The processing of time intervals in the sub- to supra-second range by the brain is critical for the interaction of primates with their surroundings in activities, such as foraging and hunting. For an accurate processing of time intervals by the brain, representation of physical time within neuronal circuits is necessary. I propose that time dimension of the physical surrounding is represented in the brain by different types of neuronal oscillators, generating spikes or spike bursts at regular intervals. The proposed oscillators include the pacemaker neurons, tonic inputs, and synchronized excitation and inhibition of inter-connected neurons. Oscillators, which are built inside various circuits of brain, help to form modular clocks, processing time intervals or other temporal characteristics specific to functions of a circuit. Relative or absolute duration is represented within neuronal oscillators by “neural temporal unit,” defined as the interval between regularly occurring spikes or spike bursts. Oscillator output is processed to produce changes in activities of neurons, named frequency modulator neuron, wired within a separate module, represented by the rate of change in frequency, and frequency of activities, proposed to encode time intervals. Inbuilt oscillators are calibrated by (a) feedback processes, (b) input of time intervals resulting from rhythmic external sensory stimulation, and (c) synchronous effects of feedback processes and evoked sensory activity. A single active clock is proposed per circuit, which is calibrated by one or more mechanisms. Multiple calibration mechanisms, inbuilt oscillators, and the presence of modular connections prevent a complete loss of interval timing functions of the brain. An important challenge for the modern neuroscience is to understand the mechanistic basis of the interval timing by the brain in the sub- and supra-second range . A steadFigure 1), several time intervals are processed within the brain, both at the conscious and subconscious levels. The temporal intervals processed at a conscious level include anticipatory interval for the arrival of ball in the world-centric view and for executing motor movements to catch a ball, both in the supra-second range. Another time interval processed is the perception of the elapsed duration during an act of catching a ball (sub-to supra-second range). The subconscious processing of time intervals helps in the determination of speed of various motor movements, necessary for the successful execution of a task. For example, elapsed duration for the contraction of a muscle will determine the speed of a muscle contraction. Time intervals processed in muscle and joint movements are generally in the milliseconds range. Time intervals in above example are absolute, duration-based, which contrasts with the beat-based timing, which is relative to a temporal regularity, such as rhythmic beats  circadian rhythm, which relies on the rhythmic expression of a set of clock genes with specific daily profiles in the suprachiasmatic nucleus and other parts of the brain , the intFigure 2). The first module is formed by a neuronal oscillator. Neuronal oscillators generate temporally regular activity from the firing of the action potentials. I propose that the oscillators are built within different circuits across the brain, consistent with the intrinsic nature of timing mechanisms  in the second module, which are endowed with special conductances, or are connected by synapses exhibiting plasticity. Special conductance properties, connections or even the nature of the input, such as recurrent excitation can produce modulation in the activity of FM neurons. The state of the circuit in the second module, determined by the frequency, or the rate of the change of frequency is proposed to code the time intervals in the brain.I propose a modular clock mechanism for the processing of the time interval in the sub- and supra-second ranges, which is based on the generic view of timing mechanism promoted by chanisms . TemporaTwo important circuits, likely to contain the second module, forming neuronal clocks, include (i) the dorsal stream responsible for sensorimotor tasks, connecting the posterior parietal cortex and the motor, premotor and prefrontal areas of frontal cortex , and ii. The staThe primary role of the oscillators in the modular clock mechanism is to represent the external physical time. In order to accurately represent the external physical time intervals, the oscillators are calibrated via tasks that involve a direct or indirect interaction with the external world. Since the oscillators are inbuilt, their intrinsic regular rhythm will be superimposed with fluctuations caused by feedback processes, as well as the sensory inputs, involved in the normal functioning of a circuit. In the next section, I will discuss (a) how the external time is represented by neuronal oscillators within circuits in the brain and (b) various types of regular neuronal activities that could potentially play role as the neuronal oscillators in the modular clocks.The proposed neuronal clock mechanism outlined above incorporates inbuilt oscillators, which generate temporally regular activity representing a unit of time. Accordingly, a temporal unit interval, called neural temporal unit, between two adjacent spikes or bursts of spikes from the output of an oscillator, is proposed to encode the external physical time information required for processing time intervals in the sub-to supra-second range in the brain. Also note that the current model shares the requirement for an oscillator or a pulse generator with a pacemaker-accumulator model . HoweverNote that the neural temporal unit will not be represented by a single value in oscillators across the brain. Instead, the neural temporal units will be represented by different values in different oscillators. This will allow mechanistic flexibility for the interaction between separate clock mechanisms . A characteristic feature of the neural temporal unit is “intrinsic regularity” during a particular task. Note that the proposed intrinsic regularity is not the same as the precise regularity of mechanical clocks. Instead, it conforms to a behavior resulting from the intrinsic regularity, dependent on the single neuron properties. The regular behavior of the proposed neuronal oscillator is further expected to exhibit statistical properties, which is not discussed in the current manuscript.According to the current anatomical and physiological data, the following classes of circuits are likely to provide the functionality of oscillators in the proposed modular clock.Figure 4A): Pacemaker neurons have a common presence in the nervous system  Pacemaker neurons : Tonic activity of slowly adapting peripheral receptors projecting into brain could also serve as an oscillator. The highly sensitive Merkel’s disks ((b) Tonic sensory input : Recording during specific tasks have shown ensembles of neurons undergoing synchronized changes of membrane potential, which are fleeting in both location and time . The inevitable result of the synchronization of the activity of neurons connected together in ensembles is the temporal regularity of synchronized bursts. I propose that temporally regular bursts of spikes, generated by synchronized activation and inhibition of an ensemble of neurons during a cognitive task, could also function as an oscillator in a neuronal clock mechanism. Experimental evidence arguing this potential mechanism is provided by studies that had shown synchronous modulation of a large number of neurons in primate brains during various timing tasks ((c) Regular synchronized activation and inhibition of ensemble of neurons . In addition to playing role as an oscillator, an ensemble of neurons with synchronized activity may also act as a feeder circuit between an oscillator and a circuit of FM neurons. Moreover, there are two types of information available for processing in ensembles of neurons with synchronized activities: (i) the interval between bursts and (ii) the activity of neurons during the bursts.The processing of neural temporal units in neuronal circuits is also inferred from psychophysics experiments. In a task designed to compare the time intervals between two sound tones of a given frequency with a standard time interval, the benefits of training disappear when the standard time interval is changed . But theIn another psychophysics experiment , particiFigure 3) is proposed to represent the “dimension of time” in neuronal circuits processing various types of temporal information, such as the time interval, speed, and rate. Activity from the inbuilt oscillator is proposed to feed into the second module, where it produces modulations in the activity of FM neurons. The modulations in the activity of FM neurons help in coding different “time intervals” by generating changes in state-dependent parameters of the circuit in the second module, represented by the frequency change and the rate of the change of frequency or their combination.“Neural temporal unit,” defined as the interval between two adjacent regular spikes or spike bursts,  or a descending activity . Short-term plasticity, associated with facilitation or depression, has been demonstrated in mammalian cortical neurons . To catch a ball, especially in a fast trajectory, the fielder is required to execute a complex set of temporally precise motor movements, which would recruit feedback mechanisms in the cerebellum. Missing the ball’s trajectory, even by a few fractions of a second, will result in a failure to execute the task. In other words, the feedback processes must tightly couple the motor actions to the requirements of the task parameters, such as the speed of a ball. The speed (ΔDistance/ΔDuration) of a ball contains information about time in an external three-dimensional surrounding. Therefore, the successful execution of a task will result in the transfer of information about “physical time intervals,” such as those based on the speed of external objects, into the neuronal circuits in the brain. Since most changes needed for the smooth control of motor movements occur over sub-second durations, it indicates that the cerebellum will play a role in representing shorter time durations. Consistent with this expectation, it has been shown that the brain uses networks involving the cerebellum for tasks involving shorter intervals . The binding property is likely to be a synchronous, but a temporally sharp change at the level of the cerebellar and the cortical oscillator circuits, produced by feedback processes controlling motor movements, matching the temporal parameters of the external task. The sharp synchronous changes will help in defining or focusing temporally sharp points on time axis within different circuits in the cortex and cerebellum in synchronicity with the external temporal events, thus, transferring the external temporal information into the circuits in the brain. It is also posited that there is a direct serial connection between oscillators in the cerebellum and the cortical areas, which would be required for the flow of information between oscillators in the cerebellum and the cortical areas. Note that the flow of external physical time information from the cerebellum to the cortical areas  would require that the neural temporal units maintain their regular interval during their transmission. The pacemaker activity has been observed in vivo and in vitro in deep cerebellar nuclei  of finger opening and release times in skilled overarm throwing in patients with cerebellar lesions . Furtherr nuclei , which mr nuclei , the preIt is argued above that external time information is represented within the neuronal circuits with the help of feedback processes, responsible for the tight coupling between the task execution and its temporal requirements. This leads us to the next question: which neurophysiological parameters help in coding the physical time as a result of the successful execution of a task? The “frequency” of neuronal firing is a good candidate for this role. Changes of activities such as the rate of muscle contraction or angular change at a joint mostly result from changes in the frequency of firing of motor neurons. Increased rate of a change in motor movements would represent shorter time