen from among identified neurons of the same or neighboring neuropils. In parallel, we may also anticipate that more detailed studies on the development of the neurons of the fly’s ON- and OFF-edge motion pathways may also help contribute additional support for our hypothesis. For example, knowing the lineage relationships between T4 and T5 may help to consolidate further the relationship we propose for these cells as evolutionary siblings, as well as it would provide support for the relationships between neurons of their input pathways. In particular, such data could help demonstrate an evolutionary relationship between T4 and T5’s upstream partner neurons. For example the lineage and genetic relationships that we may propose from morphological evidence alone, between Tm cells such as Tm3 and Tm4, or between Mi and Tm cells, such as Mi1 and Tm1/Tm2, could help to arbitrate evolutionary relationships as robustly as we hope to have demonstrated these for their target neurons, T4 and T5. Unlike the latter pair, comparative anatomy and the fossil record are, we think, unlikely to further our understanding of the evolutionary relationships of these ME cells beyond the realm of suggestion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nematostella vectensis is a well-established system for the study of development and evolution that is receiving increased attention for its regenerative capacity. Nematostella is able to regrow missing body parts within five to six days after its bisection, yet studies describing the morphological, cellular, and molecular events underlying this process are sparse and very heterogeneous in their experimental approaches. In this study, we lay down the basic framework to study oral regeneration in Nematostellavectensis. Using various imaging and staining techniques we characterize in detail the morphological, cellular, and global molecular events that define specific landmarks of this process. Furthermore, we describe in vivo assays to evaluate wound healing success and the initiation of pharynx reformation. Using our described landmarks for regeneration and in vivo assays, we analyze the effects of perturbing either transcription or cellular proliferation on the regenerative process. Interestingly, neither one of these experimental perturbations has major effects on wound closure, although they slightly delay or partially block it. We further show that while the inhibition of transcription blocks regeneration in a very early step, inhibiting cellular proliferation only affects later events such as pharynx reformation and tentacle elongation.Cnidarians, the extant sister group to bilateria, are well known for their impressive regenerative capacity. The sea anemone  Hydra, a cnidarian that belongs to the group of hydrozoans. While regenerative potential has since then been described in other groups of cnidarians , the maAnthozoa ,6). Thestostella ). They aractinia ) and eveion site . TherefoNematostella vectensis (Nv) is a well-established system for the study of embryonic and larval development [The majority of cnidarian species belong to the Anthozoa class  which diverges basally to their other cnidarian sister groups ,11,12. Telopment ,16,17,18elopment .Nematostella is a diploblastic animal comprised of an outer ectodermal epithelium and an inner gastrodermis that are separated by muscle fibers and the mesoglea  have over the past few years increased the interest to study regeneration in this cnidarian. However, studies describing the morphological, cellular, and molecular events underlying this process are still sparse and very heterogeneous in their experimental approaches [vs. adults); (ii) different amputation sites ; and (iii) isolated body parts were left to regenerate at different temperatures (between 15 and 27 °C). The potential to regenerate into a functional organism appears to be similar between the different body parts of an adult Nv [Nematostella staging system for the regenerating adult isolated physa (NRSS) that the regeneration speed is temperature-dependent [Nematostella to reform the missing body parts.Existing resources ,25,26,27iewed in , Layden proaches ,33,34,35adult Nv . The temependent . HoweverNematostella regeneration have focused on oral regeneration following sub-pharyngeal amputation. In our present study, we show that there are no apparent differences in the cellular mechanisms underlying oral regeneration after sub-pharyngeal regeneration in juveniles compared to adults. We further lay down the basic framework to study oral regeneration in juvenile Nematostella by taking advantage of the ease of animal imaging at this stage. We carefully analyze and describe morphological and cellular events that define specific landmarks of regeneration. Additionally, we propose assays to assess wound healing success and pharynx re-formation. Finally, we show the usefulness of these assays and landmarks for phenotype characterization from perturbation experiments.The majority of the studies on Nematostella cannot yet be determined at a molecular or cellular level, one can distinguish juvenile and adult polyps from their anatomy  each daWe observed a specific sequence of events during the regeneration process . First, Nematostella juveniles or adults and left them to regenerate at 16 °C , the nematosomes are forced to exit the body cavity through the amputation site  0–12 hpa, no contact between the remaining mesenteries and the surrounding oral epithelia a; Step b; Step c. The epNematostella, like other cnidarians, possesses endogenous fluorescence emitted by fluorescent proteins and/or fluorescence of the tissues. The six-week-old Nematostella juveniles display a green (excitation wave length at 488 nm) fluorescence (henceforth referred to as 488+) from a currently unknown origin  based on morphology parameters  within the same batch  that enables the analysis of animals based on their length , density (extinction), morphology (profiler), fluorescence, and the relative localization of the fluorescence along the animal body. In order to analyze the global amount of fluorescence intensity and its localization within the animals, we first defined debris  using spectral confocal imaging . Spectral imaging measures the complete fluorescent spectrum of each pixel and matches these to pre-calibrated profiles. In our experiments we calibrated the profiles to detect converted Kaede, unconverted Kaede, and endogenous fluorescence. We found that the central epithelia of the amputation site gave rise to the tentacles in 19 out of 19 cases a,a’. AddNematostella, we used EU-Click-it chemistry  to detect de novo transcription in Nematostella after amputation  throughout the body epithelia , a modified Uridine analog, incorporated into nascent RNA . In uncupithelia a,a’. Aftpithelia . Howeverpithelia b’,c’. Atpithelia . At 96 hpithelia . Staininpithelia e,e’. In pithelia .de novo transcription during regeneration in Nematostella, we treated amputated juveniles with the antibiotic Actinomycin D (AMD), an inhibitor of DNA-primed RNA synthesis [In order to determine the role of ynthesis . In untrNematostella [A recent study has used hydroxyurea (HU) to efficiently block proliferation and regeneration in tostella . Howevern = 58). At 12 hpa, almost all of the animals treated with HU are completely healed , although the process was delayed by approximately 6 h (n = 71) are not healed at 6 hpa, and a similar fraction remains open at 12 hpa  as well as 24 hpa   . This obversus AMD-treated juveniles. Similar to controls at 12 hpa, HU-treated polyps progress to Step 1 when the mesenteries are fused together and enter in contact with the surrounding epithelia at the amputation site . In AMD-treated juveniles, we never observed 488+ in 100% (52 out of 52 cases). We obtained similar results in HU-treated polyps in which 488+ never becomes detectable in 85% (53 out of 62 cases). In addition, no pharyngeal lip or tentacle bulbs were visible in either of the treatments e,f as thAt 144 hpa, HU-treated polyps still remain blocked at Step 1 h comparein vivo tools that can be used to assess wound healing and pharynx formation in juveniles after sub-pharyngeal amputation.In the present study, we describe in detail the morphological and cellular landmarks for regeneration . We alsode novo transcription first in the gastrodermis and mesenteries at 12 hpa at the amputation site. Then, a massive wave of de novo transcription is activated at 48 hpa in the epidermis, gastrodermis, and in the mesenteries at the amputation site. Similarly, cellular proliferation is first detected at 12 hpa at the wound site and increases progressively over time. It is detected in the oral parts of the mesenteries at 24–48 hpa and remains at high levels until regeneration is achieved.From a morphological point of view, we observe a stereotypic timeline of events: wound healing is completed after 4–6 hpa. Then, the mesenteries fuse together and contact the amputation site at about 12–24 hpa. This is followed by the emergence of a gap between the mesenteries and the wound site between 60 and 72 hpa. This space allows proper pharynx formation around 72–96 hpa by the means of rearrangement of pre-existing mesenteries and cell proliferation. This characteristic