Fusarium graminearum, is a devastating disease of wheat. Partial resistance to FHB of several wheat cultivars includes specific metabolic responses to inoculation. Previously published studies have determined major metabolic changes induced by pathogens in resistant and susceptible plants. Functionality of the majority of these metabolites in resistance remains unknown. In this work we have made a compilation of all metabolites determined as selectively accumulated following FHB inoculation in resistant plants. Characteristics, as well as possible functions and targets of these metabolites, are investigated using cheminformatics approaches with focus on the likelihood of these metabolites acting as drug-like molecules against fungal pathogens. Results of computational analyses of binding properties of several representative metabolites to homology models of fungal proteins are presented. Theoretical analysis highlights the possibility for strong inhibitory activity of several metabolites against some major proteins in Fusarium graminearum, such as carbonic anhydrases and cytochrome P450s. Activity of several of these compounds has been experimentally confirmed in fungal growth inhibition assays. Analysis of anti-fungal properties of plant metabolites can lead to the development of more resistant wheat varieties while showing novel application of cheminformatics approaches in the analysis of plant/pathogen interactions.Fusarium head blight (FHB), primarily caused by  Fusarium graminearum is the primary pathogen causing Fusarium head blight (FHB) or scab, a devastating disease primarily affecting wheat and barley crops in humid and semi-humid areas worldwide. FHB causes major crop losses, as well as indirect losses due to grain contamination caused by potent mycotoxins, especially deoxyvalenol (DON) [ol (DON) . Strategol (DON) ,2,3. Crool (DON) . Over onol (DON) ,5. GenetA number of secondary metabolites, including hormones, phenolic, and polyphenolic compounds, are significant in plant response to fungal infections. Hormones are metabolites that can easily circulate through parts of, or through the whole organism and that, even at low concentrations, signal and control responses, growth, and the development of organisms . Plant hFusarium [Fusarium graminearum shown recently [The roles of phenolic compounds  in fungal responses of plants have been reviewed by Lattanzio et al. . PhenoliFusarium ,29. AntiFusarium ,31,32, wrecently .Fusarium graminearum in fungal growth inhibition assays.The goal of the work presented here was to determine whether plant-produced metabolites can act as drug-like agents of the plant’s biotic response, and what are their possible protein targets. Cheminformatics analysis was used to identify plant metabolites with the highest potential to function as fungal growth inhibitors and, subsequently, the effect of several selected metabolites was tested experimentally. We present here a compilation of all previously determined resistance-related metabolites, followed by cheminformatics analysis of physicochemical properties and the relationships between these compounds. Possible protein targets of some of these compounds have been predicted using publicly available data for these, and related, molecules. The interaction potential between plant metabolites and proteins in fungus is explored computationally using high throughput small molecule docking. Subsequently, selected metabolites have been tested experimentally showing activity against Metabolic changes resulting from FHB infection in resistant or less susceptible plants have been explored in wheat ,32,33,34In order for RR metabolites to act as plant-produced antifungal agents they must have drug-like properties that can be simply represented through Lipinski “Rule-of-five” characteristics ,43. LipiThe drug-like characteristics of RR metabolites are explored in comparison to all known plant metabolites in the Golm database  and all 2 are believed to have a low capacity for unaided penetration through cell membranes, while those with TPSA less than 60 Å2 are easily absorbed by the cellular membrane [2 are believed to be favored and further regulated by molecular transporters, such as ABC transporters [The majority of RR metabolites have a larger average partition-coefficient (XlogP) value than what is seen in the overall plant metabolite set indicating their higher lipophilicity. XlogP is an atom-additive method for calculating the octanol/water partition coefficient (logP). It obtains the logP value for a compound by summing the contributions from component atoms and correction factors. Lipinski’s rule-of-five allowed range for XlogP is −0.4 to 5 and the majority of RR metabolites fit within these limits B. The tomembrane . Moleculsporters . The avesporters , possiblsporters . Overallsporters .A number of biological assays have already been performed for many of the metabolites within the RR group. Protein targets are also known for many of these and this data is available through the PubChem database . This inThe RR metabolites show activity against a number of proteins and the complete list can be obtained from the authors. A number of metabolites have more than one known protein target and for several protein targets more than one metabolite shows activity .Fusarium graminearum was recently experimentally shown [Fusarium graminearum sequence. Many other targets of tannic acid have been defined in the literature. While the activity of tannic acid against Fusarium gaminearum has already been reported, it serves as a good positive control. At the same time, tannic acid is a large phenolic compound that has structural similarity to many other, simpler phenolic acids, detected in grains infected with FHB. Examples of these include ferulic acid, p-coumaric acid, salicylic acid, gallic acid, caffeic acid, etc. These significantly smaller products of the phenylpropanoid biosynthetic pathway possibly have similar protein binding targets as tannic acid.A number of active metabolites listed in ly shown . Our anaFusarium graminearum has, to the best of our knowledge, not been explored. Luteolin, as well as several other flavones with different hydroxylation patterns, have been previously identified as RR molecules with some protein targets already identified for apigenin and kaempferol  is an extensively studied neurotransmitter in mammals, as well as a widely-distributed metabolite in plants. Serotonin is highly structurally similar to auxin (indol-3-acetic acid), one of the major plant hormones. It has been hypothesized that serotonin acts as an auxin function inhibitor, thereby regulating root development . In resited rice  where seFusarium graminearum systems. Out of 327 protein targets obtained as possible action sites for the tested RR metabolites, 211 are Homo sapiens proteins. In order to determine relevance of this finding to interactions between plants and Fusarium graminearum we have mapped the complete human proteome against protein sequences available for Fusarium graminearum . Statistical information for complete human to complete Fusarium graminearum proteome mapping is shown in Fusarium graminearum proteins obtained from the Broad Institute database [As previous testing of biological activity of compounds was primarily performed on mammalian proteins, we have utilized sequence similarity studies and homology modelling to establish protein functions in wheat and database .Fusarium graminearum sequence. Therefore, within the subset of metabolite-targeted proteins, 65% have homologs in Fusarium graminearum.For the subset of 211 human proteins shown as targets of tested plant metabolites, 138 have significant homologs in the Fusarium graminearum protein homologs based on the sequence comparison performed in this work. For example, CA Fusarium graminearum sequence FGSG_04603 has the alpha CA superfamily domain and closest similarity to human CA (BLAST score: 7 × 10−20). Carbonic anhydrase (CA) is a known target of tannic acid [The majority of proteins listed as targets in nic acid , serotonnic acid , as wellnic acid ). AlthouFusarium graminearum CA was modelled using the homology modelling method provided by Swiss-Model [Fusarium graminearum sequence FGSG_04603 was used as it was the closest homolog to several isozymes of human carbonic anhydrases of known structure.For docking ss-Model ,61,62, iFusarium graminearum CA. Human CA I in complex with the inhibitor topiramate (pdb: 3LXE) was selected for the analysis of human protein. This is the structure of one of the known target proteins of serotonin and contains an inhibitor, allowing the determination of the grid zone for docking analysis. Fungal CA homolog FGST_04603 was modeled using crystal structure pdb: 3Q31 of the fungal alpha carbonic anhydrase from Aspergillus oryzae. Carbonic anhydrase from Aspergillus oryzae showed 39.32% sequence identity with Fusarium graminearum CA, studied here.A number of X-ray crystal structures are available for CA proteins, including representatives of different classes as well as distinct isozymes from human cells. Several available crystal structures include inhibitory molecules. Docking analysis for all RR metabolites was performed for human CA for comparison and Fusarium graminearum carbonic anhydrase FGSG_04603 in space filling and skeletal format. A skeletal format compares active sites of the carbonic anhydrase proteins following structure alignment. Additionally, the inhibitor (green), in order to mark the active site of the two proteins, is shown.Structure quality has been assessed using Ramachandran plots for the model A. A compFusarium graminearum proteins. Several compounds were selected for further testing based on their binding energies that previously indicated, experimentally, relevance as antifungal agents, as well as having proven activity as inhibitors of proteins in mammalian cells that have close orthologes in Fusarium graminearum. Further testing of other proposed Fusarium graminearum CA inhibitors are underway in our group.As only a relatively small number of RR metabolites have been experimentally tested, other possibly stronger inhibitors may exist within the set. In order to assist the selection of metabolites for further experimental analysis binding energy of RR metabolites have been tested computationally using high-throughput docking analysis performed using PyRx   and AutoFusarium graminearum in fungal growth inhibition assays, including tannic acid, trans-Ferulic acid, naringenin, and N-(p-coumaroyl) serotonin. The effect of these compounds on Fusarium graminearum growth in cultures is shown in 50 values listed in Several selected metabolites from the set of bibliome and computationally-determined binders to CA were tested against Fusarium graminearum growth of the tested metabolites is tannic acid, followed by naringenin and ferlic acid. These compounds have been shown previously to be highly promiscuous inhibitors of a range of metalloenzymes in mammalian cells including CA, P450, and lipoxygenase. The majority of protein targets for tannic and ferulic acid have close sequence orthologs in Fusarium graminearum and many  are important in Fusarium graminearum growth and virulence. Structural analysis of these proteins has shown significant similarity, particularly in the binding/active-site regions, suggesting activity of similar inhibitory molecules.The strongest inhibitor of Fusarium graminearum. In addition, plant hormones, as well as metabolites listed as generally related to biotic responses are included. All metabolites determined from the literature have been mapped to the PubChem database [Metabolite information was obtained from automated and manual searching of all available literature dealing with the subject of metabolite or metabolomics analysis in wheat, barley, or oats infected with database  and CIDsdatabase , RCPI  and detected 2930 one-to-one orthologs between the two species. The average BLAST e-values for Fusarium-to-human and human-to-Fusarium mappings were 5.5 × 10−7 and 1.9 × 10−7.The OrthoPred tool was applied to the input protein sequences using bidirectional BLAST local alignments . AutoDock4 performs a rapid energy evaluation through a pre-calculated grid of affinity potentials. The macromolecule is kept rigid while torsional flexibility is allowed for the ligand molecule. The grid box for all calculations included active sites based on X-ray and literature information. Initial structures of all ligands were obtained from PubChem . Followitrans-ferulic acid, indole-3-carboxylic acid and naringenin were purchased from Sigma-Aldrich. The effects of increasing concentrations of several metabolites on the hyphal growth of Fusarium graminearum were tested on potato dextrose agar (PDA) plates. Briefly, metabolites dissolved in various solvents  were spread on PDA plates (25 mL media per plate) to obtain a final metabolite concentration of 0.01–10 mM. Equal volumes of water or ethanol were spread onto control plates according to the solvent used to dissolve the various metabolites. The plates were incubated for an hour to let the metabolites absorb into the media. A small plug of Fusarium graminearum Z-3639 was placed at the center of each plate and the plates were incubated under constant light at 27 °C for three days. The diameter of fungal colonies in each plate was measured and recorded each day after inoculation.Tannic acid, Fusarium graminearum proteome, suggesting possible targets for inhibition by RR metabolites. High-throughput computational docking analysis of interactions between RR metabolites and possible target proteins have shown that several of the RR metabolites can potentially act as significant inhibitors of carbonic anhydrase in Fusarium graminearum. Many of these metabolites result from secondary pathways stemming from tryptophan and include serotonin derivatives produced in the hydroxycinnamic acid and serotonin amide biosynthesis pathway, as well as products of IAA biosynthesis (auxin) and flavones, resulting from phenylalanine biosynthesis. Previously, experimental studies confirmed anti-fungal activity of several of these selected compounds, and herein we show that some have a significant effect on Fusarium graminearum hyphal growth. Further in planta tests investigating the ability of the compounds tested in vitro herein, as well as several other selected metabolites, to reduce FHB on wheat, are currently underway, aiming towards better understanding of possible antifungal targets, as well as focused modification of wheat to produce higher concentration of relevant RR metabolites.Cheminformatics analysis has shown drug-like characteristics for RR metabolites observed in resistant grains infected with FHB. Some of the known protein binders for several RR metabolites had close homologs in the


The treatment of the newborn diagnosed with a ureteropelvic obstruction prenatally should follow a systematic approach. Although a majority of patients can be followed without surgical intervention, controversy exists concerning appropriate follow-up. Furthermore, a significant number of patients will manifest mild disease and thus deserve abbreviated follow-up. Herein, an appropriate algorithm and a review of the literature are discussed.


