The regulation of gene expression is essential for normal functioning of biological systems in every form of life. Gene expression is primarily controlled at the level of transcription, especially at the phase of initiation. Non-coding RNAs are one of the major players at every level of genetic regulation, including the control of chromatin organization, transcription, various post-transcriptional processes, and translation. In this study, the Transcriptional Interference Network (TIN) hypothesis was put forward in an attempt to explain the global expression of antisense RNAs and the overall occurrence of tandem gene clusters in the genomes of various biological systems ranging from viruses to mammalian cells. The TIN hypothesis suggests the existence of a novel layer of genetic regulation, based on the interactions between the transcriptional machineries of neighboring genes at their overlapping regions, which are assumed to play a fundamental role in coordinating gene expression within a cluster of functionally linked genes. It is claimed that the transcriptional overlaps between adjacent genes are much more widespread in genomes than is thought today. The Waterfall model of the TIN hypothesis postulates a unidirectional effect of upstream genes on the transcription of downstream genes within a cluster of tandemly arrayed genes, while the Seesaw model proposes a mutual interdependence of gene expression between the oppositely oriented genes. The TIN represents an auto-regulatory system with an exquisitely timed and highly synchronized cascade of gene expression in functionally linked genes located in close physical proximity to each other. In this study, we focused on herpesviruses. The reason for this lies in the compressed nature of viral genes, which allows a tight regulation and an easier investigation of the transcriptional interactions between genes. However, I believe that the same or similar principles can be applied to cellular organisms too. The TIN hypothesis assumes that the process of transcription itself plays a regulatory role in the coordination of gene expression within a cluster of functionally linked genes. The mechanism of this coordination is based on the confrontation of the transcriptional machineries at the various overlaps formed by neighboring genes of a genetic modules (GM). The rationale behind the operation of the TINs lies in providing a straightforward genetic algorithm to coordinate the ON/OFF transcription pattern of functionally linked gene clusters, which would otherwise be possible only via sophisticated mechanisms, including continuous monitoring of the actual state of gene expression and modification of the transcription profiles of the genes accordingly. In a cluster of tandem genes, transcription of an upstream gene causes the inhibition of the downstream genes. This mechanism represents a unidirectional interaction between these genes . On the contrary, the various convergent overlaps allow a two-way interplay between adjacent genes, resulting in a mutually exclusive expression of the interacting partners (Seesaw model). Besides the collision-based models, another way of interference between genes occurs through the competition between the transcriptional machineries for determining the direction of the transcription from bidirectional promoters. The collision of the transcriptional apparatuses results in a non-linear effect in gene expression. The outcome of the collisions is dependent on two important factors, which are the activity of the promoter and the efficiency of the read-through. Both processes are differentially regulated and can change in time. The outcome of the interaction between two convergent genes is controlled by the frequency of transcriptional read-through; the genes with higher rate will win. Genes with initially higher activity and/or higher transcriptional read-through efficiency can significantly or entirely suppress the expression of genes with lower initial activity , for which the reason is at least threefold. First, the initiation step of transcription is a time-consuming process, which can therefore be effectively blocked via the rapidly advancing RNA polymerase II (RNAP) from a gene with high transcriptional activity. Second, the clash of the RNAP molecules occurs more frequently at the loci  of genes with lower activity in the case of convergent read-through overlaps. The reason for the unequal effect of the collisions is that the dislocation of the RNAP molecule during the transcription of a coding region results in the generation of functionless transcripts, but does not significantly influence translation in the read-through part of the transcripts. Furthermore, clashing of RNAPs outside the gene locus enables the production of a large population of normally terminated mRNA molecules (non-read-through transcripts). According to the Seesaw model, transcriptional collisions produce a self-regulatory automatism whereby the higher-rate transcription mechanically