behavior of the mesenteries to form the pharynx is accompanied by tentacle bud formation and elongation to form a juvenile that is indistinguishable from its uncut siblings. Along with the morphological timeline, we further describe While these morphological landmarks can be used to define steps of regeneration, none of them can be associated with precise temporal information. This is because within a given batch of animals we observed that regeneration speed varies considerably and not all animals reach a given intermediate step  at the same time  or late (60–96 hpa) during regeneration. In fact, we did not observe specific morphological or cellular events that are initiated in the period spanning this gap. However, it is known that cellular proliferation progressively increases  during t editing  and thus editing  that couin vivo assay to determine the success of wound healing after bisection of juveniles by taking advantage of the existence of nematosomes, free circulating aggregations of cells/cnidocytes. Upon compression of the animal with tweezers, the nematosomes exit the body cavity at the amputation site during the process of wound healing and exit through the aboral pore once wound healing is completed. However, we currently cannot exclude the fact that a reformed extracellular matrix or massive production of mucus may prevent the nematosomes from leaking through the wound site during the compression assay before the wound tissue is actually closed.We developed an n = 41). This observation suggests that the fully developed mouth contracts strongly or that the connection between the body cavity and the tentacles disperses and decreases the mechanical forces that the forceps apply to the mouth region. We also tried to assess the timing of mouth opening by feeding the juveniles. However, the size of the animals (100–150 μm) requires that we feed them with smashed brine shrimp, which makes ingestion very heterogeneous within a batch and very difficult to assess. Therefore, detailed morphological and cellular analyses are required to determine the timing of mouth reopening in juveniles  that is potentially associated with nascent nematosomes is detected ubiquitously in freshly fed animals but localized to the pharynx in juveniles that were starved for one or two weeks, reflecting potentially different metabolic states. We show that hand-sorting uncut animals by size  or bio-sorting them by size and 488+ intensity/localization results in a similar proportion of 488+ and 488− at 72 and 120 hpa . We concD. rerio ,44. In ain vivo assays to determine the timing of the wound healing and the regenerative progression, further underline that these approaches are very useful in juveniles. Furthermore, adults also contain a large amount of free circulating nematosomes, display a strong and very characteristic red endogenous fluorescence in the pharynx caused by the expression of NvFP-7R (not detected in the four-tentacle juveniles), and exhibit green endogenous fluorescence throughout the body and the tentacles [Both of the assays we present in this study, compression and emergence of 488+, enable us to determine precise events without having to fix and stain the animals. Thus, the same batch of animals can be followed over the course of the regeneration process. The perturbation experiments we carried out (AMD and HU), and for which we used the entacles . Thus, iNematostella do not display any differences in the timing of regeneration or the requirement of cellular proliferation after sub-pharyngeal amputation. Thus, it would be crucial to extend this analysis and perform a systematic comparison of the isolated body parts in juveniles and adults. This could identify potential age-related differences or a complete conservation of the cellular  mechanisms underlying whole-body regeneration in Nematostella juveniles and adults. This is of particular importance since in Hydra, the mechanism involved in head regeneration differs depending on the region of amputation (sub-tentacle crown vs. mid-body) [We show that juvenile and adult id-body) .Nematostella [We chose to analyze regeneration in juveniles for the ease of imaging morphological details that are more difficult using adult tissues . However, the morphological characteristics and the staging system we describe for juvenile sub-pharyngeal regeneration cannot be simply extrapolated to adult regeneration and, in particular, it is not intended to replace or extend the existing staging system for regeneration from isolated adult physa in tostella .in vivo fate-mapping experiments, our present study shows that, in the juveniles regenerating from sub-pharyngeal amputation, the most oral tissues of the mesenteries contribute to re-form a new pharynx.In later study, Bossert and colleagues propose (based on macro photographs) that the regenerating adult mesenteries emanate from the most aboral region of the newly formed pharynx during oral regeneration from the isolated adult physa . In contThe precise mechanisms by which mesenteries are formed during embryonic and larval development are poorly described. A first report proposes that these structures are formed by a combination of the pharyngeal ectoderm and body wall endoderm . These Nematostella showed that mRNA transcription for specific genes is upregulated as early as 1 h post-injury and that the wound signal seems to initiate from the gastrodermis [de novo transcription, in the gastrodermal tissues at the amputation site as early as 1.30–6 hpa. It is important to note that even if the size of the injury between these two studies ([vs. our present study) is different, the beginning of de novo transcription seems to correlate.A previous study on the wound healing process in rodermis . Interesstudies  of Nematostella regeneration are independent of cell proliferation. Contrary to this, the AMD treatment shows that the de novo transcription is required for those same early steps: fusion of the mesenteries to one another and to contact the epithelia at the amputation site (Steps 0–1). These treatments, HU or AMD, block all subsequent regeneration (Steps 2–4) as indicated by the absence of 488+ re-appearance, pharynx formation, and tentacle elongation. These observations strongly suggest that de novo transcription is required for the initial tissue dynamics of regeneration (from Steps 0–1), and that cell proliferation is required for later steps (Step 2–4) such as pharynx formation and tentacle elongation.In the last part of our study, we used the described morphological landmarks as a regeneration staging system, in addition to our Nematostella vectensis were cultured at the Institute for Research on Cancer and Aging, Nice (IRCAN) of the University of Nice. Adult and juvenile animals were cultured in 1/3X ASW  and maintained at 17 or 22 °C for adults and juveniles, respectively. Adults were fed five times a week with freshly hatched artemia. To obtain juveniles, spawning was carried out as described in [ribed in . Juvenil2 in 5 mL 1/3 ASW and placed on a light table for 10 to 15 min. Six-week-old polyps were cut using a microsurgery scalpel n°15 . Each cut was performed perpendicular to the oral–aboral axis of the body column.Juveniles or adults were relaxed by adding 1 mL of 7.14% MgCl2 7.14%. The polyps were then compressed laterally with the tweezers 3C GRIP . and the expulsion of the nematosomes was assayed. The wound was considered closed when, under pressure, the nematosomes did not leak through the amputated oral part. In this case, the nematosomes either leaked through the aboral opening or did not leak at all. The wound was considered as non-closed when nematosomes were expelled through the oral region. The compression rate is difficult to measure because of the variability among the polyps of the same batch . However, to standardize the process, the compression was maintained and accentuated until the nematosomes leaked. In some cases, compression was increased to its maximum and the two tips of the tweezers met.A circle was drawn on a slide with a hydrophobic pen  and the relaxed regenerating polyps were placed on this circle in a droplet of 1/3 ASW + MgCl2 for 10–15 min, animals were fixed in 4% paraformaldehyde  in 1/3 ASW for 1 h at 22 °C or overnight at 4 °C. Fixed animals were washed three times in PBT 0.2% (PBS1X + Triton 0.2%). To analyze the general morphology, Hoechst staining  at 1/5000 was used to label the DNA/nucleus, and BODIPY® FL PhallAcidin 488  staining was used at 1/200 to label actin microfilaments (cell membranes and muscle fibers).After relaxing adults or juvenile polyps in MgClde novo transcription Click-it EU (5-ethynyl-2′-uridine) kit  was used following the manufacturer's protocol. EU was used at 1 mM.To detect cellular proliferation Click-it EdU (5-ethynyl-2′-deoxyuridine) kits  were used following the protocol from [The Biosorter system  equipped with the 2000 μm FOCA flow cell was calibrated with 500 μm beads. Using the Flowpilot software (Union Biometrica), polyps were analyzed for their morphology using the extinction coefficient, their length was analyzed using the Time Of Flight (TOF) between two sensors in the FOCA, and their green endogenous fluorescence intensity (488+) was emitted after a 488-laser