durations, as the rate of change is inversely related to the time duration of occurrence of a defined change. Accordingly, a higher frequency of a neuronal activity in the nervous system will encode a shorter time duration, over which a defined activity occurs. Furthermore, as an increase in the activity of motor neurons is commonly accompanied by a general increase in the rate of the firing of neurons in various upstream circuits, a proportional increase will be seen in neuronal oscillators in the cortical and cerebellar circuits. An increased activity of oscillator circuit, according to various models, such as recurrent feedback excitation , and facFigure 5). Consistent with the proposed role in the calibration of oscillator circuits, the left parietal lobe has been shown to be associated with the perceptual and motor temporal predictability .The successful execution of motor tasks, emphasized above for their role in timing mechanisms, also depends on various sensory functions, for example, cortical representations of the spatial coordinates of the surroundings, spatial coordinates of auditory stimuli, eye-centric, and body-centric visual fields. LIP area in the posterior parietal cortex area has been shown to contain body centric; and its adjacent area 7a is shown to contain world-centric representations of visual field . Neuronstability . Along ttability , providitability . Thus, tAlthough, the role of sensory processes in transferring external temporal information via direct interaction has been emphasized, it has been argued earlier that we are able to anticipate changes in events that we cannot imitate, such as lashing waves or other inanimate events . The abiThe representation of external time into the neuronal circuits can be achieved by rhythmic external activities producing sensory stimulation, such as waves lashing a beach. Rhythmic external physical events, such as the waves lashing shores at a regular rate, will help to encode information about time represented within the periodicity of sounds and sights of lashing waves. This information would calibrate the oscillator activities within circuits processing rhythmic sensory inputs by directly or indirectly influencing their activities. A simple example of a periodicity coding time information will be the activation of an auditory neuron at two time points by consecutive single sound tones. In this example, the time interval between two sound tones is represented internally in the neuronal circuits by the time interval separating two evoked activities in auditory areas. In addition to providing information about the temporal periodicity, the periodic sensory inputs can modify or become an oscillator module for a neuronal clock mechanism.Non-regular external sensory events also appear likely to play a role in coding the temporal information in neuronal circuits by producing evoked activities at irregular intervals resulting in simultaneous sharp changes in the firing activity at variable intervals, enabling the binding between different sensory circuits, similar to the effect of feedback processes during a motor activity. Such temporally non-regular events may include the sight of an object going up in the air to fall down. Temporally non-regular event can produce synchronous evoked activities in areas of cortex with different representation of same sensory modality leading a binding of oscillators in different parts of cortex, which could play a role in the calibration of timing mechanisms. A certain kind of activity may be more likely to undergo primary processing in one or the other visual field. For example, various characteristics of a speeding car will be analyzed best by organizing the visual information in the world-centric representation in the parietal cortex. In comparison, a cricket ball being sought by a running fielder would be processed best by retrieving information from the head-centric cortical representation. Accordingly, the timing mechanism for ball movement in the head-centric area of the parietal cortex will calibrate the timing mechanism in the world-centric area 7a by virtue of the binding property, enabled by synchronously evoked activities in both cortical representations. In addition, the multisensory processing circuits in the posterior parietal cortex , may alsFigure 6).Past animal studies and clinical data have implicated the basal ganglia and nigrostriatal circuits in interval timing tasks. One study showed that Parkinson’s disease subjects (withdrawn from levodopa therapy for 12–24 h) under-estimated time intervals in a verbal estimation task and over-produced time intervals, suggesting the role of dopamine and nigrostriatal circuits in an “internal clock”. AnotherFigure 6), which are responsible for various functions depending on the cortical area involved, They include the motor movements (motor cortex), planning and working memory , and limbic functions  and a DOWN state .Striatum forms part of multisynaptic, cortical–striatal–pallidal–thalamic–cortical loops . This is indicated by the increased variations in timing tasks in Parkinson’s disease . The pacemaker activity has been observed in vivo and in vitro in deep cerebellar nuclei, forming the main output regions of the cerebellum . Furthermore, the changes that occur during a motor control are commonly in the sub-second range, which supports the role of the cerebellum in the sub-second timing.Past studies have shown that the brain uses networks involving the cerebellum for tasks involving sub-second intervals . In addirebellum , which mSince the processing of time intervals, in the absence of cues, does not involve any specific tasks, the calibration of clock mechanism will not be accomplished by a successful task completion. Instead, the mechanism for interval timing in the absence of cues would be linked to the sensory perception of the passage of time. This is based on the observed relationship between the sense of passage of time and the subjective estimation of time intervals. For example, a subjective report of the fast passage of time is often associated with the over-estimation of time intervals. The sensory perception of the flow of time is likely to be a consequence of the sensory detection of the temporal order, when there is a minimum temporal separation between adjacent multimodal or monomodal stimuli, for example by 20–30 s for auditory stimuli . During A potential mechanism of calibration in the absence of cues would be the feedback process based on the cortical–striatal–pallidal–thalamic–cortical loop. The anatomical evidence, supporting the role of cortical–striatal–pallidal–thalamic–cortical loop in the calibration of clock circuits comes from a study using the retrograde transneuronal transport of rabies viruses to examine the subcortical inputs to anterior intraparietal area in the posterior parietal cortex, which found labeling of a substantial number of neurons in the substantia pars reticulata , one of Sensory experience of external events involves simultaneity of perception of different modalities, which results from the polysensory processing in the parietal cortex, involving gain modulation . Since mFigure 5). The second-level oscillator is inbuilt within various circuits in the brain. The second-level oscillator (inbuilt) is in a binding state with oscillators in one of several calibration modules. The ability of an inbuilt oscillator to bind to the oscillators in different calibration modules is responsible for the modular nature of the clock. The effect of feedback and sensory processes on the oscillator in the calibration module would produce synchronous effects on various inbuilt oscillators due to the binding, which is proposed to lead to the representation of physical time in various brain circuits. Note that the external information can be also imported into neuronal circuits without a direct interaction with the external world  feedback processes involving motor movements, (b) binding between different circuits that result from the synchronous effects of the feedback processes or sensory processes, and (c) input of temporal periodicity resulting from a regular sensory stimulation. The calibration of the neuronal clocks involves a successful transfer of the temporal information from the external physical events into the neuronal circuits, via the successful execution of various tasks or functions. The transfer of external physical time information may sometimes involve two levels of oscillator circuits. For example, during motor tasks, there is a proposed involvement of two oscillators, which help in the transfer of external physical time information. The first-level oscillator is present in the calibration module, such as the cerebellum .Timing tasks are classified as (a) explicit interval timing, when subjects report duration by speaking, or performing delayed or prolonged motor task; and (b) implicit interval timing, when regular pattern of sensory stimuli or motor responses is used to predict non-temporal parameters, such as, velocity of object trying to land . ExpliciConsistent with the modular nature of the neuronal clocks, I propose that one or several calibration mechanisms would serve in a single clock mechanism. This feature of the modular clock model introduces redundancy in the clock mechanism of the brain. In individuals with the lesions of the cerebellum, other calibration mechanisms, mainly sensory, will take over the function of calibrating neuronal clocks. Since mechanisms based on the sensory processes calibrate or internalize time intervals of longer durations in comparison to the motor processes, the cerebellar lesions will result in greater variations during the processing of temporal intervals. Evidence for the preceding is provided by a study, which showed that the cerebellar lesions disrupted the precise timing, indicated by increased variations . FurtherThe discussion of different circuits in the current manuscript also indicates that the posterior parietal areas, pre-motor area and basal ganglia circuit  also are likely to serve as calibration modules in addition to the cerebellum. An earlier fMRI study had found significant activations in a cerebellar network during absolute, duration-based timing and a striato–thalamo–cortical network during relative, beat-based timing . Since pWhile the cerebellum is implicated in the sub-second interval timing tasks, the imaging and clinical data implicate the basal ganglia in both, the sub- as well as the supra-second interval timing tasks . A meta-Figure 5). This can be explained with the help of an example of a stochastic process, which assembles anticipatory time intervals with a random number of temporal units of a standard length, to produce the best match with a task requirement. During the production of time intervals with temporal units of a longer standard length by such process, one is likely to find an increase in variations. This type of random process may also explain the presence of greater variations in the production of time intervals of greater length, when a modular clock uses a different calibration mechanism, such as the sensory processes, instead of the cerebellar mechanisms, which is more suitable for the shorter time intervals. For similar reasons, the cerebellar lesion leads to an increase in the variability of temporal tasks (The scalar property of the interval timing, that is, the variations in interval timing increases with its increasing duration , may be al tasks . FollowiIn a recently published study, schizophrenic patients were found to underestimate anticipatory time intervals during the tracking of the movement of visual stimuli under cognitively challenging conditions . Common Emerging evidence indicates that the “correlated noise” between neurons and its structure help pools of neurons in guiding their behavior . AlthougThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Enrichment analysis is a widely applied procedure for shedding light on the molecular mechanisms and functions at the basis of phenotypes, for enlarging the dataset of possibly related genes/proteins and for helping interpretation and prioritization of newly determined variations. Several standard and Network-based enrichment methods are available. Both approaches rely on the annotations that characterize the genes/proteins included