In recent years, evidence has accumulated demonstrating that the cAMP response to different extracellular agonists is not uniform: depending on the stimulus, cAMP signals of different amplitudes and kinetics are generated in different subcellular compartments, eliciting defined physiological effects. In this review, we focus on how real-time imaging using fluorescence resonance energy transfer (FRET)-based reporters has provided mechanistic insight into the compartmentalisation of the cAMP signalling pathway and allowed for the precise definition of the regulation and function of subcellular cAMP nanodomains. Cyclic nucleotides, such as 3′,5′-adenosine monophosphate (cAMP), are small molecules used by cells to propagate extracellular information inside the cell and are referred to as second messengers. cAMP is generated by intracellular adenylyl cyclases in response to a first, extracellular message that activates a transmembrane G-protein coupled receptor (GPCR). This second messenger system is used in many types of cells, from prokaryotes to human neurons, and in each cell type its synthesis can be triggered by different extracellular stimuli with different functional outcomes. It is thus imperative to understand how a universal signal like cAMP can be versatile enough to generate cellular effects that are specific to each individual stimulus. This question demands sensitive quantification of the second messenger and accurate comparison of cAMP signal amplitude, dynamics, and subcellular location in response to different extracellular cues.In the cardiovascular system, the first message that activates the cAMP pathway is provided by a variety of biochemically diverse molecules. These include neurotransmitters, such as adrenaline and noradrenaline; peptide hormones, such as glucagon; and lipid compounds, such as prostaglandins. Each molecule binds to a distinct GPCR, coupling to different kinds of G-proteins with the ability to activate or inhibit cAMP production . Increassα subunit of a heterotrimeric G protein (PDB 1AZT) [2/s. In molecularly crowded environments, such as the cytoplasm of an adult cardiomyocyte, this can be slowed by more than a factor of ten [Interestingly, not all extracellular stimuli that activate cAMP signalling in the heart increase PKA activity uniformly and to a similar degree throughout the cell. This was first hypothesised on the basis of observations in biochemical fractions of cardiomyocytes, stimulated with either prostaglandins or β-adrenergic receptor agonists . Direct DB 1AZT) . A smallr of ten . Still, Real-time cAMP imaging techniques have greatly enhanced our understanding of the distribution and nature of intracellular cAMP signalling domains. Temporal and spatial resolution of cAMP signalling are among the main advantages that FRET-based imaging methods can offer to the field. In combination with the reversibility of the response and real-time detection of cAMP changes in living cells, this technique has revolutionised our understanding of cAMP signalling in the heart. FRET-based imaging helped replace the coarse definition of cAMP concentration gradients between biochemical fractions with our appreciation of sub-microscopic differences in cAMP signalling at different organelles. This review gives a brief account of the evolution of FRET-based imaging approaches in the study of cAMP signalling. Different probes have been instrumental in addressing specific aspects of the cAMP signalling pathway and have distinct strengths and weaknesses that are worth considering when designing experiments. We also discuss how several cardiac compartments have been characterised using real-time imaging of cAMP and present specific examples.cAMP signalling in cardiac myocytes is compartmentalised. During a signalling event, the cyclic nucleotide is not uniformly distributed through the entire cytoplasm, but it accumulates to a greater or lesser extent in distinct loci within the cell. Due to its physicochemical properties and relatively high diffusivity, this behaviour of cAMP is not intuitive, and the discovery and further analysis of cAMP subcellular compartmentation was a multi-step process that is recounted in the following paragraphs. A single cAMP compartment can be confined to a cellular substructure that involves only a small number of proteins and thus operates on the nanometre scale. Such macromolecular substructures have been termed cAMP nanodomains. cAMP domains contain functionally associated proteins, or signalosomes, which together shape the effect of the cAMP signal within the domain. Signalosomes are usually comprised of cAMP binding proteins, such as PKA and phosphodiesterases (PDEs), as well as their regulators and targets. cAMP compartmentalisation has a spatial and a temporal dimension. To adequately describe cAMP signalling in cardiomyocytes, the toolkit for cAMP detection and quantification therefore had to evolve considerably from bulk analysis of cAMP content in tissue or cell lysates to targeted, real-time, and quantitative analysis of cAMP nanodomains in living cells . Each ofSince the 1970s, detection of cAMP by calibrated amounts of cAMP binding proteins, including cAMP-dependent protein kinase purified from bovine muscle  or cAMP-Bulk biochemical quantification of cAMP in membrane and cytosolic fractions from adult rabbit cardiomyocytes revealed that selective stimulation of different families of G-protein coupled receptors elicits distinct cAMP and PKA activation profiles. Concentration of cAMP increases in both the membrane and the cytosolic fraction when β-adrenergic receptors are stimulated with isoproterenol. Meanwhile, stimulation of the prostaglandin receptor with prostaglandin E1 (PGE1) only increases cAMP in the cytosolic fraction .Biochemical cAMP detection in combination with cell fractionation provided first evidence that cAMP signalling in cardiomyocytes is not uniform and that cAMP can be compartmentalised depending on the extracellular stimulus. Biochemical detection can be as sensitive as 1 nM cAMP and is compatible with high throughput screening . HowevercAMP can activate non-selective cation channels in the plasma membrane, known as cyclic nucleotide gated channels (CNGC). Activation of these channels leads to a cation current, which can be measured using the patch clamp technique . CNGC ac2+ channels via local elevations of cAMP [This approach added more detail to the biochemical finding that β-adrenergic and prostaglandin receptors have different effects on membrane and cytosolic cAMP levels, by revealing a temporal dimension: cAMP levels near the surface membrane transiently respond to PGE1 stimulation, while bulk cellular cAMP rises to a steady level in the same time frame . Further of cAMP . While CNGC-based functional assays enabled real-time detection of changes in cAMP concentrations for the first time, CNGC biosensors are restricted to one compartment, the sarcolemma. This precludes studies of other important subcellular structures in cardiomyocytes, such as the sarcomere and sarcoplasmic reticulum. In addition, native CNGCs have comparatively low cAMP/cGMP selectivity . While mTo create a highly selective probe that can measure cAMP in living cells, Roger Tsien, Susan Taylor, and colleagues made use of the heterotetrameric structure of the cAMP effector PKA . CatalytUsing this probe, intracellular cAMP concentrations were measured in response to short β-adrenergic stimulation of isolated frog ventricular myocytes and correlated to the ensuing calcium transients . This deWhile this technique allowed for specific measurement of cAMP in living cells on a single-cell level, the requirement for production of chemically labelled PKA subunits and microinjection of correctly folded proteins renders it practically challenging. Critically, both the process of microinjection and the non-physiologically high concentrations of PKA catalytic subunits in the cell produce a considerable amount of cytotoxicity that limits the number of cell types that are amenable to this technique.While FlCRhR was an important proof of concept for the use of tetrameric PKA-based FRET in living cells, there was a need to incorporate the probe into a more ubiquitously applicable genetic system. This was generated by tagging the genes for catalytic and regulatory subunits of PKA with the coding sequence for two fluorescent proteins with overlapping emission and excitation spectra. Genetically encoded FRET sensor pairs can be co-transfected into living cells, which leads to their transient expression by the host cell expression system . Similar−/− knock out mice, as measured with the tetrameric FRET probe, was higher than in wild-type cardiomyocytes. Critically, PDE4D−/− knock out mice exhibited accelerated progression of heart failure following myocardial infarction and were highly susceptible to cardiac arrhythmias during exercise followed by low-dose epinephrine injection [This technique was widely applied to measure changes in intracellular cAMP concentrations in diverse cellular systems. Once expressed, genetically encoded PKA tetramers are compartmentalised by binding to PKA scaffolds called A-Kinase anchoring proteins (AKAPs) . AKAPs cnjection .The ability to measure real-time intracellular cAMP changes in intact, living cells also enabled studies towards understanding the integration of sympathetic and parasympathetic stimulation in cardiomyocyte cAMP signalling . Using tTagged with a peptide sequence that is post-translationally myristoylated and palmitoylated, the PKA-based sensor can be selectively directed to the plasma membrane . In HEK The development of genetically encoded cAMP sensors has strongly facilitated the spatio-temporal dissection of cAMP signalling in cardiomyocytes. However, the multimeric nature of PKA presents several technical limitations for high temporal resolution of intracellular cAMP quantification. As catalytic and regulatory PKA subunits with acceptor and donor fluorophores are transfected and expressed from two different plasmids, equal level of expression cannot be guaranteed. Additionally, each sensor subunit can potentially interact with endogenous PKA subunits, limiting the number of functioning FRET pairs. Both these effects may skew the formation of FRET-competent heterotetramers and make signal termination