excitation. For each animal, 488+ was characterized by measuring the intensity, the width, and the height of the peak in real time on live animals. The debris and small animals were excluded from the analysis based on the TOF and extinction parameters. The 488+ was localized in real time using the Profiler module that scans the profile of the object and integrates the measure of this endogenous fluorescence within this profile, allowing us to determine the relative localization of the 488+ in live polyps. For animal sorting, the optimal drop delay was determined using 500 μm beads and single-animal sorting was performed in 96-well plates filled with 100 μm 1/3 seawater.Live animals were imaged using a protocol described in . The imaNematostella juveniles were injected with mRNA encoding the photoconvertable Kaede (green to red fluorescence) protein. At six weeks the juveniles were subject to sub-pharyngeal amputation. Then 24 h later, juveniles were relaxed for 10 min with 7.14% MgCl2 in 1/3 ASW and mounted between slide and coverslip. The region of interest of the juvenile was photoconverted using a 405 nm wavelength laser on a Zeiss 510 confocal microscope. The specific region was photoconverted with the 405 nm laser at 100% power for 10 iterations (scans) and up to three repetitions at a scan speed less than 10 μs/pixel. When photoconverted, the juveniles were left to recover and regenerate for eight to nine days, then relaxed with 7.14% MgCl2 in 1/3 ASW, mounted between slide and coverslip, and analyzed for the red patch of converted Kaede on a Zeiss 710 laser-scanning microscope. To separate photoconverted cells from endogenous fluorescence, using a lambda stack, spectral profiles of converted Kaede-expressing cells, unconverted Kaede-expressing cells, and endogenous red fluorescence were analyzed using a positive and negative control . Several different red endogenous fluorescent cells were analyzed to account for variation. Finally, animals were imaged using the online fingerprinting mode and the 34-channel QUASAR detector .Fertilized Cellular proliferation was inhibited using HU  and transcription was blocked using the AMD . HU was made up fresh at 20 mM in 1/3× ASW before each experiment. A stock solution of AMD prepared in DMSO and kept at −20 °C was diluted in 1/3X ASW to use at a final concentration of 10 μg/mL prior to each experiment. Each HU or AMD treatment was performed in a final volume of 500 μL 1/3× ASW in a 24-well plate using the adequate controls (1/3× ASW or DMSO). Pharmaceutical drugs were changed every 24 h to maintain their activity for the duration of the experiments.in vivo assays for wound healing and pharynx formation using naturally existing landmarks, is a precise way to assess the phenotypes resulting from experimental manipulation during regeneration in Nematostella. This study also begins to define the cellular and molecular mechanisms underlying the intriguing phenomenon of whole-body regeneration, providing a solid basis for further developing Nematostella as a new cnidarian regeneration system.In summary, our staging system for oral regeneration after sub-pharyngeal amputation, combined with our


The cancer cells of lung adenocarcinoma with a micropapillary pattern (MPP) have been found to frequently invade lymphatic vessels, and the prognosis of patients with lung adenocarcinoma with an MPP is poor. In the present study, the cancer cells of lung adenocarcinomas containing an MPP were found to express vimentin more extensively than those in lung adenocarcinoma without an MPP. The contribution of cancer cells in the MPP component to adenocarcinoma lymphatic invasion was assessed using vimentin as a marker. Vimentin expression was analyzed in the cancer cells present in each lymphatic vessel and compared with the expression of vimentin in the cancer cells in the adenocarcinomas without an MPP component. The results showed that the cancer cells in the lymphatic vessels expressed vimentin more extensively than those in the adenocarcinoma components without an MPP, suggesting that cancer cells derived from an MPP component are present in the lymphatic vessels. By contrast, the area of the MPP component in each adenocarcinoma was <25%. These findings suggest that cancer cells in MPP components have a high capacity to invade lymphatic vessels and that their high invasive capacity may be associated with a poor prognosis in patients with adenocarcinoma with an MPP component. Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide . In the The present study included 218 patients  with pathological stage IA lung adenocarcinoma who underwent complete resection of their tumors at Toneyama National Hospital  between January 2002 and December 2010. None of the patients received neoadjuvant chemotherapy or radiotherapy. All patients underwent dissection of the bifurcation and the ipsilateral mediastinal lymph nodes, and pathological examination revealed no metastasis in these locations. Furthermore, computed tomography and magnetic resonance images showed no metastasis in any of the patients. Pathological stage was determined according to the tumor-node-metastasis classification of the International Union Against Cancer  . ClinicaThe diameters of the resected tumors were measured and the longest diameter was regarded as the tumor diameter. The tumors were fixed in 0.01 M phosphate-buffered saline containing 10% formalin pH 7.4) and several paraffin-embedded tumor blocks were generated from each tumor. Tumor sections (4-mm thick) were then generated from each tumor block. Sections were either used for hematoxylin and eosin staining or immunohistochemistry. Tumors were histologically classified according to the International Association for the Study of Lung Cancer . MPPs we and seveImmunohistochemical staining was performed using an avidin-streptavidin immunoperoxidase method with anti-human E-cadherin , anti-human vimentin  and anti-human D2-40  mouse monoclonal antibodies. Antigen retrieval was performed through incubating the deparaffinized sections in cell condition 1 solution  at a low degree (the slide temperature is controlled at 36°C). Immunohistochemical staining was then performed using an automated Benchmark system  according to the manufacturer’s instructions.The grades of immunohistochemical staining for E-cadherin and vimentin were determined according to the proportion (p) of positive cells as follows: 0, p<5% positive cells; 1+, 5≤p<30% positive cells; 2+, 30≤p<70% positive cells; 3+, p≥70% positive cells.In order to analyze the expression of vimentin in the cancer cells which had invaded into lymphatic vessels, two serial tumor sections were established and the sections were either stained for vimentin or D2-40. When cancer cells were found in a D2-40-positive lymphatic vessel in a section stained for D2-40, the grade of vimentin-positive staining of these cancer cells was analyzed in the other section stained for vimentin as described above.2 test. All statistical analyses were performed using the Excel Statistics 2012 software package  for Windows. P<0.05 was considered to indicate a statistically significant difference.Survival curves were plotted using the Kaplan-Meier method and the survival rates of the two groups were analyzed using the log-rank test. Data for more than three samples are presented as the mean ± standard error of the mean. Student’s t-test was used to compare data between two groups. The differences in frequencies between two groups were analyzed using the tet al  and those with a higher survival rate  were compared . The patEpithelial-mesenchymal transition (EMT) has an important role in cancer metastasis . During In the patients in the apparent MPP component group, cancer cell lymphatic invasion was identified in 5/18 cases. In order to assess which component of the tumor contributed to the cancer cell lymphatic vessel invasion, the level of vimentin expression was analyzed in the MPP and non-MPP components, as well as in the cancer cells in each lymphatic vessel ; Fig. 4.In the present study, the survival of patients with adenocarcinoma containing an apparent MPP component was found to be worse than that in the patients with adenocarcinoma containing no MPP component or a focal MPP component. Furthermore, adenocarcinoma with an apparent MPP component had a higher frequency of cancer cell lymphatic invasion. These results confirm the findings reported previously ,3. The pet al (Kamiya et al  reportedet al ; therefoThe present study also investigated whether cancer cells in lymphatic vessels were derived from the MPP component or the non-MPP component. The results suggested that all of the lymphatic vessels containing cancer cells had cancer cells derived from the MPP component. In each adenocarcinoma exhibiting cancer cell lymphatic invasion, the MPP component occupied <25% of the tumor area. Therefore, it is likely that the cancer cells in the MPP component have a greater invasive potential compared with those in the non-MPP component.In conclusion, the present study identified that adenocarcinoma with an MPP component had histological predominance of the papillary dominant type and the moderately differentiated type. These findings are consistent with those of previous studies ,3.


Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, play an important role in ischemic injury to the heart, yet it is not known if these MMPs are involved in the injury that occurs to the transplant kidney. We therefore studied the pharmacologic protection of transplant kidneys during machine cold perfusion.Human kidney perfusates were analyzed for the presence of injury markers such as cytochrome c oxidase, lactate dehydrogenase, and neutrophil-gelatinase associated lipocalin (NGAL), and MMP-2 and MMP-9 were measured. The effects of MMP inhibitors MMP-2 siRNA and doxycycline were studied in an animal model of donation after circulatory determination of death (DCDD).Markers of injury were present in all analyzed perfusates, with higher levels seen in perfusates from human kidneys donated after controlled DCDD compared to brain death and in perfusate from kidneys with delayed graft function. When rat kidneys were perfused at 4°C for 22 hours with the addition of MMP inhibitors, this resulted in markedly reduced levels of MMP-2, MMP-9 and analyzed injury markers.Based on our study, MMPs are involved in preservation injury and the supplementation of preservation solution with MMP inhibitors is a potential novel strategy in protecting the transplant kidney from preservation injury. In an effort to increase the number of kidneys available for transplantation in the face of ongoing donor organ shortage, the use of kidneys from more marginal donors has been increasing. This includes the use of kidneys from older donors and those with hypertension [Expanded Criteria Donors (ECD)] as well as Donation after Circulatory Determination of Death donors (DCDD). Unfortunately, both ECD and DCDD donated kidneys are associated with a higher rate of Delayed Graft Function (DGF) and poorer function at one year . AlthougMachine cold perfusion of transplant kidneys has shown benefit in terms of both early function and long term function ,5,6 and MMP-2 and MMP-9 have been shown to be involved in acute and chronic renal injury along the spectrum of basement membrane damage, to tubular atrophy, to fibrosis, to outright renal failure ,13. MMPsFinally, MMP-2 has been shown to be involved in renal ischemia-reperfusion injury in an animal model whereby warm ischemia was induced in situ for 30 to 120 minutes in an MMP-2 deficient transgenic mouse model . The degIn this study, we examined the perfusate from human perfused kidneys and documented the presence of MMPs. We then developed an experimental animal model of machine cold perfusion to study the effect of inhibition of MMPs.Twenty-four perfusates of human transplant kidneys obtained from two transplant programs between July 1, 2012 and June 30, 2013 were studied . NGAL, LAnalysis for gelatinolytic activity (zymography) showed that all perfusates containedMMP-2 and MMP-9 (molecular weights of 72 and 92 kDa respectively) . The levDelayed graft function (DGF) was associated with significantly increased MMP-9 and MMP-2 levels . In contRat kidneys (n = 5 per time point) demonstrated preservation of gross architecture under light microscopy in kidneys cold perfused for 22 hours versus those who were flushed and fixed immediately .Similar to what was observed in human kidney perfusion, NGAL, LDH and CcO were observed in the perfusate from machine cold perfusion preserved rat kidneys . The relAnalysis of perfusates for MMP-9 and MMP-2 activities (molecular weight 92 and 72 kDa respectively) showed higher activities after 22 hours of perfusion in comparison to 5 hours of perfusion. Levels of all analyzed parameters were positively correlated with the duration of cold perfusion .Changes were seen on EM as shown in tissue after 22 hours of cold perfusion revealed a marked decrease in activity of approximately 60% and 50% in mitochondrial integrity  could be investigated. The level of MMP-2 (72 kDa form) in perfusate after 22 hours of cold perfusion was approximately 3 fold higher than at the time of the first perfusate sample at 5 hours . In contIn addition to its antibacterial activity, DOXY has also been shown to have inhibitory effect against cardiac MMP-9 and MMP-2 ,8,11,19.Analysis of the inhibitory potential of DOXY on kidney MMP-9 (92 kDa) and MMP-2 (72 kDa) showed complete inhibition of the gelatinolytic activity of both enzymes in gel 2 (DOXY) .Perfusion of rat kidney with KPS-1 solution containing DOXY showed decreased levels of LDH and CcO in the perfusate and the level of both enzymes after 22 hours of perfusion was similar to the level after 5 hours of perfusion . In the Protein release into the perfusate by the kidney was also inhibited by DOXY and was similar to the control level throughout the duration of the perfusion .The levels of NGAL observed after 22 hours of cold perfusion with DOXY or MMP-2 siRNA were markedly less than that found in perfusate without additives at 22 hours .Because of the changes in mitochondria observed on electron microscopy consistent with the role of mitochondria in injury noted by other groups , the perIn both human and animal studies, our data show that proteins are released during cold perfusion, including MMP-2 and MMP-9. Markedly higher levels of total protein, NGAL, and LDH were seen in perfusate from cDCDD, compared to DBD human donor kidneys. This is in spite of a much shorter time for cDCDD donor kidney perfusion versus DBD donor perfusion . CcO was not significantly higher in the cDCDD perfusate, but this may have to do with the fact that the perfusion time was much less for cDCDD kidneys than it was for DBD kidneys. Markedly higher MMP-2 and MMP-9 release into the perfusate was also seen in cDCDD donors compared to DBD donors.In a small animal model of cold perfusion, we found that inhibition of MMP-2 during perfusion using MMP-2 siRNA led to a decrease in MMP-2 level in perfusate but also a decrease in LDH, NGAL, and CcO release after 22 hours of perfusion to a level similar to that seen at 5 hours. The use of the non-specific MMP inhibitor, DOXY, in the perfusion solution led to even greater decreased levels, suggesting that MMPs other than MMP-2 are also involved in the injury.In the electron micrographs of rat kidneys with warm ischemic injury and machine cold perfusion, changes were seen in mitochondria (swelling and loss of definition of cristae). This was supported by the CcO in the actual rat kidney tissue at 22 hours and the CcO levels in perfusate that increased with time. Indeed, while MMPs are generally thought of as acting on extracellular matrices, they also have been shown to have intracellular actions as well, including on the mitochondria .Many previous studies have documented the role of MMP’s in ischemic damage in the heart ,9,10,11 Based on our study, it is not possible to conclude on the mechanism whereby MMPs led to an increase of injury markers, however we might speculate based on the electron micrograph appearance and what is known about MMPs that the effect of MMPs on extracellular matrices and on mitochondria may be responsible.Our results suggest that certain pharmaceutical agents such as DOXY added to the machine cold perfusion solution may be helpful in reducing the amount of preservation injury to transplant kidneys. It is important to note that this study addressed preservation injury and not ischemia-reperfusion injury, which will be the topic of future studies.The fact that a clinically available drug (DOXY) exists that is capable of inhibiting MMP-2 makes it appealing to consider a studying the drug clinically in machine cold perfusion; given that deceased donor kidneys often come in pairs, it is possible to randomize one to the control arm and the other to receive DOXY in the perfusion solution. The paired nature of such a study would allow for a relatively small ‘n’.A reduction in the injury to cold preserved DCDD kidneys should result in the ability to use some of the marginal donor kidneys that would normally be rejected or discarded. If even 10% of the kidneys normally rejected by a program are able to be used, this will translate into thousands of kidneys worldwide per year, thus releasing thousands of patients from dialysis and shortening wait times significantly.Although not a main focus of our study, it is interesting that in DGF, the levels of MMP-2 and MMP-9 were approximately double compared to the non-DGF kidneys, while the measured injury markers were not significantly different between the two groups. Given that the mechanism whereby DGF occurs remains elusive, the possibility that MMPs are involved in DGF requires further study .In conclusion, this study suggests that MMP-2 and likely other MMPs play a role in transplant kidney preservation injury, and that some of this injury can be prevented by the addition of doxycycline in the preservation solution.® Machine Cold Perfuser  and perfused with Kidney Perfusion Solution-1 (KPS-1) at 3–5°C using the standard LifePort® settings (pressure 30 mmHg). After the kidney perfusion was terminated, 60–80 mL of the solution was collected and stored at -80°C.Retrieved kidneys were connected to the LifePortTwenty-four samples (15 DBD and 9 cDCDD) of perfusate from human kidneys were analysed. The standard definition of DGF was used: the need for dialysis in the first week post-transplant. Slow Graft Function (SGF) was defined as serum Cr > 265 μmol/L on post-transplant day 5 and no need for dialysis by day 5 .Proteins were precipitated with Ceramic Hydroxyapatite beads  in 2.5 mM phosphate buffer with pH 7.0. The bounded proteins were released from CHT beads with 0.5 M phosphate buffer with pH 7.0.The experimental protocol for perfusion of rat kidney is shown in The machine cold perfusion apparatus consisted of a micro-pump  with flow rate of 0.5 mL/min, operating in a 4°C cold room. The KPS-1 solution (20 mL with or without additive) was continuously infused via the renal artery and the effluent passively drained via the renal vein to be recirculated via the pump.The kidney was perfused for 22 hours; this time was chosen as longer than the usual cDCDD preservation time in order to increase the chances of having significant injury to the rat kidney. Samples of 0.5 mL were collected at 5, 10, and 22 hours and stored at -80°C. At the end of the perfusion, the kidney was stored at -80°C.For biochemical studies, frozen kidney tissue was homogenized on ice in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) containing protease inhibitor cocktail and 0.1% Triton X-100. Homogenates were centrifuged at 10,000 g for 10 min, and the supernatant was stored at -80°C until further use.NGAL level was measured using ELISA kit , and expressed as μg of protein per mL of perfusate.LDH activity was measured by LDH activity assay . Enzyme activity is expressed as the amount of NADH generated from the conversion of lactate into pyruvate.CcO activity was measured using activity assay .CcO is located on the inner mitochondrial membrane . The intProtein content of perfusates and the kidney extract in homogenized buffer was measured with the Bradford protein assay .Gelatin zymography was performed as previously described ,27. AfteSections (4 μm) of formalin-fixed and paraffin-embedded tissues were stained with Periodic Acid Schiff’s stain ,29 then Kidneys (n = 3 each for time zero and time 22 hours) were flushed with glutaraldehyde and 1 mm cubes of tissue were excised from the upper pole in each kidney for electron microscopy, post fixed in osmium tetroxide, dehydrated in graded ethanol, and embedded in an epoxy resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate and random areas photographed using digital transmission electron microscopy . These were reviewed in blinded fashion by a renal pathologist.A concentration of DOXY of 100 μM was used in the zymography gels documenting the inhibitory effect of DOXY and this was the dose used in the therapeutic arm of the rat kidney perfusions. This concentration was used in numerous studies in our lab of MMP inhibition in cardiac ischemia and found to be effective .A stock solution of a mixture containing a pool of 3 target-specific 19–25 nucleotide small interfering RNAs designed to knock down rat MMP-2 gene expression  was used . As a control, scrambled siRNA  was used.Perfusion of rat kidney was with 20 ml of KPS-1 containing MMP-2 siRNA or scrambled siRNA at concentration of 50 nM. The kidney was perfused for 22 hours and perfusate samples were collected as described above.Student’s t test or Chi-squared tests were used as appropriate.This study conforms to the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The protocol was approved by the Animal Care and Use Committee of the University of Saskatchewan (certificate # 20130073). All animal surgery was performed under isofluorane anesthesia, and all efforts were made to minimize suffering.The protocols involving human perfusates were approved by the University of Saskatchewan Biomedical Research Ethics Board (Bio # 12–44) and the Western University Health Sciences Research Ethics Board (HSREB # 101899). The perfusate that was collected for this study was fluid that is ordinarily discarded and is relevant to neither the donor nor the recipient, so the need for patient consent was waived.S1 Fig(TIF)Click here for additional data file.