in the input set; network based ones also include in different ways physical and functional relationships among different genes or proteins that can be extracted from the available biological networks of interactions.Here we describe a novel procedure based on the extraction from the STRING interactome of sub-networks connecting proteins that share the same Gene Ontology(GO) terms for Biological Process (BP). Enrichment analysis is performed by mapping the protein set to be analyzed on the sub-networks, and then by collecting the corresponding annotations. We test the ability of our enrichment method in finding annotation terms disregarded by other enrichment methods available. We benchmarked 244 sets of proteins associated to different Mendelian diseases, according to the OMIM web resource. In 143 cases (58%), the network-based procedure extracts GO terms neglected by the standard method, and in 86 cases (35%), some of the newly enriched GO terms are not included in the set of annotations characterizing the input proteins. We present in detail six cases where our network-based enrichment provides an insight into the biological basis of the diseases, outperforming other freely available network-based methods.Considering a set of proteins in the context of their interaction network can help in better defining their functions. Our novel method exploits the information contained in the STRING database for building the minimal connecting network containing all the proteins annotated with the same GO term. The enrichment procedure is performed considering the GO-specific network modules and, when tested on the OMIM-derived benchmark sets, it is able to extract enrichment terms neglected by other methods. Our procedure is effective even when the size of the input protein set is small, requiring at least two input proteins. Next Generation Sequencing (NGS) technologies enable the discovery of large sets of genetic variations characterizing the individual variability. One common problem is to dig out variations potentially related to different phenotypes, including susceptibility to diseases. A widely adopted procedure relies on the extraction of functional information from sets of genes or proteins already associated to the phenotype under investigation: this procedure allows extending the set of genes or proteins potentially associated to the phenotype and can therefore be useful for prioritizing large sets of experimental variations detected with NGS experiments. Functional association is routinely performed by means of statistical enrichment analysis over a gene/protein set of interest  have emerged in the last few years . These nPINA  is a web resource based on the integration of six protein-protein interaction databases  .The human protein interaction network was downloaded from STRING  .The network-based enrichment relies on a preprocessing phase aimed at extracting modules starting from seed sets of proteins sharing the same GO BP annotation. By construction, a module is a compact and connected subgraph of the molecular-interaction network. Given a GO BP term (our reference GO term), the corresponding module contains all the proteins directly annotated with the same term in UniProtKB (seed nodes) and some of their interacting partners (connecting nodes). The module is determined by computing all the shortest paths among the seeds and by reducing the resulting network into the minimal connecting network preserving the distances among seeds. The minimal connecting network adds to the seeds a set of connecting nodes that are more reliably related to the reference GO term. The details of module extraction are given below and the algorithmic description is available in the Additional file The output of the pipeline consists of a non-redundant ranking of GO BP terms overrepresented in the input set, ranked according to their Bonferroni-corrected p-values. It is important to notice that with a standard enrichment only GO terms already associated to input proteins can result as overrepresented. On the contrary, the network-based enrichment allows to detect statistical associations with GO terms not included in the annotations of the input protein set. Such terms represent the added-value information of the network-based enrichment analysis.The module extraction is schematized in Figure We extract the sub-network of STRING consisting of all the shortest paths between the proteins in the seed set. Seed proteins not appearing in STRING are kept as isolated nodes in the shortest path network. For the shortest paths computation, we do not make use of the edge-scores provided in STRING, i.e. we treat STRING as an undirected and unweighted graph, without self-loops. The size of the shortest path networks extracted from STRING is usually large, even for relatively small input protein sets. On average, the shortest path networks extracted for the different GO BP terms contain 15 times more proteins than their seed sets.Due to the large number of retrieved connecting nodes, a minimization is applied to the shortest path network in order to simplify its topology and thus highlight its main structure. In particular, the computational goal of the procedure is to extract from the shortest path network, the smallest distance-preserving network, i.e. the smallest subgraph that preserves the shortest distances between the seed proteins. The minimization procedure is the most computationally expensive step of the module construction, as it closely resembles the Steiner tree problem ,38. Furtseed nodes (i.e. the nodes related to the seed proteins) and connecting nodes (i.e. the remaining nodes in the shortest path network).i) The nodes in the network are split into two disjoint groups: ii) The connecting nodes are ranked according to three predefined relevance criteria. Their description is detailed in the "Ranking scores" section.iii) The ranked list is iteratively processed starting from the least important node.iv) The currently evaluated node is removed from the shortest path network only if its deletion does not increase the shortest distance between any pair of seed nodes.In the current version, the ranking of a connecting node is obtained by applying three scores , which are used as primary, secondary and tertiary sort key, respectively.Seed centrality (sc). We say that a node connects two seed nodes if it appears in some shortest path connecting them. Thus, the seed centrality measure simply counts the number of distinct seed pairs connected by a node. This property implicitly assumes that the higher the number of seed pairs a node connects, the higher the probability that such node appears in a minimal connecting network.i) Maximum semantic similarity with the reference GO term (ss). The semantic similarity measures to which extent the annotation terms of each connecting node is related to the reference GO term: a connecting node with a high semantic similarity score is more likely to be functionally related to the seed nodes. The semantic similarity is defined as the Lin's information-theoretic metric [ii) c metric . In detac metric . The maxBetweenness centrality (bc). The betweenness centrality (with respect to the nodes in the seed set) is a measure of centrality of a node in a network [iii)  network . This prAs for the shortest path network, seed proteins not appearing in STRING are kept as isolated nodes in the minimal networks. Differently from the shortest path networks, the minimal connecting networks are quite compact. On the average, they contain only 1.5 times more proteins that their seed sets. One example of a shortest path network is provided in Figure A quality filtering procedure is applied to the minimal connecting networks built in the previous step. The idea is to filter out those networks for which the GO annotations of the connecting nodes are weakly related to the reference GO term. In particular, rare BP terms (i.e. BP terms with few related proteins) tend to produce minimal networks consisting uniquely of long paths. In most of such cases, the annotations of the connecting proteins are unrelated to the reference GO, and then the resulting minimal network is unlikely to include many proteins related to the reference GO. Such network-modules are discarded and not considered for the enrichment. The quality filtering procedure makes use of the maximum semantic similarity measure, as defined above. In particular, a minimal network is retained if, with respect to the reference GO term, the average maximum similarity of the connecting nodes is significantly higher than the average maximum similarity of all the nodes in STRING, as assessed by a Student's t-test with significance set to 5%. The quality test discharges 1,205 networks out of 12,621 .We also filter out minimal networks that do not contain any connecting node. The number of GO BP terms for which we extract a non-trivial network is then 8,098.In order to benchmark the method, we extracted from the OMIM web resource  a list oThe annotation pipeline retrieves enriched GO BP terms computed with a standard and a network-based procedure. Both are performed with Bonferroni-corrected Fisher tests, considering a significance level of 5%. We benchmarked on the OMIM-derived benchmark set the level of annotation added by the network-based method from both a quantitative and qualitative point of view. The quantitative analysis highlights the ability of the network-based method in recovering new enriched functions. The qualitative analysis focuses on six cases for which the newly enriched terms add new biological insights, as confirmed by previously published experimental data.For assessing the power of the network-based enrichment, we focus uniquely on GO BP terms that are not enriched by the standard method . Results are listed in Table The newly enriched terms that are absent in the original annotations of the input genes are likely to gain new knowledge on the disease at hand. We focus the qualitative analysis on them and we detail here six case studies for which experimental validations are available for the annotations derived with our method. For all the reported cases, PINA does not return any significant association. EnrichNet enriches only terms that are already included in the annotations of the input proteins. However EnrichNet is best suited to analyze sets including at least 10 proteins, while in our case studies, four out of six cases consist of input sets comprising two to four proteins.The disease is characterized by the increase of the red blood cell mass and hemoglobin concentration, and by hypersensitivity of erythrocyte progenitors (myeloid cells) to erythropoietin. The disease is associated to three genes  and the standard method enriches two terms related to histone phosphorylation. NET-GE adds three terms, reported in Table ADHD is a psychiatric disease related to the development of the nervous system in children and adolescents. It has been linked to variations in dopamine receptors HGNC: DRD4 and HGNC: DRD5. Standard enrichment is able to highlight the connection between the disorder and the dopaminergic pathway, including the second messenger intracellular pathway based on cAMP. It also enriches GO-terms related to psychiatric functions  and to the response to several compounds . As reported in Table Susceptibility to tobacco addiction has been linked to four proteins . Standard procedure enriches the processes related to intercellular signaling and to the response to nicotine and to alkaloids. Network-based enrichment (Table THPH1 is a disorder of impaired clot formation linked to four different proteins: the coagulation factor HGNC: F13A1, the prothrombin HGNC: F2, the methylenetetrahydrofolate reductase HGNC: MTHFR, and the hyaluronan-binding protein HGNC: HABP2. Standard enrichment only extracts a GPCR signaling pathway, while the network based procedure is able to correctly identify the main impaired process, namely the platelet aggregation (Table The susceptibility to myocardial infarction is linked to 12 different proteins (see file OMIM608446.pdf in Additional file Obesity is linked to 16 different proteins (see file OMIM 601665.pdf in Additional file We describe a novel computational method, NET-GE, for enrichment analysis, which exploits the information contained into molecular interaction networks. Given a set of input proteins, our method can detect functional associations not directly inferable from the annotations of the starting protein set, and thus not detectable through a standard enrichment. The method has been benchmarked on a set of 244 different Mendelian diseases associated to more than two proteins, as reported in the OMIM database. The lists of enriched terms for the benchmark examples are available in Additional file The authors declare they have no conflict of interests in relation to this VarISIG issue article.PDL, PLM, PF and RC planned the research and discussed the result; PDL implemented the computational method; PLM and RC analyzed the biological test sets. All the authors contributed to writing and correcting the paper.Details on the method implementation.Click here for fileFigure S1: Number of enriched GO BP terms as a function of the maximum degree of the human proteins annotated with a the same term.Click here for fileDetailed results for the OMIM-derived benchmark set. The archive contains pdf documents listing the enriched terms for each one of the 244 diseases in the OMIM-derived benchmark set.Click here for file