difficult to assess. As dissociation of the PKA heterotetramer requires cooperative binding of four molecules of cAMP to the two regulatory subunits, sensor kinetics are relatively slow, which may lead to underestimation of cAMP propagation within the cell . PKA tetTo overcome cooperativity and remaining toxicity concerns, single-chain cAMP FRET sensors were developed using the cyclic nucleotide binding domains (CNBDs) obtained from a number of different cAMP binding proteins.Several generations of unimolecular FRET sensors based on exchange protein directly activated by cAMP (Epac) are available to date. Epac is a guanine nucleotide exchange factor (GEF) for Ras-like small GTPases. Binding of cAMP to a unique binding domain induces a conformational change in the inactive Epac protein to expose both the catalytic domain  and a taThe uniform cytosolic distribution, particularly of the sensors based on a single cAMP binding domain, uncoupled the site of cAMP detection from any particular cellular structure and allowed for detection of bulk cytosolic cAMP. However, the site of cAMP detection could be chosen deliberately by fusing these sensors to specific targeting sequences or domains. Directing the sensor to nuclei, mitochondria, or the mitochondrial matrix, for example, allowed for measurement of the dynamic local changes in cAMP concentrations in response to PGE1 or adrenergic stimulation , revealiAn Epac-based single-chain FRET sensor, in its untargeted form, was used in combination with scanning ion conductance microscopy (SICM). After local receptor stimulation of either β1- or β2-adrenergic receptors, cAMP was found to be differently distributed across the healthy cardiomyocyte membrane. As a consequence of β2-adrenergic receptor redistribution in heart failure, such cAMP compartmentation was altered .Epac-based sensors are versatile and easy to use, but their properties may need to be adjusted to suit a particular cellular system. Some of the Epac-based sensors, including the popular Epac1-camps, have high cAMP sensitivity . This cThe sensitivity of Epac-camps sensors to cAMP is a concern in cardiomyocytes with high basal cAMP concentrations, and so a sensor that sandwiches an alternative cyclic nucleotide binding domain between its fluorophores was developed. Here, fluorescence donor and acceptor proteins are fused to the cAMP binding domain of the hyperpolarization-activated, cyclic, nucleotide-gated potassium channel 2 (HCN2) .With this sensor, contributions of different PDE isoforms to cAMP hydrolysis in adult mouse cardiomyocytes after adrenergic stimulation were assessed, which ranked the activity of PDE4 above PDE2 and PDE3 . MeasuriWhile this sensor addresses some limitations of the Epac-based cAMP sensors in cardiomyocytes, the main issue of comparability between targeted sensors remained unresolved.cAMP Universal Tag for imaging experiments) was developed based on the CNBD of the PKA regulatory subunit IIβ. The unique feature of this new sensor is that CUTie enables fusion of the targeting domain distal to the FRET fluorophore pair. This is attained by fusion of the FRET donor to the C-terminus of the CNBD and insertion of the FRET acceptor in an intra-domain loop of the CNBD, leaving the N-terminus free for the targeting domain. In this configuration, targeting domain and FRET module are physically separated from each other and steric hindrance on the conformational change required for energy transfer is minimised. As a result, the dynamic range of the sensor in different compartments is now comparable [To minimise potential interference of the targeting domain with FRET, a novel cAMP sensor named CUTie  and 380 nm (Ca2+-free), and follow the activation of both pathways simultaneously [In cardiomyocytes, cAMP signalling is closely linked to calcium signalling. It is possible to use ECFP/EYFP FRET sensors in combination with Fura-2, a calcium imaging dye with absorption peaks at 340 nm (Caaneously . To streaneously . In the aneously . Both seThe most extensively studied effector of cAMP in the heart is protein kinase A (PKA) . A familSimilar to cAMP probes, these sensors are theoretically targetable to any cellular compartment or signalosome. As such, they are useful reporters of the functional effects of cAMP signalling on effector proteins within a pre-defined compartment. Tethering of AKAR reporters to AKAP-binding domains, for example, shortened the response time of the sensor to adenylate cyclase stimulation, whereas tethering to a nuclear localisation sequence prolonged it . This emThe use of AKAR reporters is extremely effective in real-time quantification of PKA-mediated phosphorylation in a given cellular compartment. It is important to keep in mind that this is an indirect measure of the cAMP signal in this compartment, as signal intensity not only depends on the cAMP concentration but also on the availability of functional PKA to mediate AKAR phosphorylation, and on the level of counteracting phosphatase activity.Development of genetically encoded cAMP sensors greatly enhanced our ability to describe the biochemical properties of cAMP in living cells through real-time visualisation of cAMP responses. The sensors allowed for more accurate definition of the diffusive properties of the small signalling molecule in a cellular context. Physical barriers in the structurally highly complex adult cardiomyocytes, for example, considerably slow down diffusivity of cAMP compared to the loosely structured neonatal cardiomyocyte . AccuratTranscriptional regulation of nuclear genes can alter metabolic and developmental programmes, and changes in transcriptional profile are strongly associated with pathological conditions, such as cardiac hypertrophy . Major tAs detected by Epac-based cAMP FRET sensors targeted with a nuclear localisation sequence (NLS), cAMP concentration in the nucleus increases after stimulation of β-adrenergic or prostaglandin receptors in human embryonic kidney (HEK 293) and airway smooth muscle (ASM) cells ,37,69. TThe direct mechanism of nuclear PKA activation does not seem to be conserved in cardiomyocytes. PDE inhibition in neonatal rat ventricular myocytes does not accelerate nuclear AKAR phosphorylation, despite rapid nuclear cAMP accumulation after co-treatment with adenylate cyclase activators and PDE inhibitors . DisruptThe sarcolemma has distinct microscopic domains. T tubules b reach dUsing plasma membrane-targeted cAMP sensors, it was established that proximity to the plasma membrane can have a temporal effect on the onset of cAMP signalling. For example, in one study the response time of the Epac-based cAMP sensor ICUE1 targeted to the plasma membrane was 40% reduced over its diffuse cytoplasmic counterpart upon β-adrenergic stimulation . To addrNot only the agonist, but also the receptor subtype affected the propagation of cAMP in cardiomyocytes. While β1-adrenergic stimulation lead to Epac-based cAMP detection throughout the entire cell, β2-adrenergic stimulation responses were locally confined by an unknown mechanism . β2-adreDownstream of β1-adrenergic signalling, but not β2-adrenergic signalling, PDE4B-mediated degradation of cAMP is activated in cardiomyocytes . PKA inhIn adult cardiomyocytes, mitochondrial fatty acid beta-oxidation is the major source of energy. For this reason, cardiomyocytes are tightly packed with mitochondria . This ecAMP signalling plays important roles in mitochondrial calcium handling, mitochondrial metabolism, and protection against cytochrome c-mediated apoptosis in cardiomyocytes. Close proximity c,d betweIschaemia-reperfusion injury of the heart is associated with a dramatic change in mitochondrial morphology. Changes in mitochondrial structure impact their function and have been shown to induce cyotochrome c-dependent cardiomyocyte apoptosis . InteresTo specifically describe mitochondrial cAMP signalling, FRET-sensors were fused to different mitochondrial targeting domains. An Epac-based FRET sensor, fused to the mitochondrial PKA scaffold protein AKAP1, showed a similar FRET response to β-adrenergic stimulation as in the cytosol . AKAP1 bIn contrast to cAMP generated at the sarcolemma, calcium release from the ER or capacitative calcium influx from the extracellular medium could activate the matrix cAMP sensor . The solUsing OMM-, and matrix- and untargeted Epac-based sensors, the outer mitochondrial membrane cAMP nanodomain was subsequently dissected further. It was confirmed that this domain on the mitochondrial surface is supplied by plasma membrane adenylate cyclases and is independent of any cAMP generated in the mitochondrial matrix by sAC . Instead2+) currents from the SR. Cardiac Ca2+ homeostasis is vital in maintaining the ability of the cardiomyocyte to contract and relax, as Ca2+ is a necessary co-factor for the force-generating module in the sarcomere [The sarcoplasmic reticulum (SR) of mammalian adult cardiomyocytes is a highly differentiated organelle and the main regulator of calcium transients in these cells. Its proximity to cadiomyocyte T tubules c,d facilarcomere . 