makes gene expression topple over from one kinetic (sub)class to another, subsequently maintaining the switched state for a given period of time. In other words, the expression of a gene is not only dependent on the presence or absence of transcription factors and the epigenetic state of the given locus, but also on the activity status of adjacent genes and the control of poly(A) recognition by factors such as ICP27 in herpesviruses . The TINHox genes are a set of transcription factor genes that are instrumental in regulating the timing and route of animal body formation during development. They belong to the homeotic gene family, which is characterized by a DNA sequence of approximately 180 base pairs called the homeobox encoding the homeodomain, a DNA-binding segment of proteins. Hox proteins act like genetic switches, turning off and on other genes with a mechanism whose key parameter is the gene dosage. The Hox genes themselves are regulated by more posteriorly acting Hox proteins and several other types of transcription factors. Many Hox genes have remarkably conserved functions. For example, the Pax6 gene is involved in eye development both in insects and vertebrates, and is even expressed in the photoreceptor cell of the Cnidarian species. Vertebrate Hox genes are organized into four clusters , while invertebrates contain only a single set of Hox genes. One of the most striking properties of the Hox genes is that they are activated in a collinear fashion; that is, the relative order of their expression along the anterio-posterior axis correlates with the position of the genes in the gene cluster.The recent view held that transcription is controlled by the combinatorial action of transcription factors and other regulatory proteins through binding to the enhancer and promoter sequences of the genes. This notion had its roots in the biochemical perspective that proteins comprise the structural and core catalytic framework of the cell. RNAs were thought to play subsidiary roles compared to the two truly important molecules: DNA, which carries the genetic information, and proteins, which execute nearly every function in the cell. However, this picture has been dramatically changing in recent years with the realization that RNAs have far more important functions in cells than was considered before . Alpha-hCaenorhabditis elegans genes arose from gene duplication , and revealed that a large fraction of the DNA is arranged in neighborhoods of functionally related genes. Intriguingly, they further found that these functional gene clusters were generated more frequently through the reorganization of the genome than through gene duplication. Genes of a cluster may form interaction networks, in which the proteins interact with each other directly by forming multimeric proteins, or serving as upstream and downstream components in the signaling pathways (Hox genes). Alternatively, co-localized genes can feature in the same biochemical pathway without their protein products participating in direct interactions, e.g., enzymes taking part in a common metabolic pathway, β-globins carrying oxygen in the muscle, or in fetal or adult blood, or tRNAs and MHC proteins involved in the translation and immune defense, respectively. In either case, the clustering of genes appears to confer a selective advantage for the formation and maintenance of gene clusters. Besides the coordinated gene expression, the selective pressure may also favor the long-term linkage (co-inheritance) of clustered genes. For example, the existence of large regions of linkage disequilibrium has been reported among inbred mouse lines, which were shown to be correlated with the biological function . However, the adaptive origin and maintenance of genetic networks in complex eukaryotes has been called into question in several publications  has been reversed during evolution . In various arthropod species, genomic reorganization events have generated antiparallel gene pairs within the Hox gene cluster . Herpesviral genes exhibit a characteristic modular organization, which includes two convergently oriented gene clusters, each containing parallelly overlapping genes . Questions arise as to whether this modular genomic organization is common in cellular organisms too and if so, whether it has any functional significance or represents a mere curiosity without any apparent function.In sum, the tandem arrangement of functionally related genes on the DNA appears to represent a common organization principle in both cellular organisms and viruses with large genome sizes. Mammalian Current technological advances have allowed the investigation of overall expression of human and mouse genomes with an exceptional resolution. These analyses have revealed that the major parts of these genomes are transcribed and that protein-coding sequences account for only a small proportion of the total transcriptional output . There icis-NATs. In contrast, trans-NATs are encoded outside the genomic loci that