The intra-class correlation (0.78) was acceptable, indicating good reliability. The results suggest that PDAQ is a valid and reliable measure of diabetes nutrition recommendations. Because it is quick to administer and score, it may be useful as a screening tool in research and as a clinical tool to monitor dietary adherence.Nutrition therapy is essential for diabetes treatment, and assessment of dietary intake can be time consuming. The purpose of this study was to develop a reliable and valid instrument to measure diabetic patients’ adherence to Canadian diabetes nutrition recommendations. Specific information derived from three, repeated 24-h dietary recalls of 64 type 2 diabetic patients, aged 59.2 ± 9.7 years, was correlated with a total score and individual items of the Perceived Dietary Adherence Questionnaire (PDAQ). Test-retest reliability was completed by 27 type 2 diabetic patients, aged 62.8 ± 8.4 years. The correlation coefficients for PDAQ items  Over 347 million individuals have diabetes and it is estimated that by the year 2030, 552 million people will be living with diabetes . In CanaWhile clinical outcomes such as A1c and blood pressure can easily be monitored by the medical team treating the diabetic patient, assessing dietary intake and creating a longitudinal record of dietary intake is not as practical . Howevern = 27, Cohort II) were recruited through poster and database of the Alberta Diabetes Institute to measure the test and retest reliability of the PDAQ with a one-week interval. The inclusion and the exclusion criteria were the same as for the intervention study. The University of Alberta Human Research Ethics Board approved both studies. Written informed consent was obtained from all participants.Data from the Physical Activity and Nutrition for Diabetes in Alberta (PANDA) intervention study  were used to test internal consistency and validity. Briefly, 73 participants were enrolled in the dietary intervention (Cohort I). Participants were recruited through a variety of avenues including posters, word-of-mouth, contact via a list of potential participants maintained by the Alberta Diabetes Institute and via an article about the project in a local newspaper. The inclusion criteria were: people diagnosed with type 2 diabetes and able to read and speak English. The exclusion criteria were: having severe gastrointestinal issues, type 1 diabetes or kidney disease. Anthropometric measures , A1c, blood pressure, serum lipids, 24-h recall repeated on three successive days, and PDAQ were obtained at baseline and three months. Subsequently, additional type 2 diabetes patients  oils, and high fat foods. One item addresses appropriate carbohydrate spacing. The response is based on a seven-point Likert scale to answer the question phrased as “On how many of the last 7 days did you…?”  using an internet-based questionnaire (WebSpan), which has been shown to reduce assessment error and bias . Daily rp < 0.05. Descriptive statistics were used to summarize demographic data. The mean ± SD was calculated for continuous variables, and percentage for categorical variables. Comparison of demographic characteristics between the first cohort  and the second cohort (used to do test-retest reliability) was assessed by Chi square and unpaired t-tests as appropriate. Spearman rank-order correlation coefficients were calculated between PDAQ questions to determine if the perceived adherence to CFG question score (Question (1)) correlated with the scores of Questions (2) through (9).Statistical analyses were performed using SPSS version 22.0 , Statistical significance was set at n = 64). Normality of nutrient intake distributions was checked statistically. If the normality assumption failed, data were log10-transformed. The questions of the PDAQ were individually correlated with specific information derived from the three 24-h dietary recalls . Specifically, the question related to CFG was correlated with the mean number of servings of the four food groups. The question related to vegetables and fruits consumption was correlated with the mean servings of vegetables and fruits. The question related to consumption of foods with low glycemic index was correlated with the mean glycemic index score. The question related to consumption of foods high in sugar was correlated with the average daily intake of added sugar. The question related to intake of foods high in fibre was correlated with the servings of whole grains. The question related to spacing carbohydrate throughout the day was correlated with the total carbohydrate spacing score. The question related to eating fish or foods high in n-3 fatty acids was correlated with the number of foods in the dietary recall that were high in n-3 fatty acids. The question related to using healthy oils was correlated with the intake of monounsaturated fatty acids. The question related to eating foods high in fat was correlated with the intake of total fat. The correlation coefficients were interpreted by using Dancey and Reidy’s categorisation [Validation: After screening the food intake data for implausible dietary intake or incomplete three 24-h dietary recalls, nine participants were removed (risation .Reliability: the intra-class correlation coefficient (ICC) was calculated as an indication of test-retest reliability and internal consistency was measured by Cronbach’s α coefficient .versus second cohort except for employment status (p = 0.008).A total of 73 participants were enrolled in the PANDA study, which provided data for validity testing and internal consistency, and 27 participants were separately recruited for test-retest reliability testing. The characteristics of the participants are reported in n = 39) and female  participants. A significant positive correlation was found between PDAQ score and age with r = 0.46, 95% CI , and inversely with weight with r = −0.36, 95% CI .The score for the PDAQ was normally distributed and ranged from 10 to 54. PDAQ scores were not statistically significant different between male ), as well as with Vegetables and Fruits servings ). In contrast, total PDAQ scores were negatively correlated with added sugar intake ) and saturated fat intake ).Total PDAQ scores were associated with nutrient intakes from the average of the three 24-h dietary recall and correlated moderately with HEI-score (p < 0.05). Perceived eating of the recommended servings of Vegetables and Fruits more days per week was associated with higher intake of Vegetables and Fruits reported in 24-h recalls (p < 0.05). Reported consumption of foods with a low glycemic index more days per week predicted lower glycemic load (p < 0.05). Reporting eating of foods high in sugar  on more days was associated with higher added sugar intake (p < 0.01). Perceived eating of foods high in fiber  predicted higher intake of whole grains (p < 0.001). Reported consumption of foods high in fat  on more days predicted higher fat intake (p < 0.01). No significant association was found for spacing carbohydrate, foods high in n-3 fatty acids, and healthy oils versus the actual intake.To test the validity, we associated individual items of the PDAQ with nutrient intakes from the average of the three 24-h dietary recalls adjusted for total calories . Followir = 0.60, 95% CI ), higher intake of foods with low glycemic index ), higher intake of foods high in fiber ), more likely to space carbohydrate throughout the day ), and higher intake of fish high in n-3 fatty acids ). Conversely, there were negative correlations between perceived adherence of following CFG and intake of foods high in sugar ) and foods high in fat ).n-3 fatty acids, and healthy oils) as well as the total PDAQ score  between Vegetables and Fruit and short food frequency questionnaires [Other authors have developed short questionnaires to assess intake of various foods or nutrients in the general population whereas the PDAQ is targeted to specific nutrition recommendations for diabetes. The correlation obtained for Vegetables and Fruits intake between PDAQ and 24-h recall is comparable to previous studies that found moderate correlation  suggesting that the PDAQ score is stable over time. Test-retest administration of PDAQ produced good correlations for questions related to Vegetables and Fruits, foods high in sugar and fibre, fish or foods high in n-3 fatty acids, and healthy oils; meanwhile, questions related to spacing carbohydrate and foods high in fat had moderate correlations . The question related to CFG had poor test-retest correlation (r = 0.21). Low and moderate ICC values in some individual scores are due to the intra-individual variability [We determined that PDAQ had acceptable internal reliability since Cronbach’s α was 0.78 . The test-retest correlation coefficient for the entire questionnaire was acceptable (on r = 0.. Low andiability .The correlations produced in validity tests between following CFG more days per week and each subscale shows that the PDAQ ranked subjects quite well. We showed that reporting consistent following of CFG is more likely to be positively associated with the intake of low-caloric density foods, and negatively associated with high-caloric density foods. This finding indicates that the PDAQ is a good instrument to measure adherence to CFG recommendations. We were particularly interested in examining PDAQ’s ability to assess intakes specifically mentioned in the CDA Nutrition Therapy Guidelines that may not be captured using scores like the Healthy Eating Index. PDAQ subscales for low GI foods, high sugar, fiber and fat foods were moderate predictors of intake substantiated by the 24-h recall data.We also found correlations between PDAQ and demographic or biological variables. The positive relationship found between PDAQ and age is similar in direction to a previous study that examined the association between HEI and age . The dieSeveral other short questionnaires for dietary assessment have been developed. Calfas and colleagues  conducteOne strength of our study is that we used three internet-based, 24-h dietary recalls to estimate dietary intake, a method that has less bias than some others  and was Following the CDA nutrition therapy guidelines is important for improving health outcomes in people with type 2 diabetes, but there is a need to develop practical and quick tools that help clinicians and researchers to assess adherence to these guidelines. We suggest that the PDAQ may be useful to accomplish this objective and that it can be implemented in research. It may be worthwhile to test the PDAQ in a clinical setting.