2+ from the SR to enable contraction  [2+-ATPase (SERCA) [2+ handling by the SR is often dysfunctional in heart diseases, including heart failure [Calcium cycling at the SR is heavily regulated by PKA. PKA phosphorylates the channels that release Cars, RyR) ,88, as w (SERCA) . Ca2+ ha failure .An Epac-based cAMP sensor, fused to full-length phospholamban using a flexible linker, was used to investigate propagation of adrenergic signals to the SR. The SR compartment was found to be predominantly under β1-adrenergic control . β2-adreThis could be increasingly relevant as transverse aortic constriction (TAC), which causes pressure overload-induced cardiac hypertrophy in the heart, reduces any differences in cAMP between SR and cytosol observed after adrenergic stimulation . Phospho2+ [The sarcomere is the proteinaceous force generator within the cardiomyocyte that mediates muscle contraction. It contains more than 600 structural, regulatory, and associated proteins . The mai2+ . In doinTaking into account the important functional role of PKA activity in sarcomere contraction, it is interesting that this cellular compartment undergoes the smallest change in cAMP in response to adrenergic signalling in healthy cardiomyocytes. Comparing cAMP levels quantified with CUTie sensors targeted to the sarcomere with full length cardiac troponin I to sarcolemmal and SR-targeted sensors, low level adrenergic stimulation generated only about 60% as much cAMP at the sarcomere than at the other compartments . CalibraAKAPS are intracellular scaffolding proteins that provide a platform for the assembly of PKA-containing signalosomes. AKAPs were first discovered in the 1970s and 80s  and thenTargeting FRET sensors to distinct AKAPs can be utilised to directly describe cAMP fluctuations around defined PKA signalosomes in cardiomyocytes. CUTie sensors were used to quantitatively compare cAMP concentrations in the vicinity of AKAP79 at the plasma membrane or AKAP18δ at the sarcoplasmic reticulum with cytosolic cAMP concentrations after adrenergic stimulation of cardiomyocytes. These studies revealed that cAMP concentrations around AKAPs can be detectibly higher than in the bulk cytosol .AKAPs interact with PKA through AKAP-binding domains on the regulatory (R) subunits. Of note, different isoforms of R subunits preferentially interact with distinct AKAPs. By tethering Epac-based cAMP reporters to the dimerization/docking domains of RI and RII, it was possible to specifically dissect molecular components that shape cAMP pools around these different PKA isoforms in cardiomyocytes . Both coGenetically encoded, tetrameric PKA-RII containing FRET probes naturally localise to AKAPs in cardiomyocytes. These probes identified a prominent role for PDE2 in shaping the cAMP response to catecholamines . InhibitTo dissect the role of AKAPs on cAMP signal compartmentation themselves, cytosolic FRET responses were compared in neonatal cardiac myocytes from wild type and AKAP5 knock out mice . While tThe use of FRET-based cAMP and PKA activity sensors has greatly enhanced our understanding of spatial organisation in the cAMP signalling cascade, as well as the mechanisms that generate a uniquely patterned cAMP response through cardiomyocytes as they react to defined stimuli. Localised FRET sensors have refined our ability to monitor cAMP signalling events. The sensors constantly evolve , thus inFRET-based sensors for cAMP and downstream PKA activity have evolved from a highly demanding technique into a universally applicable tool over the last two decades. In the future, fine tuning this approach by targeting sensors to additional compartments, adjusting their sensitivity accordingly, and improving their dynamic range through engineering the fluorophores in a FRET pair will undoubtedly continue to refine our understanding of cAMP-regulated cardiomyocyte compartments.


Ictaluridae, specifically the blue and channel catfish, have received very little research attention directed toward their gut-associated microbiota using 16S rRNA gene sequencing. Within this study we utilize multiple genetically distinct strains of blue and channel catfish, verified via microsatellite genotyping, to further quantify the role of host-genetics in shaping the bacterial communities in the fish gut, while maintaining environmental and husbandry parameters constant. Comparisons of the gut microbiota among the two catfish species showed no differences in bacterial species richness (observed and Chao1) or overall composition (weighted and unweighted UniFrac) and UniFrac distances showed no correlation with host genetic distances (Rst) according to Mantel tests. The microbiota of environmental samples (diet and water) were found to be significantly more diverse than that of the catfish gut associated samples, suggesting that factors within the host were further regulating the bacterial communities, despite the lack of a clear connection between microbiota composition and host genotype. The catfish gut communities were dominated by the phyla Fusobacteria, Proteobacteria, and Firmicutes; however, differential abundance analysis between the two catfish species using analysis of composition of microbiomes detected two differential genera, Cetobacterium and Clostridium XI. The metagenomic pathway features inferred from our dataset suggests the catfish gut bacterial communities possess pathways beneficial to their host such as those involved in nutrient metabolism and antimicrobial biosynthesis, while also containing pathways involved in virulence factors of pathogens. Testing of the inferred KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways by DESeq2 revealed minor difference in microbiota function, with only two metagenomic pathways detected as differentially abundant between the two catfish species. As the first study to characterize the gut microbiota of blue catfish, our study results have direct implications on future ictalurid catfish research. Additionally, our insight into the intrinsic factors driving microbiota structure has basic implications for the future study of fish gut microbiota.The microbiota of teleost fish has gained a great deal of research attention within the past decade, with experiments suggesting that both host-genetics and environment are strong ecological forces shaping the bacterial assemblages of fish microbiomes. Despite representing great commercial and scientific importance, the catfish within the family  Recently the aquaculture industry has seen a great deal of interest in pre-, pro-, and synbiotic supplementation to modulate the gut microbiota of fish, primarily due to their anticipated efficacy in improving growth rates, feed conversion ratios, and replacing the use of antibiotics to maintain fish health. This interest has in turn stimulated much research on the microbiome of cultured teleost fish species. Over the past decade, the physiological utility of the gut-associated microbiota of teleosts has been proven, with many studies implicating the microbiota in host functions such as lipid metabolism and absorption , vitaminOncorhynchus mykiss fed three different diets, Of these variables known to shape the gut-associated microbiota of fish, host genetics is of particular interest. If a strong correlation among fish genotype and a desirable gut microbial community with beneficial function can be established, it would suggest that the host traits are heritable. Therefore, a superior performing microbiota could be achieved through selection within aquaculture breeding programs . To dateIctaluridae, particularly the channel catfish Ictalurus punctatus and blue catfish I. furcatus, represent an ideal system for further evaluating this relationship between host genetics and the gut-associated microbiota. Channel catfish currently represent the greatest market share of the US freshwater aquaculture industry (approximately 65%) (I. punctatus ♀ × I. furcatus ♂) produce offspring with desirable phenotypes including superior disease resistance and fillet yield (n = 5) collected from a recreational fishing pond  , with anet yield . In addiet yield , physiolet yield , and mucet yield . Many ofing pond . In the ing pond . Togetheing pond . More reI. punctatus  and three strains of I. furcatus  were selected for inclusion in this study. The USDA103 strain is a channel catfish research strain known for high-feed intake that was developed by the US Department of Agriculture – Agriculture Research Services (USDA-ARS) through selection for growth performance , Stoneville, MS, United States, in accordance with the experimental research protocol approved by the Institutional Animal Care and Use Committee (64-F-006-6803).-1, ∼26°C, pH ∼8.5, dissolved oxygen >5 ppm) . Fish densities within each tank were maintained relatively equal over time through random culling; however, no size selective grading was conducted. All fish were fed to apparent satiation daily using the same commercially available diet . Fish were euthanized in a solution of 300 mg L-1 tricaine methanesulfonate  buffered with sodium bicarbonate in water taken from the culture tanks. Fish length and weight were recorded prior to rinsing the ventral surface of the fish with 75% ethanol, then excising the entire intestinal tract and its contents using sterile procedures. Intestinal samples were immediately flash frozen, transported to the laboratory and stored at -80°C until further processing.In early July, eggs from multiple families within each of the selected strains were collected from outdoor spawning ponds at NWAC and rinsed in a 100 ppm povidone-iodine solution at the time of collection as per industry standard practices . Eggs we® DNA Isolation Kit  was then used to isolate DNA following the manufacturer’s recommendations. DNA was checked for quality and concentration using a NanoDrop 2000c  and gel electrophoresis. DNA samples were then submitted to the Roy J. Carver Biotechnology Center  for preparation and sequencing of 16S rRNA V4 gene amplicons. Sequencing libraries were prepared using 505f and 806r target-specific PCR primers , yet the resulting sample sizes were uneven at the level of fish strain .Raw demultiplexed sequences were processed in R  using thTo examine the possible function of the microbiota detected in the gut of these catfish and determine whether microbiota functions differed among host genetics, piphillin was used to normalize our amplicon data by 16S rRNA gene copy number and infer metagenomic contents . Followin = 20) from each strain . Host genetic data, in the form of microsatellite genotypes, were generated by isolating DNA from whole blood samples and amplifying 22 known microsatellite loci using locus-specific primers  and normality (Shapiro–Wilk test) followed by a nested one-way ANOVA (fish strain nested within fish species). Microbiota alpha-diversity indices (observed and Chao1) among the gut-associated fish samples were also analyzed by nested one-way ANOVA after testing assumptions, which led to a log transformation of Chao1 data. Additionally, a Wilcoxon rank-sum test was conducted to test the alpha-diversity between all gut-associated samples and the environment-associated samples (diet and water). To account for differences in library size, differential abundance analysis of the detected microbiota was conducted on a proportional (relative abundance) taxonomy table agglomerated at the genus level using analysis of composition of microbiomes (ANCOM) (-5), using default parameters and a FDR-corrected significance threshold of 0.05. To test whether