In this paper, we study the power saving mechanism of NRT-VBR and BE services for mobile handsets (MHs) to prolong their battery lifetime  in the fourth generation networks. Because the priority of NRT-VBR and BE is lower than that of real-time VBR (RT-VBR) or guaranteed bit rate (GBR) services, we investigate an extended sleep mode for lower priority services  in an MH to conserve the energy. The extended sleep mode is used when the MH wakes up from the sleep mode but it cannot obtain the bandwidth from base station (BS). The proposed mechanism, named  The fourth generation (4G) personal communication networks provide telecommunication and data access together for nomadic users by integrating telephone and computer networks into a single system extended sleep duration for energy conservation. In this paper, we present an MH PSM named extra power saving scheme (EPSS) to prolong the lifetime of the MH battery by avoiding those unnecessary and uncertain waiting time for available bandwidth. From the performance evaluation, we find that the proposed mechanism can achieve better power saving efficiency as compared with the 4G communication protocols when the traffic load is large.On the other hand, because the energy of the MH battery is limited, how to prolong the lifetime of the MH battery to support multiple services has become an valuable issue. Many power saving mechanisms (PSMs) have been broadly studied such as adaptive traffic coalescing (ATC) scheme The rest of this paper are organized as follows. A detailed description of the unnecessary energy wasting because MHs wait for available bandwidth in 4G systems and the solution of EPSS is given in Section. The analytical model of the proposed EPSS is presented to show how to obtain the estimated extended time and derive the energy consumption in Section. Section shows the validation of the analytical model and related numerical experiments. Finally, the conclusion is given in Section.Latest 4G communication techniques, the IEEE 802.16m In order to prolong the battery lifetime of an MH, the PSM turns off an MH radio interface when the MH is in idle duration. The IEEE 802.16m PSM provides three types of PSCs for different service types to conserve the battery energy. The PSC type I is used for NRT and BE traffic, PSC type II is used for unsolicited grant service (UGS) and RT-VBR traffic, and PSC type III is used for multicast and management traffic, respectively. Similarly, the LTE-A PSM uses discontinuous reception and discontinuous transmission (DRX/DTX) mechanism to conserve the battery energy in downlink and uplink connections, respectively. When an MH is in active mode, it can receive the packets from the BS regularly. When an MH is in sleep mode, it turns off the transceiver power and wakes up periodically in a period of time . In the listening window, the MH listens the message from the BS to confirm whether the BS has any packets to the MH. If the BS has any packets to the MH, it must inform the MH in the listening window to let the MH keep awake and receive the packet. If the MH does not receive a positive notification from the BS, it will go back to another sleep cycle.Based on preceding described PSM, when the MH is in sleep mode, the BS cannot communicate with the MH except in the pre-negotiated listening window. If the BS has packets to the MH, the BS has to send a positive notification to inform the MH. Because the positive notification does not include the information of the bandwidth allocation message, the MH must keep awake until it receives the downlink bandwidth allocation information. This leads the MH to take more unnecessary energy consumption to wait, especially the scheduler cannot allocate available bandwidth for the nrtPS/non-GBR transmission. We find that both of IEEE 802.16 and LTE-A do not have effective solution for this problem. According to the aforementioned increasing trend of nrtPS/non-GBR services in Section, this problem will become more series in the future.As mentioned in Section and Subsection, when an MH receives a positive notification in the listening window, the MH keeps awake to wait for the downlink bandwidth allocation message to receive upcoming packets. To alleviate the energy wasting of MHs in case the downlink bandwidth are not available, EPSS modifies the positive notification message  mechanism are treated as sessions. In order to increase bandwidth utilization, the number of allowed sessions could be greater than the number of total resource spaces per frame, i.e., The system is modeled as a one BS to The PSM of EPSS is modeled as the Markov chain model An MH wakes up periodically to listen AAI_TRF-IND during In the state transition diagram, all the states are represented as a set Erlang-B formulaIn EPSS, the MH will enter shown in ). As menflow balance property of steady state in Markov chain of EPSS, we have Since the transition probability from is  (see ), the stThe sleep mode probability can be obtained by summing up all probabilities of sleep mode states, then according to (5), we can obtain Following the steady state property, in state Solving To solve Substituting (6) and (8), into (9) leads to Then Substitute (11) into (8) and (5), we can obtain the steady state probability The energy consumption can be derived by using preceding obtained probabilities In comparison with EPSS, we use the similar model and obtain the steady state probability of the legacy IEEE 802.16 PSM In a similar way, we can substitute the probability The delay of access network bandwidth is an important metric of performance. In order to estimate  system.  shows thAccording to the process, the steady-state probabilities To solve Based on the derivation of Let Substituting (17) into (18), we get Because the downlink transmission arrival rate of nrt-PS to the BS is From (20), the BS can estimate the expected time duration The simulator is developed by the authors to evaluate the energy consumption of the legacy IEEE 802.16 PSM and EPSS. The simulation model is based on all system assumptions presented in Section. There are The transmission of arrival in a session is assumed exponentially distributed with mean rate To study the effect of the power saving achieved by EPSS, we investigate the relationship between Other system parameters are set as follows: the initial sleep window shown in . The solshown in  that theIn this subsection, we use some numerical examples to illustrate the effects of the number of MHs  illustra , and .  shows thIn contrast, initially e than .  shows th shows th addition plots  a served.  shows th shows thmple, in (a), EPSStrast to (c), [Figtrast to (d) showsAnother affected result, the mean downlink transmission delay shown in . As we cen  (see ) because illustraIn the following, we present a realistic traffic study of buffered video streaming and web browsing to investigate the effect of EPSS on energy conservation. The traffic model refers to the statistical results in The buffered video streaming traffic we consider here is the behavior of people watching the YouTube video clip. The resolution of video clip is 480p  has at least a resource block quota for service when The simulation results are shown in . Figure shows thsaw from , EPSS ca demonstrle load ((a)), theshown in (b), EPSSof EPSS. (c) showsextra power saving scheme (EPSS), to let MHs enter the extended sleep mode for extra power saving when the downlink traffic load is saturated. We show the impact of EPSS on energy consumption In this paper, we invent a new power saving scheme, called


Paracentrotus lividus, the Hbox12 homeodomain-containing repressor is expressed by prospective dorsal cells, spatially facing and preceding the onset of nodal transcription. We report that Hbox12 misexpression provokes DV abnormalities, attenuating nodal and nodal-dependent transcription. Reciprocally, impairing hbox12 function disrupts DV polarity by allowing ectopic expression of nodal. Clonal loss-of-function, inflicted by blastomere transplantation or gene-transfer assays, highlights that DV polarization requires Hbox12 action in dorsal cells. Remarkably, the localized knock-down of nodal restores DV polarity of embryos lacking hbox12 function. Finally, we show that hbox12 is a dorsal-specific negative modulator of the p38-MAPK activity, which is required for nodal expression. Altogether, our results suggest that Hbox12 function is essential for proper positioning of the DV organizer.Dorsal/ventral (DV) patterning of the sea urchin embryo relies on a ventrally-localized organizer expressing Nodal, a pivotal regulator of the DV gene regulatory network. However, the inceptive mechanisms imposing the symmetry-breaking are incompletely understood. In DOI:http://dx.doi.org/10.7554/eLife.04664.001 Embryos begin as a collection of identical cells. As the embryo develops further, the cells in different regions must take on different structures and roles in order to form the complex tissues and organs seen in the fully developed organism. Therefore, a key task in early development is to inform cells where they are in a developing embryo. Signaling proteins released by special groups of organizing cells are responsible for providing the information about where a cell is located. Networks of genes controlled by these proteins then inform embryonic cells of where they are and what they should, or should not, become.One such signaling protein is called Nodal, and is needed to perform a number of tasks in the developing embryo, including helping to form the basic tissues of the organism. Many animals depend on Nodal to develop correctly—from mice and humans, to zebrafish and sea urchins.During sea urchin development, Nodal establishes where the mouth of a larva forms, setting up what is called the dorsal/ventral axis of the embryo; this separates the front and back of the embryo. To do so, the Nodal protein is mostly produced at the front of the embryo. Although much is already known about the network of genes that the Nodal protein controls, the genes and proteins that ensure that the initial source of Nodal is present at the right time and place are largely unknown.hbox12 gene function is impaired, the Nodal protein is produced in both the front and the back sections of the embryo. Conversely, if Hbox12 is introduced into regions where Nodal is present, the amount of Nodal decreases. Furthermore, disrupting Hbox12 prevents any signs of the dorsal/ventral axis forming.Another protein called Hbox12 was also thought to be important for setting up the dorsal/ventral axis. Now, Cavalieri and Spinelli reveal that Hbox12 regulates Nodal during the development of a sea urchin embryo. In the early developing sea urchin, the gene that produces Hbox12 is activated in the region of the embryo that will become its back, directly opposite where Nodal is present. This activation normally occurs just before the gene that produces Nodal is turned on. If the nodal gene. By doing so, Hbox12 sets up the dorsal/ventral axis by restricting Nodal to the cells that will make up the front half of the embryo.Cavalieri and Spinelli propose that Hbox12 inhibits the production of Nodal by briefly inactivating