This also happens in vivo in live intact snails, where early exposure to miracidia also elicits non-random repositioning of genes. We reveal differences in the nuclear repositioning between the response of parasite susceptible snails as compared to resistant snails and with normal or live, attenuated parasites. Interestingly, the stress response gene heat shock protein (Hsp) 70 is only repositioned and then up-regulated in susceptible snails with the normal parasite. This movement and change in gene expression seems to be controlled by the parasite. Other differences in the behaviour of genes support the view that some genes are responding to tissue damage, for example the ferritin genes move and are up-regulated whether the snails are either susceptible or resistant and upon exposure to either normal or attenuated parasite. This is the first time host genome reorganisation has been seen in a parasitic host and only the second time for any pathogen. We believe that the parasite elicits a spatio-epigenetic reorganisation of the host genome to induce favourable gene expression for itself and this might represent a fundamental mechanism present in the human host infected with schistosome cercariae as well as in other host-pathogen relationships.Schistosomiasis is an infectious disease infecting mammals as the definitive host and fresh water snails as the intermediate host. Understanding the molecular and biochemical relationship between the causative schistosome parasite and its hosts will be key to understanding and ultimately treating and/or eradicating the disease. There is increasing evidence that pathogens that have co-evolved with their hosts can manipulate their hosts' behaviour at various levels to augment an infection. Bacteria, for example, can induce beneficial chromatin remodelling of the host genome. We have previously shown  Bilharzia is a parasitic disease endemic in many parts of the world. The schistosoma parasite that causes Bilharzia infects humans but uses a fresh water snail as a secondary host. These two organisms have co-evolved together, and as such the parasite will have mechanisms to overcome the host defences. Understanding this delicately balanced relationship is fundamental to controlling or eradicating the disease. We have studied how this parasite can influence how the DNA within the snail behaves. We have shown snail genes have specific locations within the cell nuclei. Further, we have revealed that specific snail genes related to a schistosome infection change to a new non-random nuclear location as they are turned on or up-regulated. We have snail strains that are susceptible or resistant to the infection of parasites and we can also take live parasites and make them unable to complete an infection by irradiating them. In this unique study, we have shown a gene that is involved in stress pathways moves to a new nuclear location and becomes turned on, but only in susceptible snails, infected with fully functional parasite. Our data suggest that this gene is regulated by the parasite, which has control over the host's DNA, so that the gene is moved to an area where it can be actively expressed. We have uncovered a novel mechanism whereby the spatial organization of a host organism is interfered with by a pathogen. This type of control is probably found in other host-pathogen relationships. Biomphalaria glabrata is an intermediate host for Schistosoma mansoni parasite, which causes one of the most prevalent parasitic infections in mammals, known as intestinal schistosomiasis (Bilharzia). The disease is endemic in 77 countries of the tropics and subtropics, and effects over 230 million people yearly whom are infected with S.mansoni and other schistosoma species The fresh water snail Biomphalaria snails vary in their compatibility to schistosomes such that some display resistance to infection while others are susceptible S. mansoni larvae and an infection will develop following miracidial exposure ferritin, actinin vitro tissue culture model to support the intramolluscan stages of S. mansoni has also aided the investigations into B. glabrata's relationship with S. mansoniBiomphalaria glabrata embryonic (Bge) cell in vitro co-culture system to determine spatio-temporal effects on specific genes in the nuclei of Bge cells that have been co-cultured with S. mansoni miracidia in situ hybridisation with specific bacteria artificial chromosomes (BAC) probes and image analysis. The intranuclear movements of genes correlated with the temporal kinetics of gene expression, meaning that genes were repositioned within nuclei with their up-regulation in expression after a signal was transmitted thorough the cell that parasites were in the vicinity. Co-culturing with live attenuated miracidia on the other hand failed to elicit similar gene expression and gene loci repositioning, indicating that normal but not attenuated schistosomes provide stimuli that evoke host responses in vitro system was a response found also in vivo in whole organisms and thus a true parasite induced mechanism it was critical to study this phenomenon in live snails. In the study presented here we utilise the B. glabrata fluorescence in situ hybridisation technique using snail ex vivo cells to determine spatio-temporal effects on three genes in the resistant and susceptible snails after early exposure to S. mansoni miracidia. We find the same phenomenon exists in vivo and snail genes relocate in snail nuclei soon after exposure to schistosomes, concurrent with the up-regulation of their corresponding transcript. We believe that we are seeing in the intact snail, as previously observed in the Bge- parasite co-culture in vitro system, a physical schistosome- specific reorganisation of the host's genome in response to the parasite and by the parasite, in a manner suggesting that early direct influence on a susceptible but not resistant snail's transcriptional activity is controlled by the parasite. This is the first time that this type of pathogen-induced spatio-epigenetics has been observed in an intact whole organism and should be investigated in other co-evolved host:pathogen relationships.actin, ferritin, and Hsp 70 that were previously found to be up-regulated after an infection of the snails with S. mansoni miracidia in situ hybridisation (FISH) of labelled BAC probes in interphase nuclei of B. glabrata snails from the susceptible (NMRI) and the resistant (BS-90) snail lines. The gene signals can be seen as small concentrated loci in the interphase nuclei and all genes displayed two gene signals, one for each allele  being located at an intermediate position within nuclei. The nuclear location of the genes was similar between the NMRI and BS-90 snail strains  rather than quiescent or senescent cells and we would argue this makes them as a population more responsive. The ovotestes were easier to identify and remove than other tissues ensuring that the population of cells we analysed were similar. We have shown previously, in the pig, that chromosomes are found in similar locations between cells in culture and cells in frozen tissue in vitro induced situation. However, Bge cells have been shown to possess an abnormal complement of chromosomes, aneuploidy, which may impact gene positions in these cells Three genes, lele see . In ordelele see . The scrlele see . All of B. glabrata with S. mansoni miracidia modulates host gene expression resulting in the upregulation of certain genes including actin, ferritin, and Hsp 70actin and ferritin were shown to be non-random repositioned in the in vitro co-culture experiments with the parasite and Bge cells S. mansoni miracidia. Considering that genes and chromosomes can move within a matter of minutes to hours within the nuclear environment Infection of actin at different time points in the NMRI and BS-90 snail lines. In the NMRI (susceptible) snails the actin gene loci moved from the nuclear interior towards a more intermediate position within the nuclei at 30 min, and subsequently moved back to the nuclear interior