the potential function of the gut microbiota differed between the two catfish species, the raw KEGG pathway output from piphillin was analyzed by DESeq2 using default parameters, after flooring fractional counts to the nearest integer  . ANCOM w integer . The inf integer . The pac integer  was used integer  at the l integer . To testSD) grew significantly slower than that of the three strains of channel catfish (221.43 ± 19.26 g) up to 193 dph, but no intraspecies differences in fish size were detected . Fish remained healthy throughout the study and showed no visual signs of disease. After applying the filtering steps outlined in the section “Materials and Methods,” a total of 2,735,946 high-quality 16S rRNA V4 gene amplicon sequences were obtained from the 42 gut-associated microbiota samples, yet a high level of variation in library size was still present after filtering . The three environment-associated samples (water and diet) contributed another 278,734 amplicon sequences to the dataset, after filtering. A total of 290 unique bacterial taxa were identified from the gut-associated microbiota samples, with an additional 266 unique taxa found in the environment-associated samples alone.Fish weight indicated that the three strains of blue catfish , and no significant differences in alpha-diversity were detected by nested ANOVA across fish species or strain (Table 2). Significant differences were, however, detected by Wilcoxon rank-sum tests comparing the observed richness and Chao1 estimates of the environment-associated samples (water and diet) to the gut-associated samples (Table 2). In terms of beta-diversity, the permutational test of multivariate dispersion (PERMDISP2) at the level of fish species revealed a significant difference between blue and channel catfish, with the blue catfish gut-associated samples showing slightly greater intra-species variability using weighted UniFrac . Principle coordinate analysis (PCoA) of beta-diversity shows some separation among the overall bacterial communities detected in gut-associated samples; however, within species and strain variation was greater than interspecies differences . As expected, unweighted UniFrac distances show the gut-associated samples both within and between species to be more similar in composition to one another than to that of the environment-associated (diet and water) samples .In this study, the alpha-diversity (observed and Chao1) was relatively low for the gut-associated samples . Bacteria from the phylum Fusobacteria dominated the microbiota of each strain of blue catfish , as well as the USDA503 channel catfish (61.9%), while bacteria from the phylum Firmicutes accounted for the greatest relative abundance in the Delta Select and USDA103 channel catfish strains . Interestingly, Fusobacteria were much less abundant within the diet and water microbiota samples . Firmicutes, however, were detected at rather high levels in the diet-associated microbiota sample , potentially explaining the results found in the gut microbiota samples from the Delta Select and USDA103 strain individuals. This is further supported by the reduced beta-diversity distance between the channel catfish gut samples and the feed sample using weighted UniFrac . The most abundant bacterial phylum detected in the environment-associated microbiota was Proteobacteria, accounting for 40.3 and 90.2% of the diet and water microbiota, respectively. All phyla that were detected in the gut-associated microbiota samples were also present in the environment-associated samples, however, multiple phyla were detected in the environment-associated samples that were not present in the gut-associated samples. Taxonomic data at the genus level for the gut microbiota are shown in Supplementary Figure Figure 6). Firmicutes bacteria within the genus Clostridium XI were shown to be consistently more abundant in channel catfish individuals , while Cetobacterium within the phylum Fusobacteria were found to be more abundant in blue catfish .The dominant bacterial phyla detected within the gut-associated microbiota of the ictalurid catfish in this study were Fusobacteria, Proteobacteria, Firmicutes, and Bacteroidetes, with other less abundant phyla detected as well . Of the 288 inferred metagenomic KEGG pathway features, only two were found to be differentially abundant between the two fish species by DESeq2 (FDR ≤ 0.05), with one feature representing the biosynthetic pathway for beta-lactam antibiotics and the other feature being associated with the potential pathogenicity of Vibrio bacteria .The metagenomic functions inferred by piphillin to be associated with the gut microbiota detected in this study showed the predominant bacterial pathway features to be related to microbial metabolism, nutrient processing, antimicrobial biosynthesis, and to a lesser extent pathogenicity (SD) . Additionally, genetic distances among the three domestic strains of channel catfish  were less than that detected among the three domestic strains of blue catfish  .To quantitatively measure the relationship among the host genetics of each strain of ictalurid catfish in this study, microsatellite genotype data from across the host genome were collected and analyzed for pairwise genetic distance (Rst). To reduce the effect of PCR artifacts such as null alleles, individual samples were required to have ≥85% valid genotype results (non-zeros) across the 22 microsatellite loci, and 2 loci were removed from the dataset because ≤90% of the total samples contained valid genotypes at those loci. Filtering yielded genotypic data across 20 microsatellite loci for 20 individuals from each strain. Population level observed and expected heterozygosity across the 20 included microsatellite loci can be seen in Supplementary Figure Figure 9). No significant correlations between host Rst genetic distance estimates and gut microbial beta-diversity were detected using either test or beta-diversity metric.Linear regression analysis and Mantel tests were used to evaluate the relationship among mean pairwise host genetic distance (Rst) and mean pairwise gut microbiota beta-diversity (weighted and unweighted UniFrac) in the six strains of ictalurid catfish . It is likely that the shared water chemistry, husbandry practices, and nutrient inputs administered throughout this study acted together to indirectly shape the gut bacterial communities. These common selective forces appear to have moderated any differences in microbiota composition that may have been present due to factors shaped by host genetics. Previous studies have shown environmental parameters such as water salinity to be a strong influencer of microbial ecology .When considering the factors affecting the colonization dynamics of fish, dispersion of gut-associated microbiota between hosts should also be considered, as the aquatic environment in which fish live serves as an ideal medium for transferring or inoculating microbes . The disCetobacterium somerae, which was also detected as a dominant microbe in the present study , contrary to the results found in the channel catfish within our study . The ANCOM test, which utilizes log-ratio tests to account for the compositional nature of microbiota data  significant genetic differences were present in the strains and species of catfish . While a modest number of individuals from each strain were genotyped (n = 20), previous research has shown that roughly 25 individuals are sufficient to capture population level allele frequencies for reliable estimation of genetic distances between populations .The host genotyping conducted within this study showed that despite the lack of significant differences in the gut microbiota  might suggest that the greater the genetic distance between populations the more likely those populations are to have disparity in their gut microbial communities. In a study comparing the gut microbiota of multiple wild populations of threespine stickleback Gasterosteus aculeatus with varying levels of geographic and genetic separation, R = 0.651, P = 0.02) among host genetic distance (Rst) and gut microbial beta-diversity (unweighted UniFrac). Similarly, Despite the lack of significant correlations in this study between host genetics and microbiota composition, the slightly positive slope of the linear regressions and the moderate R values of the Mantel tests using either UniFrac metric  research, demonstrating that a common environment and life history can diminish genetic-based differences in gut microbiota composition and function.To summarize, our study is the first comparison of the structure and potential function of the gut microbial communities of multiple strains of the commercially and scientifically important blue and channel catfish. Findings show that despite rather clear distinctions in genome-wide microsatellite-based genetic distance, there were no significant differences in gut-associated microbiota composition among strains and species of these ictalurid catfish. Our results show that the environment-associated (diet and water) microbiota is starkly different than that of the fish gut-associated samples. However, shared environment, diet, and husbandry practices appeared to moderate the microbiota composition associated with hosts of divergent genetic backgrounds. This suggests that environment may have an indirect yet strong influence on shaping the bacterial communities of teleost fish. While previous studies have shown strong correlations among host-genotype and gut microbiota beta-diversity, the present study suggests such results may be have been influenced by other uncontrolled co-variates such as geographical location  and dietary inputs. Results from this study have implications on future basic research on teleost microbiota assemblage dynamics as well as on applied catfish (JB, BP, KS, and BS conceptualized and designed this work. JB and GW were involved in sample processing and data collection. JB conducted the primary investigation, bioinformatic and statistical analysis of the data, and wrote the manuscript. KS, BP, GW, and BS provided resources and analysis tools. KS, BP, GW, and BS reviewed and edited the manuscript. BP and BS were responsible for funding acquisition. All authors approved the final manuscript.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.