another protein that is required to activate the Most complex organisms have asymmetric bodies, and failure to establish these body asymmetries can result in disease and other disorders in humans. Deciphering how the dorsal/ventral asymmetry in the sea urchin embryo is established should improve our understanding of how the mechanisms that form body shapes have evolved.DOI:http://dx.doi.org/10.7554/eLife.04664.002 Patterning of the embryonic ectoderm along the dorsal/ventral (DV) axis, also known as oral/aboral axis, has been extensively studied in various species of sea urchins. DV polarity is not firmly established in the unfertilized egg, but rather relies on a combination of inherited maternal information and inductive interactions among early blastomeres, becoming morphologically recognizable from the gastrula stage onward . The ectThe genetic landmark of polarization along the secondary axis is the zygotic expression of the TGF-β superfamily member Nodal on the future oral side, which behaves as an organizing centre imposing DV polarity in all three germ layers of the embryo . Targetsnodal expression is growing rapidly , the embryos developed normally  as well as the aboral ectoderm marker tbx2/3 was also reduced , the distribution of PMCs into the blastocoel was disorganized  lineage tracer and allowed to develop up to the 16-cell stage. Then, animal and vegetal halves from these embryos were separated and recombined with their complementary halves derived from control uninjected embryos  developed into larvae that were indistinguishable from controls  appeared to be constituted by a uniformly thickened epithelium and no discernable ciliary band was identified (msp130 probe (hd-injected embryos (28%) showed a slightly less severe phenotype , which endomesodermal territories appeared red-fluorescent . At this stage, all the n > 500; . By contpression .phbox12-HD-GFP-injected embryos showed rather severe radialization coupled to the presence of multiple ectopic spicules . This number did not differ from that observed in control uninjected plutei. In striking contrast, pigment cell population was greatly decreased in more than one half of phbox12-HD-GFP injected embryos observed at the same stage. In particular, specimens showing the milder phenotype differentiated less than 16 pigment cells   tracer. As expected, embryos injected with the hd-GFP mRNA and observed at the hatching blastula stage showed a diffused green fluorescence, indicating that the synthetic transcript was efficiently translated throughout the early embryo   was succy embryo . Since, hd-GFP mRNA or Mo-nodal alone, and observed at 48 hpf. Once again, the former embryos exhibited a typically ventralized phenotype  developed into normal pluteus larvae with a harmoniously patterned DV axis .According to current models, the activation of p38 MAPK . As mentpression . By thismplished , and thehd mRNA, efficiently supplanted the p38 asymmetry, as shown by the uniform nuclear GFP staining in roughly 80% (n > 250) of the resulting embryos observed at the 60-cell stage .It should be emphasized that at the 60-cell stage p38 is normally activated in a sector encompassing about 250° . On the nodal in hd-injected and hbox12 overexpressing embryos, respectively. Most importantly, our findings also strongly support the hypothesis that Hbox12 defines the future dorsal side of the embryo by transient inactivation of p38 activity at a key time point, thereby allowing the asymmetric expression of nodal on the opposite side.To sum up, these results very well correlate with either the ectopic or reduced expression of During early development of bilaterian embryos symmetry breaking is imposed through establishing of distinct polarities, which are precursors of the larval axes. Although polarization is morphologically not apparent in the zygote, fates of different embryonic regions are patterned on the axial coordinate system by a combination of maternally inherited factors and differential zygotic gene expression .nodal by the early blastula stage and behaves as a DV organizing centre. Production of the Nodal ligand by these cells is pivotal for both defining of their ventral identity and accomplishing the DV patterning program . After surgery, the embryos were returned to regular sea water and reared until the desired stage.Recombination of animal and vegetal halves were carried out as described . BrieflyTransplanted and non-chimeric injected embryos at the desired stage were harvested, mounted on glass slides and examined under a Leica DM-4500B upright fluorescent microscope. Digital images were captured and processed using Adobe Photoshop CS6.cytochrome oxidase or the mbf1 mRNA, which are known to be expressed at a constant level during development  or BCIP (for the fluo-probe) as chromogenic substrate.Chromogenic whole mount in situ hybridization procedure was performed as described , with Dirocedure . In this review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  axis of the sea urchin embryo. This manuscript presents generally compelling experimental evidence that the sea urchin Hbox12 gene encodes a transcriptional repressor whose early expression in the dorsal animal domain of the embryo is required for the proper dorsal-ventral patterning of the embryo by restricting Nodal to the ventral domain. The work contributes to our understanding of early symmetry breaking events in embryonic development and because Nodal has an important role in patterning of other deuterostome embryos this study should be of interest to the broader research community. However there are several important points which need to be addressed in a revised version of the paper before making a final decision on acceptance for publication.hbox12 or hd-En mRNA caused a dose-dependent attenuation in the level of nodal transcript at a very similar extent”. However, as mentioned in 1) The morphological evidence presented strongly supports the conclusions. The analysis of gene expression is somewhat less compelling. It is stated “microinjection of equal amounts of either 2) It is stated ”We conclude that in the double hd-GFP and Mo-nodal injected embryos, clonal knock-down of nodal was able to restore the asymmetrical production of the Nodal ligand, which in turn sufficed to resume the entire DV axis.” While this is a reasonable interpretation, no direct evidence was presented for the abundance or localization of the Nodal  in this experiment, so the statement is conjectural and should be modified.hbox12 gene is annotated in the S. purpuratus genome as pmar1 and has been extensively characterized by Oliveri and Davidson. Pmar1 is expressed in micromeres and functions as a critical component of an endomesodermal specification regulatory network. This paper is essentially proposing that there is a radical difference in the function of this gene in two closely related species of urchin. The authors need to make an attempt to provide a reasonable explanation for what many would consider a contradiction. Hence, address a rationale for what appear to be differences in fundamental molecular patterning mechanisms in closely related species.3) The hbox12. An underlying assumption of this paper is that expression is restricted to presumptive dorsal ectoderm. The cis-regulatory DNA drives mosaic expression of GFP in dorsal ectoderm, as previously reported, but a conventional in situ hybridization or antibody staining would be reassuring to clearly demonstrate that there is a sound basis for the assumed localization of transcription.4) There is a need to see clear data on the localization of the expression of 5) General point, it is difficult to come to the conclusions that the authors report without analyzing the distribution of the tissue specific genes that have only been quantified by qPCR; it is essential to have both. Conventional loss of function experiments are not provided data, presumably because there are multiple genes. The RNA overexpression only partly overcomes this problem and the overexpression phenotype is not fully described. For instance, there is a description in the text, and a figure , but the6) Specific expression analysis this would help other parts of the paper as well, for instance in the Results section, the defects are not clearly dorsal-ventral polarity and tissue specific markers should be used to establish this. Similarly the authors assert a dorsal-ventral defect without demonstrating alterations in the distribution of tissue markers.7) A minor point, a curious outcome of the experiments is the effects on the pigment cell lineage. As this lineage arises from mesoderm, the effect is not fully explained. A clear demonstration that the early commitment of macromere derived cells is the proximal effect by using tissue specific gene probes; the standard in the field would be valuable. The loss of pigment and the suppression of GCM could come about in several ways: one cannot assume it is at the level of specification. 1) The morphological evidence presented strongly supports the conclusions. The analysis of gene expression is somewhat less compelling. It is stated “microinjection of equal amounts of either hbox12 or hd-En mRNA caused a dose-dependent attenuation in the level of nodal transcript at a very similar extent”. However, as mentioned in(but not in the Results or Discussion), the reduction in nodal transcript was actually less than significant in the quantitative PCR assay used. The authors reasonably point out that this may be because many of the embryos injected with these mRNAs ultimately did not show loss of dorsal-ventral patterning. This lack of consistency in injected embryos is of some concern and somewhat unusual. The lack of significance  should be made clear in the text (and in the legend towhich mentions the gray area but not the lack of significance).As far as qPCR data is concerned, we would like to emphasize that the 3-fold significance threshold does not correspond to a technical limit of our experimental conditions. Rather, it is the criterion proposed by Davidson  and widely adopted by the rest of the sea urchin community. However, less than 3-fold difference could be significant when strengthened by WMISH results, and this is exactly our case.hbox12-injected embryos developed as aberrant larvae , we predicted that the qPCR data on nodal, gsc and tbx2/3 expression would refer to an heterogeneous population of embryos (affected and not affected).Since only about half of the hbox12- injected embryos at the mesenchyme blastula stage, in which DV polarity is not yet morphologically noticeable. In close agreement with our hypothesis, a significant fraction (∼47%) of hbox12-injected embryos showed reduced expression of the mentioned genes. Most importantly, such a fraction was reasonably congruent with that of embryos that ultimately displayed unambiguous defects in DV polarization at later stages.Thus, we performed WMISH on control- and These results were included in the current version of the manuscript (in the Results section and 2) It is stated “We conclude that in the double hd-GFP and Mo-nodal injected embryos, clonal knock-down of nodal was able to restore the asymmetrical production of the Nodal ligand, which in turn sufficed to resume the entire DV axis.” While this is a reasonable interpretation, no direct evidence was presented for the abundance or localization of the Nodal  in this experiment, so the statement is conjectural and should be modified.As suggested, the statement has been modified.3) The hbox12 gene is annotated in the S. purpuratus genome as pmar1 and has been extensively characterized by Oliveri and Davidson. Pmar1 is expressed in micromeres and functions as a critical component of an endomesodermal specification regulatory network. This paper is essentially proposing that there is a radical difference in the function of this gene in two closely related species of urchin. The authors need to make an attempt to provide a reasonable explanation for what many would consider a contradiction. Hence, address a rationale for what appear to be differences in fundamental molecular patterning mechanisms in closely related species.pmar1 nor that hbox12 and pmar1 are orthologous genes. In fact, we believe that hbox12 and pmar1 are indeed distinct genes playing distinct roles in two evolutionary