after 5 hours. Interestingly however, no major repositioning was observed for the actin gene loci in the BS-90 (resistant) snails. The repositioning of actin in the susceptible snails correlated with a 16-fold maximum increase in its expression at 2 hours after exposure and 1.5 hours after gene repositioning. The elevation in gene expression was initiated at 30 minutes after exposure, indicating close correlation with spatial repositioning. In the resistant BS-90 snails there was a 22-fold increase in gene expression after only 30 min of infection. Although there was no major movement of the actin gene in these snails, the actin gene loci were already at a more intermediate nuclear location in the susceptible snails after movement had been induced. T-tests between the position data for 0.5 hours in the susceptible snails and 0 hours in the resistant snails show only shell 2 being statistically different at the 95% confidence interval and between 0.5 hours in the susceptible snails and resistant snails only shell 3 was statistically different at the 95% confidence interval. Although there are some differences we suggest that actin was already in a prime position for transcription in the resistant snails. No repositioning of gene loci or change in gene expression was observed for actin in snails infected with attenuated miracidia.Hsp 70 gene in timed samples prepared for FISH are displayed in Hsp70 skewed towards shell 5, the nuclear interior and displaying statistically significant differences for shell 5 at 2 hours and shell 3 at 5 hours. This repositioning event of Hsp 70 gene loci correlated directly with the upregulation of gene expression as shown by qRT-PCR. In the resistant BS-90 snails however, Hsp 70 gene loci do not alter their intermediate nuclear position and this is in conjunction with the qRT-PCR data showing no change in gene expression. There are no statistical differences between the positions of the Hsp70 genes after infection with attenuated parasites when compared to before infection until 24 hours, where there is a statistical difference for shell 1. In the resistant snails there is no change in the location of the Hsp70 gene nor meaningful increase in gene expression at 2 or 5 hours. Furthermore, there is little gene expression in the resistant snails and there is only a slight difference of gene position with statistical significant differences for shell 1 at 5 and 24 hours only between the resistant and susceptible snails. No evidence for the relocation of the Hsp 70 gene loci and no induction of Hsp 70 expression were detected by qRT-PCR when the two snail lines were exposed to attenuated miracidia, indicating that these results reflect a spatio-epigenetic influence from the parasite in susceptible snails. Heat shock proteins have been found as important genes for the parasite to infect its host since in experiments where heat shock protein function is inhibited with a drug geldenamycin the susceptible snail is rendered resistant to parasitic infection Hsp 70 expressions in a parasitic infection is a further step in understanding how the presence of heat shock proteins permits an infection.The data for the positioning of the ferritin gene behaved differently to the other two genes since it moved in all situations in susceptible, and resistant snails, when exposed to either normal or attenuated parasite. ferritin gene loci and corresponding gene expression. In the NMRI susceptible snails ferritin repositioned from the nuclear interior to a more intermediate position at 30 min post -exposure. Interestingly, ferritin gene loci also repositioned at 30 min in these snails exposed instead to attenuated miracida, correlating with a 37-fold increase in the expression of this gene. A similar response was seen in BS-90 resistant snails after exposure to attenuated miracidia. In these snails, ferritin repositioned to the nuclear periphery, which once again correlated with its up-regulation as determined by qRT-PCR. There was a dramatic alteration in gene positioning in BS-90 snails after 30 minutes exposure to normal miracidia with the majority of gene loci located in the second shell of the eroded nuclei, more towards the nuclear periphery. This again correlated with an 8-fold increase in the expression of ferritin message at 5 hours. There is only one statistical difference between the ferritin gene movement in the susceptible snails infected with normal parasite as compared to attenuated parasite and that is for shell 2 at 0.5 hours, when we see the correlated upregulation in gene expression. Although a differential temporal response for ferritin was observed between the susceptible and the resistant snails, relocation of ferritin gene loci and the induction of gene expression in snails infected with attenuated miracidia suggested that this reflected a response to the presence of the miracidia from injury as has been seen in human tissues The B. glabrata has greatly increased, establishing it as the molluscan model organism, especially in host: pathogen studies. While the majority of studies using this model system concentrate on the molecular biology of the snail in order to understand the snail – parasite relationship, only our group examined the genome organisation in this host: pathogen interaction. Thus, in our earlier study in vitro co-culture assay employing Bge cells and schistosome miracidia, we revealed that a profound reproducible reorganisation of the snail genome occurs in response to the presence of normal but not attenuated miracidia B. glabrata cells were revealed which interestingly differed between parasite resistant and susceptible snail lines. Repositioning of gene loci for actin, ferritin, and Hsp 70 all correlated with up-regulation of their expression as determined by qRT-PCR. This fits with an evidence -supported hypothesis in the mammalian genome behaviour field that certain genes, probably those that are either being switched on, or are vastly up-regulated, move to an area of the nucleus where transcription is more active B. glabrata at the present time it is not possible to say if whole chromosomes or genes on chromatin loops have made the transition but the movement is probably initiated by chromatin remodelling with respect to acetylation and mediated thorough nuclear motor proteins. Pathogen chromatin remodelling in the host genome, regulated by a pathogen, has been observed for many bacterial infections Following the initiation of the snail genome project actin gene loci repositioned from the nuclear interior to an intermediate position between the nuclear interior and periphery in the susceptible snails after the infection, but did not alter their position in the resistant snails. The repositioning of gene loci in the susceptible snails correlated with the expression of the gene 1.5 hours after the repositioning event. However in the BS-90 snails there was a rapid response to parasite exposure with a large fold increase in the expression of actin after 30 min. Although in the BS-90 snails no repositioning was observed for the gene loci at any of the 0.5, 2, 5, and 24 hour time points, alterations in the organisation of interphase nuclei can be a very rapid process of less than 15 mins after a change in status in a cell actin gene loci in the BS-90 cells before infection were in a similar nuclear position to the actin loci in NMRI cells after exposure which could mean that this gene does not need to move far to reach an amenable transcription factory. Actin protein is a major constituent of the cytoskeleton and is involved in a number of cellular processes actin has been reported in plant- fungal pathogen interactions The Hsp70 relocation and expression are tightly temporally correlated at 2 and 5 hours in susceptible snails which is shown to be significant using t-tests. This relocation and expression is only found in susceptible snails with normal but not attenuated parasite. We know that the heat shock proteins are intimately involved in a positive outcome for the parasite since exposing the BS-90 snail to heat changes it from a resistant snail to becoming susceptible to parasite infection Hsp 90 genes makes the susceptible snail NIMR resistant. We also know that a rise in temperature in snails increases Hsp 70 gene expression and this correlates with the Hsp 70 gene loci changing their location . It is therefore possible that a stress response that includes Hsp 70 expression permits an infection and this is controlled through an epigenetic pathway such as spatial positioning and/or transcriptional regulation of the gene. Evidence for this is found in the resistant BS-90 snails where we find neither Hsp 70 gene movement nor gene expression similar to the susceptible NIMR snails when exposed to attenuated parasites (We feel one of the most interesting findings in this study is that arasites .actin) prior to an alteration to its transcriptional activity, occurring 1.5 hours after relocation. Thus, we have some evidence to add to the debate on what comes first; gene expression or relocation. Although, in order for a gene to be moved to a new location there needs to be signal and this is probably epigenetic via chromatin modification followed by a nuclear motor activity. The snail genome does carry marks of CpG island methylation In this study we