Schmitz and colleagues recently investigated DNA methylation patterns in diverse flowering plant species, finding substantial variation in the extent and distribution of methylation in angiosperms.http://www.doi.org/10.1186/s13059-016-1059-0Please see related Research article:  Arabidopsis thaliana and more recently in maize, two species that diverged at the monocot–eudicot split approximately 150 million years ago. In their recent article, Robert Schmitz and colleagues analyzed whole-genome bisulfite sequencing data from 34 diverse flowering plant species to vastly expand our knowledge of plant methylome patterns [For the past decade, plant research has improved our understanding of the distribution and function of eukaryotic DNA methylation at a genome-wide scale. Significant progress has been made in understanding DNA methylation in the model plant Plants have relatively high concentrations of 5-methylcytosine (5mC) compared to non-plant species . This isDNA methylation is established by chromomethyltransferase (CMT) enzymes and the de novo methyltransferase DRM2. CMT enzymes interact with methylated histone H3K9, a mechanism by which repressive histone methylation and DNA methylation reinforce one another . CG methArabidopsis. This model organism continues to be essential for deciphering the genetic players that underlie methylation patterning and function. Methylation research in maize has revealed some features that differ from Arabidopsis. For example, there is a high concentration of 100-bp regions with >25 % CHH methylation in gene flanking regions, known as CHH islands, which may protect silent TEs from the activity of adjacent genes [Arabidopsis has a compact genome with relatively few TEs; in contrast, maize has a very large genome with 85–90 % TEs. It is unclear whether some of the unique methylation features observed in these species are truly unique or if they instead represent insufficient sampling across the flowering plant phylogenetic tree.Single-base resolution methylomes were first described for Arabidopsis, maize, rice, and poplar: (1) methylation is highest in the CG context and lowest in CHH, which reflects the different mechanisms by which these types of methylation are maintained; (2) repeats are highly methylated in the CG context; (3) gene bodies with typical CG methylation patterns are moderately expressed; and (4) CG methylation concentrated around the transcriptional start site is associated with repressed transcription.Armed with whole genome bisulfite sequencing data from leaf tissue of eight previously studied species, as well as 26 newly generated flowering plant methylomes, Schmitz and colleagues analyzed the distribution and extent of CG, CHG, and CHH methylation in relation to TE content, gene expression, and genome size . Many ofBeta vulgaris) has higher methylation levels than any of the other species assayed, with particularly high CHH methylation, seemingly driven by a high percentage of genes that contain repetitive elements. Among repeats, there was substantial interspecies variation in the amount of CHG and CHH methylation, and only CHG methylation correlated with genome size across all species. CHH islands in gene flanking regions were not restricted to maize and were found in many other species. Yet, the positive correlation between CHH islands and gene expression in maize was not universal. It remains unclear if all regions annotated as CHH islands are comparable across- or even within-species, owing to the fairly broad definition of CHH islands.Results from the large-scale methylome analysis by Schmitz and colleagues also demonstrated some surprising patterns. For example, results showed that maize is not an extreme example of a highly methylated genome. Beet  have overall low levels of CHH methylation, particularly in the inner regions of repeats, but the CHH methylation that is present is concentrated at high levels in smaller regions of the genome.Schmitz and colleagues analyzed multiple species from the same family, a powerful aspect of the study that allowed broader phylogenetic conclusions to be drawn. For example, Eutrema salsugineum, which has the lowest levels of CHG methylation and no CG gene body methylation, lacks a functional CMT3 enzyme [Arabidopsis have linked methylation variation to CMT2 [What causes interspecies methylation variation? In some species there may be differences in the activity of, or mutations in, DNA methylation machinery. Schmitz and colleagues have shown previously that 3 enzyme . Genome- to CMT2 , which iThe study by Schmitz and colleagues provides several intriguing findings that warrant follow-up study. Outside of the grasses, multiple dicots  also had low levels of CHH methylation, independent of genetic relatedness . The autResults from the study also showed that conserved non-coding sequences, which are home to gene regulatory regions like transcription factor binding sites, are less methylated than expected. Schmitz and colleagues suggest that these regions remain methylation-free because DNA methylation might negatively impact transcription factor binding, as has been shown recently in vitro . An alteIt is not yet clear if there are functional consequences of the differences in methylation and, if so, what they might be. However, the data presented by Schmitz and colleagues generats many hypotheses for future investigation. The rules of DNA methylation are not yet fully written.


Mastectomy in the context of breast malignancy can have a profoundly negative impact on a woman’s self-image, impairing personal, sexual and social relationships. The deep inferior epigastric perforator (DIEP) flap and implants are the two commonest reconstructive modalities that can potentially overcome this psychological trauma. The comparative data on clinical outcomes and costs of the two modalities is limited. We aim to synthesise the current evidence on DIEP versus implants to establish which is the superior technique for breast reconstruction, in terms of clinical outcomes and cost-effectiveness.A comprehensive search will be undertaken of EMBASE, MEDLINE, Google Scholar, CENTRAL and Science citation index databases (1994 up to August 2017) to identify studies relevant for the review. Primary human studies evaluating clinical outcomes and cost of DIEP and implant-based reconstruction in context of breast malignancy will be included. Primary outcomes will be patient satisfaction and cosmetic outcome from patient-reported outcome measures , complications and cost-analysis. The secondary outcomes will be duration of surgery, number of surgical revisions, length of stay, availability of procedures and total number of clinic visits.This will be the first systematic review and meta-analysis in available literature comparing the clinical outcomes and cost-effectiveness of DIEP and implants for breast reconstruction. This review is expected to guide worldwide clinical practice for breast reconstruction.CRD42017072557.PROSPERO The online version of this article (10.1186/s13643-017-0628-y) contains supplementary material, which is available to authorized users. Breast cancer is the commonest malignancy in women and a major cause of cancer-related mortality . While mA number of reconstructive techniques exist for breast reconstruction. The two most frequently employed techniques include the autologous deep inferior epigastric perforator (DIEP) flap and implant-based reconstruction . The choOn first glance, implant-based reconstruction is a simpler technique compared to free flap reconstruction, requires less training and time and can be performed by many more surgeons . HoweverConversely, DIEP flap is often now considered the gold-standard autologous flap reconstructive technique. This is because it results in less abdominal donor site morbidity compared to the traditional transverse rectus abdominus myocutaneous (TRAM) flap, by preserving the continuity of the rectum abdominis muscles . CompareThus, an extensive search will be undertaken in the MEDLINE (Ovid SP), EMBASE (OvidSP), Google Scholar, Cochrane Central Register of Controlled Trials and Scientific Citation Index databases to identify primary studies on DIEP (intervention) and implant-based reconstruction (comparator) in context of patients with breast malignancy. Data extracted will be used to evaluate which technique is superior in terms of clinical outcomes and cost and thus inform worldwide clinical practice.This systematic review is intended to evaluate the current evidence on the clinical outcomes and cost of deep inferior epigastric perforator (DIEP) flap versus implants for breast reconstruction post cancer-related mastectomy, to determine which technique is more cost-effective and clinically superior.This protocol has been registered with the National Institute of Health Research (NIHR) Prospective Register of Systematic Reviews PROSPERO CRD42017072557. We have adhered to and completed the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 2015 statement .(1) exp Breast Neoplasms/ OR ((breast adj6 cancer*) or (breast adj6 neoplasm*) or (breast adj6 carcinoma*) or (breast adj6 tumour*) or (breast adj6 tumor*) or (breast* adj4 reconstruct*))(2) exp deep inferior epigastric perforator flap/ OR DIEP flap* OR DIEAP flap* OR ((Deep and inferior and epigastric and perforator) adj2 flap*) OR Deep and inferior and epigastric and perforator and flap*)(3) exp breast implant/ OR breast adj3 implant* OR exp silicone prosthesis/[(1) AND (2)] OR [(1) AND (3)]; publication date: January 1994–August 2017We will conduct a comprehensive search of the MEDLINE (OVID SP), EMBASE (OVID SP), Google Scholar, CENTRAL and Science citation index databases from 1994 up to August 2017 to identify studies relevant to the review. A combination of Medical Subject Headings (MeSH) terms and free text will be used, combined with Boolean logical operators to construct the search strategy. Explode function will be used to capture narrower terms. No language or study design restrictions will be applied. The reference list of all included articles will also be screened for relevance. A sample search strategy, for EMBASE (OVID SP), is shown below and a similar strategy will be adapted for the other databases:Stage 1: Title and abstract screening will be carried out by two researchers acting independently. Any discrepancies will be resolved by consensus. If any doubt remains, the article would usually proceed to full-text review.Stage 2: The full-text versions of the studies included in Stage 1 will be downloaded and screened for eligibility by two researchers independently. Discrepancies will again be resolved by consensus. If this is not possible, a third author will be consulted.Studies extracted following database searching will be populated into an Endnote X7 library . The screening will be carried out in two stages using pre-specified screening criteria by two independent reviewers. Inter-rater reliability will be calculated using Cohen’s kappa score.All primary human studies evaluating clinical outcomes and cost of DIEP and implant-based reconstruction in context of breast malignancy will be included. The intervention is the DIEP flap and the comparator is implant-based reconstruction. The inclusion and exclusion criteria are highlighted below.aStudies involving adult patients between 18 and 90 years old.bUnilateral DIEP flap or implant-based breast reconstruction due to breast cancer .cClinical studies .Review articles, case reports, simulation studies, clinical studies in non-human subjects, patients with segmental or partial mastectomy, technical descriptions of operative repair with no outcome measures, breast reconstruction not related to cancer, other autologous flap techniques.Duplicates will be excluded and studies will be screened for bias. The Cochrane’s risk of bias tool will be used for randomised controlled trials . Bias wiPatient satisfaction and cosmetic outcome from patient-reported outcome measures Complications Cost-analysisThe primary outcomes will be:Duration of surgeryNumber of surgical revisionsLength of stayAvailability of proceduresTotal number of clinic visitsThe secondary outcomes will be:If the data is appropriate for quantitative synthesis, then risk ratio with 95% confidence interval (CI) will be used to determine dichotomous outcomes (complications). Continuous outcomes  will be determined by weighted or standardised mean differences with 95% CI. Subgroup analysis may be performed for patients with different breast cancer types and for breast implant materials, dependent on sufficient data sets. Where possible, we will utilise results from an intention to treat analysis.first authoryear of publicationstudy designstudy settingstudy populationparticipant demographics complications measures of patient satisfaction (PROMs e.g. BREAST-Q)economic dataData, from the full-text articles, will be extracted by two independent authors using a standardised extraction form. Any discrepancy will be resolved by consensus or with referral to a third author. If any data is missing or further information is required, the primary authors of the manuscript will be contacted directly. The following data will be extracted:An assessment of heterogeneity will be performed using Review Manager 5.3 provided by The Cochrane Collaboration ; if the I2 statistic [I2 statistic is high, indicating high heterogeneity, a random effects model will be employed. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach [Statistical heterogeneity will be quantified by the tatistic . If the approach  will be approach . SensitiThe aim of this review is to evaluate the clinical outcomes and cost of DIEP flap versus implants for breast reconstruction in context of breast malignancy. Despite many centres ascribing DIEP flap as the gold-standard reconstructive modality, data on clinical outcomes and cost-effectiveness is limited. Therefore, it is important to determine which of the two techniques is clinically superior and more cost-effective as this will guide clinical management. To our knowledge, this is the first systematic review to compare the clinical outcomes and cost of DIEP versus implants.Based on the results of this systematic review, independent recommendations will be made to plastic surgeons, researchers, policy makers and plastic surgery societies. The results will be disseminated at international meetings in the fields of Plastic, Reconstructive and Aesthetic Surgery and published in a high-impact peer-reviewed journal.