distant species of sea urchins. To meet the Referee’s request, we have included a paragraph in the Discussion section to explain this point.In the former version of the submitted manuscript we did not mention neither 4) There is a need to see clear data on the localization of the expression of hbox12. An underlying assumption of this paper is that expression is restricted to presumptive dorsal ectoderm. The cis-regulatory DNA drives mosaic expression of GFP in dorsal ectoderm, as previously reported, but a conventional in situ hybridization or antibody staining would be reassuring to clearly demonstrate that there is a sound basis for the assumed localization of transcription.hbox12 was not arbitrarily assumed, but rather derived from previous results based on in situ hybridization  and cis-regulatory analysis .The dorsal-specific expression of hbox12 transcripts, we performed double-staining WMISH with probes for hbox12 and nodal . As clearly shown in To definitively confirm the dorsal localization of 5) General point, it is difficult to come to the conclusions that the authors report without analyzing the distribution of the tissue specific genes that have only been quantified by qPCR; it is essential to have both. Conventional loss of function experiments are not provided data, presumably because there are multiple genes. The RNA overexpression only partly overcomes this problem and the overexpression phenotype is not fully described. For instance, there is a description in the text, and a figure , but the only substantiation of changes in tissue distribution is a low quality in situ RNA hybridization for a mesenchyme gene. If the contention is that there is a change in the distribution of ectodermal domains, this really should be substantiated with tissue specific gene or antibody localizations with specific markers.As suggested, we answered to this point by performing WMISH with specific probes for ectodermal domains and implemented the current version of the manuscript accordingly .6) Specific expression analysis this would help other parts of the paper as well, for instance in the Results section, the defects are not clearly dorsal-ventral polarity and tissue specific markers should be used to establish this. Similarly the authors assert a dorsal-ventral defect without demonstrating alterations in the distribution of tissue markers.P. lividus reproductive cycle starts with the New Year but eggs are not suitable for microinjection and/or micromanipulation before the end of March. This would delay the publication of our paper quite a lot.This point refers to the results derived from the transplanted chimeric embryos. We are well aware that the suggested additional analysis of tissue markers in chimeras would improve our paper but, unfortunately, we face two types of difficulties that make these experiments quite unfeasible, at least in this period of the year. This is because currently we are not in the spawning season. In Sicily, the In addition, there is also a severe adverse technical limitation related to the fact that several tens of micro-injection/manipulation sessions would be required in order to obtain relevant numbers of chimeras to be processed for in situ hybridization with various probes. This would introduce a high extent of variability due to mixing different batches of embryos (in our hands the number of chimeric embryos is about ten for each experiment), that might hinder the interpretation of the results.On the other hand, we would like to underlie that, in our opinion, the morphological evidence presented fit well with all the other results and support our conclusions.7) A minor point, a curious outcome of the experiments is the effects on the pigment cell lineage. As this lineage arises from mesoderm, the effect is not fully explained. A clear demonstration that the early commitment of macromere derived cells is the proximal effect by using tissue specific gene probes; the standard in the field would be valuable. The loss of pigment and the suppression of GCM could come about in several ways: one cannot assume it is at the level of specification.gcm suppression in HD-expressing embryos may not necessarily reflect a failure in the specification of secondary mesenchyme cells. However, this appear to us the most plausible explanation essentially because hd-injected embryos are subjected to overexpression of nodal, and it is commonly accepted that Nodal signaling antagonizes the specification of pigment cells in unperturbed embryos. In agreement to this notion, it has been demonstrated by other authors that ubiquitous expression of nodal mRNA provokes loss of gcm transcription in the resulting albino embryos . A circumstantiated explanation has been included in the Results section.We fully agree with the concept that loss of pigment and


MicroRNAs (miRNAs) are important regulators of gene expression and like any other gene, their coding sequences are subject to genetic variation. Variants in miRNA genes can have profound effects on miRNA functionality at all levels, including miRNA transcription, maturation, and target specificity, and as such they can also contribute to disease. The impact of variants in miRNA genes is the focus of the present review. To put these effects into context, we first discuss the requirements of miRNA transcripts for maturation. In the last part an overview of available databases and tools and experimental approaches to investigate miRNA variants related to human disease is presented. This miThis review provides an overview of the current knowledge of the influence of genetic variants on miRNA biogenesis and function. First, miRNA biogenesis and requirements of the miRNA processing enzymes are described, followed by a discussion of isomiRs and their functional implications. In the second part, we highlight the effect of genetic variants on the expression and functioning of miRNAs. In the last part, approaches that are used to identify miRNAs involved in diseases are discussed, with a focus on genetic strategies. Furthermore, we present available tools, databases, and experimental approaches to aid functional characterization studies of disease-associated miRNA variants.miRNA genes can be classified in several categories according to their location in the genome. A majority of the currently known human miRNA genes deposited in miRBase are intergenic (68%). Of the intragenic miRNAs, most are intronic . The remaining genes are located in repeats, lncRNAs, UTRs, or coding regions of host genes . miRNA gIn the canonical biogenesis pathway, primary miRNA transcripts (pri-miRNAs) are cleaved in the nucleus by the Microprocessor complex, a complex consisting of ribonuclease Drosha and its cofactor DGCR8, to release the shorter precursor miRNA (pre-miRNA). This precursor molecule is transported to the cytosol by the nucleocytoplasmic transport protein Exportin-5, where it is cleaved by the ribonuclease Dicer to result in a mature miRNA duplex consisting of the mature 5p and 3p strands. The duplex is loaded onto an Argonaute protein, the core component of the RNA induced silencing complex (RISC). One of the strands is discarded, while the other mediates posttranscriptional regulation by base pairing to target mRNAs.There are different types of target sites. Canonical sites for miRNA targeting are seed matches (nt 2–7 of the mature miRNA) with an adenine opposite from the first nt of the mature miRNA and/or complementarity to the eighth nt of the miRNA. This seed match can be supplemented with complementarity to nt 13–16 (3′ supplementary sites). Extended sites of complementarity at the 3′ end of the miRNA can also compensate for seed mismatches (3′ compensatory sites) . Though Mirtrons, miRNAs encoded within short introns of host genes, bypass processing by the Microprocessor complex. By splicing the mirtron is released and can be, if needed after 5′ or 3′ tail trimming, transported to the cytosol to be further processed by Dicer. In the study of Drosha, a nuclear ribonuclease, is the enzyme that cleaves the miRNA hairpin from the primary transcript, whereby it generates a precursor with 3′ end overhangs of 2 nt . An esseIn vitro cleavage assays showed that Drosha was able to process pri-miR-30a and pri-miR-16-1 with as little as ~20–25 nt of flanking regions , consisting of an upper stem  and a lower stem (~11 bp), a terminal loop region and flanking segments that are usually single-stranded or have large bulges or internal loops . Essenti regions . Howeverrequired . The prerequired . These pTwo different models have been proposed to explain how Drosha determines where to cleave the pri-miRNA. One study proposed that the Microprocessor mainly determines the distance from the terminal loop region of the hairpin and cleaves ~22 bp upstream . AnotherNext to the structural prerequisites of the hairpin, position-specific sequence motifs present in the hairpin flanking segments of the primary transcript  and in the loop region (UGU or GUG motif) can enhance processing efficiency in human cells. Nearly 80% of the human pri-miRNAs that are conserved between human and mice contain at least one of these motifs .The transport of pre-miRNAs from the nucleus to the cytosol is mediated by Exportin-5 in the presence of cofactor RanGTP . The RNADicer, a ribonuclease that processes dsRNA into duplexes with a length of ~22 nt, processes pre-miRNAs into mature miRNA duplexes . Dicer cThe mature miRNA duplex is loaded onto an Argonaute protein by the RISC loading complex. This complex matures to RISC when one of the strands, termed the passenger strand, is removed . The othIn addition to the biogenesis key proteins described above, a number of other proteins are known to be involved in the regulation of miRNA biogenesis. The following examples highlight the relevance of the terminal loop region in this regulation. The first example is the protein KSRP. This protein binds directly to the terminal loop of a set of miRNAs and enhances their processing by Drosha and Dicer . Anotherin vivo, with sequences and lengths closely related to, but different from the canonical miRNA sequence reported in miRBase. Such alternative sequences are termed isomiRs . Sequence variability in miRNA genes can therefore influence both the expression level as the functionality of the miRNA and consequently will result in differential regulation of their target genes.in silico analyses of single nucleotide polymorphisms (SNPs) in human miRNA genes have demonstrated that miRNA genes have lower SNP densities than their flanking regions or the human genome  or of the polycistron (clustered miRNAs) could result in aberrant expression patterns. Next, variants within the miRNA transcript can have an effect on miRNA maturation in multiple aspects. They can change the binding affinity of the miRNA hairpin to biogenesis enzymes or accessory proteins . Variants can lead to altered processing accuracy or to changed frequency of alternative cleavage sites of biogenesis enzymes. They can also lead to altered strand loading bias into RISC. These changes in maturation can all result in altered expression of the canonical miRNA and its existing isomiRs, resulting in deregulation of target genes. It may also result in production of novel isomiRs, which can lead to altered functionality of the miRNA. Lastly, genetic variants within the mature sequence can affect target specificity by generating isomiRs.Given the potentially huge impact of genetic variants on the tightly regulated miRNA repertoire, and the importance of miRNA-mediated gene regulation, it is not surprising that genetic variants have been found to be causal for or associated with human diseases . VariantThough bio-informatics approaches have been used to predict miRNA gene promoters , many miSNP rs57095329 is located in the promoter region of MIR146A, 17 kb upstream of the pre-miR-146a sequence. This variant was found to be associated with systemic lupus erythematosus (SLE). The risk allele reduces the binding to transcription factor Ets-1, another SLE susceptibility gene, and results in decreased promoter activity. Consistent with this mode of action, risk allele carriers have lower expression of miR-146a-5p .SNP rs999885 is located in the promoter region of the protein-coding gene MCM7. This variant was associated with a decreased