have shown that relocation of genes correlated with change in their transcriptional status and for the first time we have provided evidence for the movement of a gene (B. glabrata snails from the susceptible line (NMRI) and the resistant line (BS-90) were incubated overnight in sterile distilled water containing 100 µg/mL ampicillin at room temperature in an attempt to reduce contaminating resident bacteria in the snails. Individual snails were exposed to either attenuated or normal miracidia (10 miracidia per snail) at five different time points  post-infection. Attenuated miracidia were obtained by irradiation (20 Krad) as described by Ittiprasert et al 2009. Following parasite exposure the snail ovotestes were dissected and incubated in hypotonic potassium chloride solution (0.05 M) for 30 min, during which time the tissues were macerated with a needle to obtain single cells. The cells were then centrifuged at 163 g for 5 min and fixed with methanol and acetic acid (3∶1 v/v) for 15 min at room temperature. The fixed cells were washed five times with methanol and acetic acid (3∶1 v/v) and stored at 4°C until further use. The cells of the ovotestes were chosen since they contain more proliferating rather than quiescent or senescent cells than other areas of the snail and so maybe as a population be more responsive to an infection. The ovotestes are also easier to isolate than other tissues ensuring that similar cells are being compared. Cells were dropped onto wet glass microscope slides to prepare interphase nuclei samples for fluorescence in situ hybridisation (FISH).Adult B. glabrata adult snails (∼8–12 mm shell diameter) either unexposed (0 min) or exposed was extracted individually by RNAzol RT  according to the manufacturer's manual. With this reagent the contaminated residual DNA was eliminated as previously described ferritin, actin, Hsp 70 and myoglobin, a house keeping gene B. glabrata irrespective of their susceptibility to the parasite. While others prefer actin, in our hands, this transcript is differentially regulated upon infection depending on whether or not snails are resistant or susceptible to the parasite. Furthermore actin is a nuclear motor protein and maybe used in moving chromatin around after an infection and is not used as a control gene in Q-PCR in studies interested in genome reorganization. Using custom designed primers that were synthesized by Operon Biotechnologies, the gene specific primers (GSPs) for Q-RT-PCR, showing no cross hybridization to S. mansoni, were designed using Primer-Blast algorithm in GenBank (Ta = 58°C). Twenty-five microliters of each RT-PCR mixture contained 100 ng total RNA, 12.5 µl Brilliant II SYBR green PCR master mix, 150 nM of each gene specific primers (GSPs): actin, ferritin and Hsp 70 and 1 µl of blocking reverse transcriptase. Individual snail RNA samples were run in triplicate, and all assays contained a no template negative control to rule out non- specific amplification from contamination in the buffers. The Q-RT-PCR data were normalized using myoglobin (Mb) as housekeeping gene as previously described (7). The sequences of each primer are: 1) actin (Acc. no. CO501282); actin-F 5′-GTCTCCCACACTGTACCTATC-3′, actin-R 5′-CGGTCTGCATCTCGTTTTC-3′, 2) ferritin (Acc. no. AW739595); ferritin –F 5′-GGAGGAGAGAGAACATGC-3′, ferritin-R 5′-CACCAATCTGCTTGATGGAC-3′, 3) Hsp 70 (Acc. no. L44127); Hsp 70-F 5′-AGGCGTCGACATTCAGGTCTA-3′, Hsp 70-R 5′-TGGTGATGTTGTTGGTTTTACCA-3′ and 4) myoglobin ; Mb-F 5′-GATGTTCGCCAATGTTCCC-3′ and Mb-R 5′-AGCGATCAAGTTTCCCCAG-3′ferritin, actin and Hsp 70 during different time points following exposure were normalized relative to myoglobin expression as mentioned above. Fold change of gene expression were calculated by the comparative Ct method with the formula indicated below Total RNA from the whole body of P-values <0.05 (N = 10 for each group) showing differentially expressed transcripts between the two groups that were significant. In these experiments, we performed Q-RT-PCR, in triplicate, from RNA samples isolated from 10 individual snails per time point for each group with 2 biological replicates.The gene fold change difference between unexposed and exposed snail groups was compared by Student's t-test, with B. glabrata genes for fluorescence in situ hybridisation (FISH) were derived from clones of B. glabrata bacterial artificial chromosome (BAC) libraries (BB02 stocks), containing actin, ferritin, and Hsp 70 coding sequences DNA probes for the actin, ferritin, Hsp 70) were captured and the position of the gene assessed using the erosion script analysis t test.Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) were observed using the Olympus BX41 fluorescence microscope and UPlanFLN 100x/1.30 oil immersion objective. Digital images were captured using a grayscale digital camera  and the Smart Capture 3 software . Fifty images of nuclei for each gene (B. glabrata actin Acc.no. CO501282, B. glabrata ferritin Acc. No. AW73959, B. glabrata Hsp 70 Acc. No. L44127, B. glabrata myoglobin Acc. No. U89283.


Most cellular proteins function as part of stable protein complexes. We recently showed that around 38% of proteins associate with mRNAs that encode interacting proteins, reflecting the cotranslational formation of the complex between the bait protein and the nascent peptides encoded by the interacting mRNAs. Here we hypothesise that these cotranslational protein-mRNA associations can be used to predict protein-protein interactions.eti1 mRNA, which codes for a protein of unknown function and uninformative sequence. Based on this protein-mRNA association, we predicted that the Exo2 and Eti1 protein are part of the same complex, and confirmed this hypothesis by coimmunoprecipitation and colocalization of the proteins. Similarly, we show that the cotranslational interaction between the Sty1 MAP kinase and the cip2 mRNA, which encodes an RNA-binding protein, predicts a complex between Sty1 and Cip2.We found that the fission yeast Exo2 protein, which encodes an exonuclease of the XRN1 family, coimmunoprecipitates with the Our results demonstrate that cotranslational protein-mRNA associations can be used to identify new components of protein complexes. To address this question we recently used a method called RIP-chip  to identify mRNAs associated with specific proteins. We found that ~38% of a set of proteins with varied structures and functions interacted with mRNAs known or predicted to encode proteins that interact with the bait proteins. We also discovered that these associations required the presence of the protein encoded by the mRNA as well as active translation. These results indicated that the interaction between the bait protein and the protein encoded by the associated mRNA takes place cotranslationally . The immunoprecipitated protein is associated with the nascent peptide together with the mRNA that is being translated, allowing the identification of the nascent peptide through its mRNA . As hypothesised, we found that the two proteins coimmunoprecipitate in vitro and colocalise in vivo. This work demonstrates that genome-wide protein-mRNA interaction datasets can be used to predict protein-protein associations.Although most cellular proteins are part of protein complexes, little is known about how the formation of protein complexes occurs  Figure A. This aip2 mRNA, which epyp2, which encodes a protein phosphatase known to physically interact with Sty1[cip2, which codes for an RNA-binding protein that regulates the response to oxidative stress[cip2 mRNA requires active translation of cip2, as it is disrupted when translation is inhibited using puromycin and when the cip2 initiation codon is mutated[cip2 mRNA, and thus that both proteins are part of the same complex . SPAC12G12.09 encodes a predicted protein of 977 amino acids that lacks any known domain and does not display homology to any other protein. We named this gene eti1, for exo two interacting (see below). To confirm that these interactions reflected cotranslational assembly, we incubated cells expressing Exo2-TAP with puromycin to disrupt active translation, and used them for RIp-chip experiments. This treatment led to a complete loss of interaction between Exo2 and both ppo1 and eti1 mRNAs  in vegetatively growing cells  and of S phase initiation (cdc18)[Our results demonstrate that cotranslational mRNA-protein interactions detected using RIP-chip can be used to predict protein-protein associations. The nature and behaviour of the proteins we identified suggest that the interactions may have biological importance. For example, there is evidence that Sty1 regulates the phosphorylation of Cip2: Cip2 is phosphorylated in response to oxidative stress, and this phosphorylation does not take place in  mutants. Our resStandard methods were used for growth and manipulation of fission yeast cells. All strThe authors declare that they have no competing interests.JM and CD designed the study, performed the data analysis and wrote the paper. All experiments were carried out by CD. Both authors read and approved the final manuscript.Microarray data: The data sets supporting the results of this article are available in the ArrayExpress repository with accession number E-MTAB-1856. Additional supporting data includes four figures (Additional file Click here for file