The sub-network included the regions of the fronto-thalamic circuits in the left hemisphere. The network measures of the left superior frontal gyrus, pars orbitalis , left thalamus , and right thalamus  were shown, through graph theoretical analysis, to be significantly negatively correlated with severity of suicidal ideation. The reduced functional connectivity in left orbitofrontal-both thalamic regions with suicidal ideation in MDD were inversely proportional to the severity of suicidality independent from depression severity. These findings suggest problems with decision-making and information integration in MDD patients with suicidal ideation.Despite recent developments in neuroimaging, alterations of brain functional connectivity in major depressive disorder (MDD) patients with suicidal ideation are poorly understood. This study investigated specific changes of suicidal ideation in functional connectivity of MDD patients. Whole brain functional connectivity in 46 patients with MDD  and 36 age- and gender- matched healthy controls were compared using resting-state functional Magnetic Resonance Imaging (fMRI) analyzed with network-based statistics (NBS) and graph-theoretical methods. Decreased functional connectivity in a characterized sub-network was observed in patients with MDD and suicidal ideation (FDR-adjusted  And major depressive disorder (MDD) is the most researched associative factor with suicide, and is noted in 60% of psychological autopsy reports3. Fifty eight percent of patients with a current MDD episode report suicidal ideation, with 15% attempting suicide4.Every 40 seconds, one individual dies from suicide6. Still, 86% of suicides occur in people with a low risk of suicidal ideation, suggesting that a suicide attempt over a specific period may not accurately reflect the risk of imminent suicidality, a prime target to prevent suicide7. It is important to differentiate between suicide attempt and suicidal ideation.Not every suicide attempt is fatal, and suicidal ideation does not always lead to suicide. A previous suicide attempt may be the most important predictor of completed suicide9. MDD accompanying suicidal ideation is related to a higher rate of previous suicide attempts10, poorer treatment response11, and is thought to have different neuropsychological correlates that discriminate it from MDD without suicidal ideation12. Measuring severity of both depression and suicidal ideation in MDD patients with suicidal ideation may help determine whether severe depression status predicts depression with suicidal ideation. Considering that severity and intensity of suicidal ideation can fluctuate over time, vary according to biological, psychological, and environmental factors14, suicidal ideation should be assessed as a continuous measurement, and real-time neuroimaging could be a promising approach to examine neural network alterations as correlates of the degree and intensity of suicidal ideation15.Suicidal ideation is a distinct phenomenon that is not equivalent to depression severity indicators, other dimensions with underlying biology, impact on disability, and risk factors17. But few studies have focused on MDD with suicidal ideation, and these studies addressed only verbal fluency with functional near-infrared spectroscopy18 and cognitive control with fMRI19. Few researches reported changes in the fronto-limbic connectivity during the resting state in MDD group with suicidal ideation21 but further researches should be accumulated to figure out associative characteristics of functional connectivity to with suicidal ideation.Neuroimaging studies have demonstrated that several areas of the brain are associated with suicide attempts at a neuroanatomical level, involving the dorsolateral and orbitofrontal cortex22. In this view, neuroimaging of MDD is a prudent modality to use. MDD patients have been classified into different groups based on dysfunctional connectivity findings from resting state fMRI23. Functional connectivity analysis in MDD patients with suicidal ideation would be a useful approach to focus attention on high-risk subgroups24.A recent study highlighted the value of considering MDD as a spatiotemporal disturbance of resting activity related to ruminations and enhanced self-focus27. Network-based statistics (NBS) analysis28 is a useful approach for localizing dysfunctional brain connectivity and is frequently applied to clinical applications30. Unfortunately, however, there are few data on suicidal ideation in MDD, using resting state functional magnetic resonance imaging (fMRI) with graph theoretical and NBS analyses. Therefore, the aim of this study was to investigate the functional organization of whole-brain networks according to suicidal ideation in MDD patients by NBS analysis and graph theoretical analysis using fMRI. We focused on the comparison between MDD patients with suicidal ideation and patients without suicidal ideation, but we also compared healthy control to rule out the pathologic functional connectivity change coming from depression itself, which can find clear associative functional connectivity in suicidal ideation. We had three hypotheses. First, MDD patients with suicidal ideation will show different functional connectivity in the whole-brain networks compared with MDD patients without suicidal ideation. Second, the degree of the change in functional connectivity in those networks will correlate with the severity of suicidal ideation independently from depression severity. Third, functional connectivity associated with suicidal ideation will differ between MDD patients and healthy subjects.Graph theory-based connectivity analysis has been successful in investigations of organizational changes in patients by modeling the whole brain as a networkTable There was also no significant difference in demographic profiles between the two depressive groups and the healthy control group.Figure The connectivity of all 11 edges of the sub-network were significantly correlated with the suicidality score. In addition, nine edges were also significantly correlated with the SSI scores. The directions of all significant correlations were negative Table . There wNode strengths, clustering coefficients, and regional efficiencies of all three hub regions were significantly correlated with SSI scores. Furthermore, all these network topological measures were also significantly correlated with suicidality scores, except the node strength of the orbital part of superior frontal gyrus. In addition, the betweenness centrality of the orbital part of superior frontal gyrus was significantly correlated with the BIS motor scores. The directions of the correlation coefficients between the network topological measures and suicidality scores/SSI were negative, while the other was positive Table . There wFigure To the best of our knowledge, this is the first study comparing resting state functional connectivity in whole-brain network between MDD patients with and without suicidal ideation. We observed that a distinct brain network characterized functional connectivity differences identified in patients with suicidal ideation versus patients without suicidal ideation. This network includes the regions of the orbitofrontal cortex, especially the left superior frontal gyrus, pars orbitalis, left middle frontal gyrus, pars orbitalis, and right olfactory cortex. Additionally, the functional networks consisting of the left middle temporal gyrus, left postcentral gyrus, and subcortical regions including both caudate, both thalami, and right putamen were significantly different in MDD patients with suicidal ideation. There was significant correlation between suicide severity and functional connectivity measures in the subnetwork in the NBS analysis. These functional networks were completely distinct from the functional networks distinguished between MDD patients and healthy subjects.31 and information integration32 with self-attribution33. The primary regions apparent from NBS analysis were the left superior frontal gyrus, pars orbitalis, and both thalami; the hub regions are presented in Table 34. Another hub region that showed reduced functional connectivity was both thalami, which are in the central area that is involved with transmitting information through the brain, and communication with many associative brain regions and global multifunctional pathways35. The thalamus also contributes to homeostatic brain rhythms that function in basic inhibition and coordination36. In the mood-related neural network, the thalamus receives strong dopaminergic projections, playing a critical role for information integration, which is responsible for depression and suicidal behavior38.Regions associated with suicidal ideation among MDD patients observed in our study are involved in decision making40. Decision-making represents a higher level of executive function based on information integration, leading to cognitive flexibility and appropriate social judgement with behavioral control41. These impairments of cognitive actions have clinical implications for suicide in depression. The present results strengthen the view that the alterations in functional networks involved in decision-making with information integration are associated with suicidal ideation. Care is needed in interpreting these results, given that the study was designed to indirectly investigate the association between decision-making, suicidal ideation, and sub-network alterations due to lack of a decision-making task.Poor decision-making coupled with maladaptive information integration is linked with suicidal ideation or behavior42. The hub nodes were limited to reduced fronto-thalamic functional connectivity, consistent with previous studies that demonstrated impaired fronto-thalamic circuitry in suicidal MDD patients through DTI44. Dysfunctions in these circuits may also result in the loss of prefrontal cognitive control through the subcortical area. These disturbances might increase the risk for dysregulation of emotional responsivity and vulnerability to suicidal ideation and behavior.Reduced functional connectivity in fronto-subcortical circuits involving the regions was observed between the left superior frontal gyrus, pars orbitalis, and both caudate; between the left superior frontal gyrus, pars orbitalis, and right putamen; between both thalamus and left middle frontal gyrus, and pars orbitalis; and between the right thalamus and left postcentral gyrus Fig. . Fronto-46. Along with the left postcentral gyrus, three of these regions provide information from a person’s previous experiences and build mental simulation to make decisions. Deductive reasoning based on previous information and simulation is related to self-attribution, and negative attributional style is associated with suicidality that successfully resolves after treatment47. Hence, reduced functional connectivity in the left lateralized middle frontal and temporal networks could be related to suicidal ideation by altering the appropriate deductive reasoning and self-attribution in depressive patients. However, this research was not designed to evaluate asymmetry, and so the findings should be carefully interpreted.Presently, the left lateralization related to suicidal ideation was limited to the left middle frontal gyrus, pars orbitalis, left middle temporal gyrus, and left postcentral gyrus. The left middle frontal gyrus and middle temporal gyrus contribute for deductive reasoning, which has been most often observed with left lateralization49.Our correlation