risk of chronic hepatitis B infection, but also with an increased risk of hepatocellular carcinoma (HCC) in individuals with chronic hepatitis B virus infection. The miR-106b-25 cluster, coding for miR-106b, miR-93, and miR-25, is located in the 13th intron of MCM7. HCC patients that carry the risk allele have higher expression of the miRNA transcript in non-tumor liver tissue than non-carriers . RecentlTo the best of our knowledge, variants in splice sites that affect miRNA expression in human disease have not been published yet. Nevertheless, the effect of host gene splice variants on mature miRNA expression has been demonstrated by mutagenesis experiments .Variants within the pri-miRNA can affect miRNA expression levels by inducing changes in the maturation process, as described above. This effect can take place at several steps in the maturation pathway: Drosha processing, Dicer processing, and/or altering strand preference.in vitro processing assay (An example of a variant with and effect on the Drosha processing step is rs2910164. This variant is located in the seed sequence of miR-146a-3p and was predicted to lead to a mismatch in the hairpin stem. The heterozygous variant genotype was associated with increased risk of papillary thyroid carcinoma (PTC). After overexpression of pri-miR-146a with the G allele or the C allele in cells, the C allele resulted in nearly twofold reduction of pre-miR-146a compared to the G allele. The reduced production of pre-miR-146a from pri-miR-146a with a C allele was also confirmed by an ng assay . An examng assay .A variant affecting Dicer processing is rs546098287, located within the seed sequence of miR-96-3p. This variant was identified as the causative mutation in an Italian family with non-syndromic hearing loss . Two segAn example of a variant influencing the relative strand abundance is a variant in MIR133A2, identified in a patient with atrial fibrillation. The variant is located at the 3′ end of the miR-133a-3p sequence and was predicted to alter the secondary structure of the hairpin at the base of the miR-133a duplex. miR-133a was found to be highly expressed in atrial tissue, with the 3p strand being the dominant one, while miR-133a-5p represented less than 1% of all the miR-133a reads. Functional validation experiments demonstrated that the variant resulted in increased miR-133a-5p expression, but had no significant effect on miR-133a-3p expression. The variant thus increases the relative abundance of the 5p strand compared to the 3p strand .The cases described above are all variants located either within the precursor sequence or close to it. A study by Beside resulting in altered expression, miRNA variants can also result in altered target specificity by creating isomiRs.An interesting example of a variant creating a polymorphic isomiR is rs2910164 in miR-146a-3p. This variant also affects Drosha processing as described above . The isoDirect evidence of altered targeting was shown in a study investigating the effect of eight seed SNPs and three mature sequence SNPs (among which one deletion) identified in dbSNP in nine miRNA genes. Four of the tested miRNAs  partially or completely lost their potential to suppress their original target due to a SNP in their seed sequence. The seed sequence variant rs2620381 in miR-627-5p not only resulted in loss of targeting of SEMA3F, but also in gained capacity to suppress ATP6V0E1 . In addiTo study if and which miRNAs play a role in the pathogenesis of a disease, two main strategies can be used: a miRNA profiling approach and a genetic approach.In the miRNA profiling approach, the affected tissue of a group of patients is subjected to miRNA expression profiling and compared to the expression profile of either the same tissue in healthy individuals, tissue of patients with a different disease or subphenotype, or to adjacent non-affected tissue of the same patients . The tesIn the genetic approach, the starting point is a linkage or an association analysis (genome-wide or candidate gene approach) including but not necessarily limited to miRNA genes between patients and control individuals . When a Both strategies have limitations and benefits. General considerations regarding miRNA profiling were reviewed by The main advantage of a genetic approach is that when a variant is associated with disease risk, it gives an indication that the miRNA involved has a primary effect in the disease pathomechanism , which cannot be determined by expression profiling. This advantage also has a downside: while the association or linkage analysis may indicate a miRNA variant is likely to be involved in the disease, it is not at all clear whether the identified variant has a functional effect and whether the expression of that miRNA is altered in the disease state. Another asset of the genetic approach is that diseases for which the main affected tissue is not accessible can still be studied because genomic DNA can be isolated from more accessible patient material, such as blood or saliva. There is, however, still a chance of missing a mutation, if the patient is mosaic for the mutation and the load of the mutation is lower in the examined tissue than in the affected tissue.Both approaches are complementary and should both be used to determine the involvement of a miRNA in relation to a disease: genetic approaches can pinpoint the causal or risk factors involved in the disease, while expression studies reveal whether and how the expression of the disease-associated miRNAs is altered in the affected tissue. Neither approach is sufficient to determine both. An example of a study combining both approaches is the study of in silico approaches. In this section, we will focus on available databases and tools for the impact prediction of genetic variants on miRNA function (Tables 1 and 2). Different types of resources for miRNA research have been reviewed by To provide direct evidence that a genetic variant in or near a miRNA gene and the deregulated miRNA expression in disease tissue are linked, detailed functional validation is required. Functional validation is labor-intensive, costly, and time-consuming, therefore researchers usually prioritize the potential candidate variants using A first step in the assessment of the relevance of a genetic variant or a set of genetic variants is determining whether the variant is located near or in a specific region of a miRNA gene. For a single variant of interest, the variant location may be assessed in a genome browser to determine the position of the variant compared to neighboring miRNA genes. To evaluate the exact location of the variant relative to the different miRNA gene regions , one also has to compare this location with the information provided about the gene in miRBase . This pr1. Required input is the genomic location of a (known or novel) variant or of a list of variants.Online repositories compiling information about known SNPs in miRNA genes, such as miRNASNP , miRNA S1 automates this type of analysis, predicting structural impact for lists of variants, needing only the genomic location and alternative allele of the variant.The next possible step in determining the potential relevance of a genetic variant is predicting its effect on the secondary structure of the miRNA. For known SNPs within miRNA genes, structure prediction results of the miRNA hairpin with and without the variant can be found in the miRNASNP database . The difin silico. Both PolymiRTS database  into cell lines to assess whether there is a difference in promoter activity due to the variant and whether the variant promoter has different binding affinity to transcription factors .To assess the impact of a genetic variant on miRNA biogenesis and/or targeting, cell lines overexpressing wild type and mutant miRNAs are usually established. The constructs should include the full precursor gene and upstream and downstream flanking sequences. The inclusion of flanking regions is crucial: the biogenesis machinery in the cell may not efficiently recognize or process the transcript when the flanking regions are absent or too small, as evidenced by the study of If a variant is located in the mature miRNA sequence, targeting may be affected. Investigating which genes and pathways are affected due to the variant may provide insight in the disease mechanism by identifying new pathways and/or by confirming pathways previously hypothesized to be involved in the disease. Investigation and validation of direct interactions between miRNA and target RNAs can be done using reporter assays. Hereto, the potential binding site is cloned downstream of a reporter gene and cells are cotransfected with the miRNA and the reporter vector to assess whether the miRNA (or the variant miRNA) can target the site and thus reduce the reporter gene activity . While tInvestigation of variant induced targeting alterations can also be performed on a large scale by subjecting the wild type and variant miRNA expressing cells to transcriptome and/or proteome profiling. Single genes or proteins can be analyzed for differential expression. Pathway analysis can also identify which set of genes are influenced by the variant miRNA and may lead to biological meaningful findings about the variant miRNA and the pathways in the cell it affects in the disease state . If a vain silico target prediction is required to assess the interactions between the identified RNA sequences. These approaches thus provide enrichment for actual target RNA sites bound to Argonaute and limits the target prediction to these sites and to the detected miRNAs.Different crosslinking and immunoprecipitation (CLIP) approaches were developed and applied for global miRNA–RNA target interaction identification by Argonaute precipitation, such as HITS-CLIP and PAR-CLIP . In thesA method that can identify direct miRNA-target interactions on a large scale is the crosslinking, ligation, and sequencing of hybrids (CLASH) technology . SimilarTogether, these approaches can be used to assess what the effect of the genetic variant is on the expression and the functionality of the miRNA.in silico and experimental approaches to investigate the effect of these variants on miRNA expression and function. This in turn could then be correlated to changes in miRNA and target gene abundance in the affected tissue of patients, as a first step in the understanding of the role of the miRNA in the disease pathomechanism.In this review we summarized the current knowledge of the miRNA maturation process, focusing on the prerequisites of the miRNA transcript to be processed. From these requirements it is clear that the RNA structure, and thus also the underlying sequence determining the structure, are of paramount importance for maturation into mature miRNAs. Thus genetic variants in miRNA genes can have large effects, not only on the miRNA gene itself, but due to its role as a fine-tuner of gene expression and translation, also on its downstream targets. In line with this, different miRNA variants have been found to be involved or hypothesized to be involved in human disease. We also highlighted the biological relevance of genetic variants located within or near miRNA genes and provided an overview of As we are now coming to realize that the miRNA repertoire is much more complex than previously appreciated with the recognition of the wide-spread presence of isomiRs and their functional relevance, the possibilities of intronic miRNAs being transcribed independently from their host genes and even alternative splicing, many more questions arise. Though many aspects of miRNA biogenesis and functioning have already been unraveled, more investigations will need to be done to be able to fully grasp the regulation and the genesis of the correct expression pattern of the full miRNA repertoire in the cell. We also anticipate that, especially for the investigation of genetic variants in disease, we will need to undertake comprehensive and integrative approaches to be able to fully appreciate how certain genetic variants can undermine this regulatory system and lead to disease, while others remain completely harmless.All authors contributed to the conception of the work. SC wrote the manuscript. MS, PD, and JD provided input and revised the manuscript. 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.