study, with network measures and hubs, provides a complementary view of the left superior frontal gyrus, pars orbitalis, and both thalamic regions. We applied graph theoretical analysis and measured the capacity of the overall information segregation, represented as functional connectivity. By identifying the brain’s modular structures, this graph theoretical analysis can detect groups of functionally associated regions responsible for specific functions. This approach (termed connectomics) has been applied to differentiate characteristics of healthy brain networks from other networks with disorders50. Clustering coefficient is a measurement of local segregation of network and is equivalent to the fraction of a node’s neighbors50. Regional efficiency represents the level of intermodular connectivity, which is responsible for communicating information between modules. The betweenness centrality of a given node is defined as the number of shortest paths between any two nodes going through this node, and represents the level of influence on information transformation51. These results suggest an impaired function of coordinating brain networks in the left superior frontal gyrus, pars orbitalis, and both thalami in patients with higher suicidal severity, and could be an indicator of pathological changes in this region44.Node strengths, clustering coefficients, and regional efficiencies displayed significant negative correlations with the severity of suicidal ideation Table , and the52. These negative correlations also highlight reduced orbitofrontal-thalamic functional connectivity as an independent finding related to suicidal ideation, free from other depressive features and impulsivity. However, careful consideration of the findings is needed because of the possibility of false-negative results due to multiple tests and the small sample size.We did not find any significant network measure associated with other depressive symptom severity scales, anxiety scales, and impulsivity scales. In between impulsivity scales, only the motor scale representing impetuous action was significantly associated with reduced functional connectivity in MDD patients with suicidal ideation. The results suggest a possible association with impetuous action in suicidality of MDD and support the previously published association of the BIS motor scale with suicidal ideation in bipolar patients53. Other observations that have been noted in meta-analyses are decreased connectivity between posterior DMN and central executive network54, especially emphasized in the right hemisphere55.Interestingly, no overlapping regions representing significant alterations were evident upon a comparison of MDD patients with healthy controls, and alterations upon comparison of MDD patients with or without suicidal ideation Fig. . Reduced57.The complete independent functional connectivity evident in this research suggests that the sub-networks are specifically involved in the development of suicidal ideation, and are not associated with depression status or severity of the disorder. Patients with premorbid reduced functional connectivity in the sub-network might be more at risk to develop suicidal ideation when they experience depressive episodes. Another view is that suicidal ideation has its own neurobiological changes that are independent of specific psychiatric disorders. Beyond the diagnosis, two recent studies generalized the discovery, prioritization, validation, and testing of few suicidality related markers across major psychiatric disorders to understand commonalities and differences of suicide58 including suicidal ideation59, was not evaluated in our study as covariate, but should be considered as an important covariate in further study associated to suicidality. Brain imaging studies that examine superficial phenomena related to suicide-related behavior and more diverse molecular biology techniques for the pathogenesis of suicidal behavior are needed. Interpretation with fMRI data, especially negative findings, should be done carefully, recognizing the potential for signal dropout, geometric distortion, and susceptibility artifacts in the orbitofrontal cortex because of its close proximity to the air-filled sinuses61.The methodological limitation of our study is the cross-sectional design, which prohibits drawing a conclusion of a causal relationship. Smoking history, as significant risk factor for suicidal behaviorIn conclusion, reduced functional connectivity in orbitofrontal-thalamic regions in MDD patients with suicidal ideation suggests difficulties in decision making and information integration. Reduced functional connectivity correlated only with suicidality, and was independent from severity of depression, anxiety, and impulsivity. The correlation between functional connectivity and suicidal ideation score was significant after controlling the effects of those scores as covariates. This could suggest our finding with pure independent suicidal pathology associated with reduced fronto-thalamic functional connectivity. It could suggest another perspective on suicidal prevention independent from severe depression leading to suicide, but more focused approach on suicidal risk itself. Supplemented evaluation of attribution style and executive function accompanied with neuroimaging focused on default node network are suggested avenues of future research.62, and HAM-D 17-item score ≥1663. A board certified psychiatrist evaluated the results of the full version of the MINI64, which was repeatedly applied to each patient by another psychologist blinded to the study. Exclusion criteria were: any psychotic disorder , bipolar affective disorder, neurological illness including significant cognitive impairment or Parkinson’s disease, mental retardation, significant medical conditions, epilepsy, history of dependence on alcohol or drugs, personality disorders, or brain damage.Subjects were recruited from April 2011 to April 2013 through the outpatient clinic of the Depression Center of the Samsung Medical Center. These 46 patients comprised 23 MDD patients with suicidal ideation and 23 MDD patients without suicidal ideation who were matched demographically for age, gender, and education year. No patient took any psychotropic medication within two weeks of participation in the study or fluoxetine within four weeks. Inclusion criteria were: age ≥ 18 years, current unipolar major depressive episode as verified by Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV-TR) criteria for MDDThirty-six healthy volunteers with no history of psychiatric disease were recruited from advertisements. We excluded participants with a positive family history of a mood disorder. The study protocol was approved by the ethics review board of Samsung Medical Center, Seoul, Korea. And all methods were performed in accordance with the relevant guidelines and regulations approved by the same ethics review board. All subjects were fully informed and consented prior to participation.64. Subjects replying “yes” to the question “Did you think about suicide during the past one month?” were classified as the ‘suicidal ideation group’ (n = 23), and those replying “no” were classified as the ‘no suicidal ideation group’ (n = 23). The severity of suicidal ideation was measured by both the suicidality score and the SSI65. BIS was applied to evaluate self-report impulsivity66. The severity of depression was measured by the 17-item HAM-D63. MDQ67 was used to assess bipolrarity in depressed patients68. The same trained rater applied HAM-D and MDQ.From the baseline visit, suicidal ideation was assessed with the suicidality module of the MINI and suicidality score was assessed with total number of points in the suicidality module of the MINIAll imaging was performed on a Philips 3.0-T Intera Achieva MRI scanner  within one week after the baseline visit. Functional images were obtained using a two-dimensional echo planar imaging-sensitivity encoding (EPI-SENSE) sequence with the following parameters: voxel size 2.86 × 2.89 mm; slice thickness of 4 mm, repetition time (TR) of 3000 ms, echo time (TE) of 35 ms, flip angle of 90°, and matrix size of 220 × 220 pixels. Images were reconstructed to 128 × 128 over a 220 mm field of view. During the scan, all subjects were instructed to rest quietly with their eyes closed and not to fall asleep. A whole-brain three-dimensional fast field echo T1-weighted structural image was acquired with scan parameters: 1 mm sagittal slice thickness, over-contiguous slices with 50% overlap, no gap, TR of 9.9 ms, TE of 4.6 ms, flip angle of 8°, and matrix size of 240 × 240 pixels. Images were reconstructed to 480 × 480 over a 240 mm field of view. All axial sections were acquired parallel to the anterior commissure-posterior commissure line.http://www.fmrib.ox.ac.uk/fsl). Motion correction was carried out using MCFLIRT69, and temporal high pass filter using Gaussian-weighted least-squares straight line fitting with cut-off of 126 s was applied70. Volumetric regions of interests (ROIs) were defined based on automated anatomical labeling (AAL) atlas, which includes 40 cerebral cortices and five subcortical regions for each hemisphere72. All time-series signals in each ROI were averaged, so that 90 node-averaged time-series signals in every subject were made.Data from resting state fMRI scans were preprocessed using fMRI Expert Analysis Tool (FEAT) of the FMRIB Software Library  or as median and interquartile range, and according to the normality of the distribution, Student’s t test, one-way ANOVA, Wilcoxon rank-sum test, or Kruskal-Wallis test was applied.28. In the NBS analysis, we first perform two-sample t-test for all edges, resulting 4005 t-statistics for each subject. Then we randomly re-assigned the entire subject sample t-statistics for all permutations. We computed the maximum component size of sub-networks whose edges had bigger t-statistics than a certain initial threshold for all permutations, and formed a null-distribution of maximum component sizes. We checked whether the maximum component size of sub-networks in original set was bigger than those of randomly re-assigned sub-networks at certain significance level. The initial threshold and the number of permutation (N) were 4.20 and 10000, respectively.NBS was used to detect the significantly different sub-networks between MDD patients with and without suicidal ideation, and between healthy participants and total MDD patients, respectively. NBS is a mass-univariate testing method based on a network component, rather than an individual link, that controls family-wise error rate (FWER)78.Hub regions extracted from the sub-networks were identified by comparing MDD patients with and without suicidal ideation. Since hub regions have nodal degrees larger than two standard deviations away from the network mean, they signify the most affected regions. The network topological measures on these hub regions, including nodal strength, regional efficiency, clustering coefficient, and betweenness centrality, were computed as the measures using the Brain Connectivity ToolboxIn conclusion, correlation analyses were performed between the functional connectivity of the sub-networks and the neuropsychological measures. Correlation tests were also performed between the network topological measures on the hub regions and the neuropsychological measures. For the correlation analyses, correlation coefficients were calculated using Spearman partial correlation after controlling the effects of age, gender, and education years.