There are a number of errors in the legend for There is an error in the sixth sentence of the fifth paragraph of the Introduction. The correct sentence is: Based on observed ERP latencies, including a source-related positivity over the prefrontal scalp region starting at 800 ms after word onset, they concluded that voice information was retrieved after word information, suggesting a hierarchical system.There is an error in the fourth and fifth sentences of the last paragraph of the Introduction. The correct sentence is: In addition, we predict a late frontal deflection in the source task at a somewhat earlier latency than the one found by Senkfor and Van Petten [19]. Moreover, though this modulation has typically been investigated post-retrieval, recent work by Hayama et al. [20] has indicated that the traditionally right-lateralized frontal deflection might actually index decision/judgment processes.


Many methods are available for the manipulation of brain function and behavior. From pharmacological tools through electrophysiological techniques to neuroanatomical procedures, an arsenal of approaches has been successfully utilized to answer the question of how the brain works and what may be behind its dysfunction in human disorders. Why do many scientists prefer to use genetics then? There may be two principally different reasons for this choice: one relates to evolution, the other to practical or mechanistic points. Ultimately, all behavioral features of an organism, and thus all functional aspects of its brain, must pass the test of natural selection. Natural selection operates at the level of genes. In other words, albeit often complex and difficult to disentangle, the effects of genes on brain function and behavior is not questionable. The second reason is more practical, and concerns how efficiently a biologist can answer mechanistic questions. The cause of human CNS disorders is often multifaceted, and is usually expected to involve numerous unknown environmental factors. Simply put, the number of such environmental factors could be staggering, whereas the number of genes one can manipulate is only about 25,000. Identifying a gene is also much simpler than identifying environmental agents possibly involved in brain dysfunction. A good example for this reasoning is the discovery of the involvement of Amyloid Precursor Protein (APP) misprocessing in Alzheimer's Disease. Only <5% of Alzheimer cases are familial, i.e., heritable, yet it was the discovery, for example, of APP mutations and the mutations in APP processing enzymes that propelled the understanding of the disease forward. Another mechanistic reason why genetic manipulation is often preferred to other methods is the specificity of the former. Modern recombinant DNA methods allow one to precisely silence a single gene, or change its expression in a brain region specific manner, or even modulate neuronal function in particular circuits and at particular time points. This level of control, temporal and spatial resolution, as well as target specificity are often not achievable with methods other than genetics. The current special topic will present numerous such genetic techniques, the first, and perhaps the most famous of which being homologous recombination-based gene targeting in embryonic stem cells.Tsien; Gerlai). The latter papers describe what we have learned over the past three decades about the utility and the limitations of gene targeting in brain research, and what the potential future of this technology may be. The special topic also extends the question of how gene targeting is employed for the discovery of gene function to how it may be used to generate animal models of human brain disorders (Leung and Jia). In the past decade, novel genome engineering tools have also been developed. These new methods now compete with the true and tried ES cell based gene-targeting technique. For example, the Transcription activator-like effector nuclease (TALEN) system now allows one to custom design restriction endonuclease-like enzymes that will cut specifically at the desired nucleotide sequence . Similarly, the Clustered regularly-interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system now also enables the researcher to create double stranded cuts at pre-specified nucleotide sequences of choice . Importantly, both the TALEN and the CRISPR/Cas9 system can be employed in species other than the mouse or the rat, the two laboratory model organisms to which ES cell based gene-targeting methods of the past were restricted. Last, the special topic also reviews the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology . Although not a gene targeting method per se, DREADD is a recombinant DNA manipulation-based approach that combines genetics with pharmacology and allows one to specifically target particular neuronal populations and conduct a high temporal resolution analysis of their function.The double selection scheme that allowed efficient identification of homologous recombination-based replacements of endogenous genes with a targeting vector in embryonic stem cells has revolutionized reverse genetics. The method, developed by Mario Capecchi and his colleagues in the mid-80s, which became known as gene targeting or the knock out technology, has since been widely used in studies investigating the function of genes in practically every sub-discipline of biology. This special topic presents a collection of papers that focus on how gene targeting has been utilized in neurobiology, i.e., in the analysis of the biological mechanisms of brain function and behavior. It reviews classical (first generation) knock out methods and second generation (inducible and cell type restricted gene expression and targeting) techniques, including the CRE-recombinase system, the evolution of the technology as well as the evolution of its use . The investigator now has an array of modern recombinant DNA-based tools with which he or she can manipulate brain function and/or behavior. The number of such tools is continually growing, and the context in which they are employed is increasing in breadth. This special topic only captures a fraction of these methods and the different ways they may be employed. Nevertheless, it attempts to paint an objective picture discussing the pros and cons of these tools. As such, it may be a useful read for the novice neurobehavioral geneticist and the expert neurobiologist alike. It may be of value to scientists of other fields of biology as well who wish to employ these techniques for purposes other than the investigation of the brain. Last, we feel the papers may be useful for both instructors and students who are interested in the rapidly evolving field of neurobehavioral genetics and modern behavioral neuroscience.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DPOP (ΔPOP or Delta-POP) is a noninvasive parameter which measures the strength of respiratory modulations present in the pulse oximeter waveform. It has been proposed as a noninvasive alternative to pulse pressure variation (PPV) used in the prediction of the response to volume expansion in hypovolemic patients. We considered a number of simple techniques for better determining the underlying relationship between the two parameters. It was shown numerically that baseline-induced signal errors were asymmetric in nature, which corresponded to observation, and we proposed a method which combines a least-median-of-squares estimator with the requirement that the relationship passes through the origin (the LMSO method). We further developed a method of normalization of the parameters through rescaling DPOP using the inverse gradient of the linear fitted relationship. We propose that this normalization method (LMSO-N) is applicable to the matching of a wide range of clinical parameters. It is also generally applicable to the self-normalizing of parameters whose behaviour may change slightly due to algorithmic improvements. The modulation may manifest as amplitude, baseline, and/or frequency perturbations of the signal, depending on the physiological origin. Through sophisticated signal processing it has been shown that these perturbations may be used to determine frequency of respiration (respiratory rate) using commercially available hardware [ strength of respiratory modulations present in the pleth waveform [max and AMPmin are the maximum and minimum amplitudes of the cardiac pulse waveform in the pleth during a respiratory cycle. This is illustrated in The respiratory modulation of the pulse oximetry photoplethysmograph (“hardware . DPOP (Δwaveform . It is dwaveform (1)DPOP=With Institutional Review Board approval and written informed consent, a convenience sample of adult patients was enrolled at the Ohio State University Wexner Medical Center comprising mechanically ventilated patients requiring the placement of an intra-arterial line who had been scheduled for elective surgery. Each patient was fitted with a finger sensor . Synchronized acquisition of the pulse oximeter and arterial pressure signals was performed during the whole procedure and saved for later analysis. Further details of the acquisition system, patient exclusion criteria, and the data are provided in , 15. The all data acquired during the clinical trial. This is more representative of the data that will be fed into a pulse oximeter in practice and hence contains significant noise. It is this kind of data we have a specific interest in within our development of robust parameters for use in medical devices. We also know that when there are no respiratory modulations present in the blood pressure (BP) waveform, there will be no respiratory modulations in the pulse oximeter waveform. Hence, assuming no other signal components, the relationship should pass through the origin. The regression line plotted in An example of the least-squares fit for the DPOP-PPV data pairs is shown in Amin and a range of baseline gradient induced amplitude measurement errors . The determination of fluid responsiveness is performed by comparing the parameter value against a predefined threshold, for example, 13% for PPV , 23, 24.However, now that we have determined the linear relationship that passes through the origin . Whether a higher or lower DPOP value occurs at a threshold value of PPV , indicaIn addition, we have detailed a technique for normalizing the parameters using the inverse of the gradient of the best fit line. This method of normalizing DPOP in terms of PPV is advantageous in that the clinician may change between the two parameters seamlessly as there is no need for separate parameter-dependent thresholds. The LMSO-N method is also useful in self-normalizing any parameter against itself. For example, the method could be used to normalize the algorithm output of the parameter to the previous version of the same parameter, thus maintaining consistency across algorithm revisions.Although we have discussed our methods in terms of the relationship between DPOP and PPV, other relationships both in terms of fluid responsiveness parameters  and other medical parameters may benefit from this technique.