specify the target mRNAs. Cis-NATs are entirely complementary to their mRNA partners, whereas trans-NATs generally display imperfect complementarity to their target mRNAs (trans-NATs are the microRNAs (miRNAs) and the endogenous small interfering RNAs (siRNAs), although these transcripts can be encoded in cis, too. MiRNAs comprise short non-coding transcripts, which are post-transcriptional regulators that bind to complementary sequences on target mRNAs and modulate gene activity by interfering with mRNA stability or translation  represent a group of ncRNAs that have complementarity to other transcripts. Many loci of chromosomes contain transcription units on both DNA strands. Often, one transcript encodes a protein, whereas the RNA from the complementary DNA strand is non-coding. These latter RNA molecules are termed et mRNAs . NATs caet mRNAs . NATs hanslation . Small Rnslation . The humnslation .Recent attention has focused on miRNAs. However, the most abundant and least annotated class of ncRNAs is the long non-coding RNAs . Antisense RNA expression has also been reported in some other HSV genes , they are thought to be monocistronic. Hox genes are tandemly arranged in an order collinear with their expression along the anterior–posterior axis. Extensive polycistronism has been reported in mammalian Hox gene clusters, where Hoxc6, Hoxc5, and Hoxc4 are co-transcribed  in picornaviruses and hepatitis C virus  is an lnc-RNA found in Drosophila, which silences the expression of the Ultrabithorax (Ubx) gene through TI. Furthermore, the amount of transcripts synthesized from tandem genes in the yeasts genome decreases when the transcriptional termination sequences of the upstream gene are removed , resulting in the confrontation of their transcriptional machineries at the overlapping region. Furthermore, it is proposed that this form of genetic regulation occurs at a genome-wide scale and, therefore, plays an essential role in controlling global gene expression in both cellular organisms and viruses. Convergently overlapping genes can be protein-coding genes or RNA genes, or the combination of them. True convergently overlapping protein-coding genes, especially those with ORF overlaps, are rare in various genomes. However, the TIN hypothesis proposes that in many gene pairs, transcription from convergently positioned genes is not terminated with perfect efficiency at their poly(A) sequences. Instead, transcription occasionally continues at the locus of the opposing gene, which leads to the generation of long RNA molecules containing mRNA sequences from the given gene and antisense RNA sequences from the oppositely oriented gene. The process of alternative polyadenylation can also lead to differential transcriptional overlaps. The long transcripts, generated by the read-through mechanism, appear to be the mere by-products of this process; however, it cannot be excluded that they are further utilized at other levels of genetic regulation. Likewise, downstream-positioned genes of polycistronic RNAs, if untranslated, are also superfluous by-products of the transcriptional read-through between tandemly oriented genes. The potential use of these polycistronic RNAs, if at all, is unknown. Another way of TI is based on the competition of transcription apparatuses on the divergent promoters to determine the direction of transcription. According to the TIN hypothesis, transcription of neighboring genes can depend on each other in either a one-way  or two-way direction (convergently overlapping genes). The interaction between the transcriptional machineries provides a mechanism for the coordinate control of the expression of functionally linked genes using a simple genetic algorithm: “keep a gene silent while the adjacent gene is transcribed through the process of transcription itself,” which would be a very complicated task to accomplish in a large gene cluster by means of transcription factors alone. A TIN regulates the transcription switch between two or more genes and acts thereafter to maintain the turned-on state of a gene and the turned-off state of the neighboring genes for a given period of time. The interdependence of gene activities provides an in-built automatism for producing a tightly choreographed succession of expression patterns of co-localized genes. To coordinate gene expression at a broader scale, GMs themselves can also interact with each other in various ways, including the TI within the divergent overlaps of upstream genes of adjacent GMs or the transcriptional read-through from distant genes belonging to other GMs. The operation of TINs will now be discussed in the alpha-herpesviruses, especially in the PRV, which is a useful model organism for studying the molecular pathogenesis of herpesviruses  and transcription factors, but also via the activity or silence of upstream genes and the control of the transcriptional read-through efficiency. In the Waterfall model, the downstream genes do not exert any direct effect on the transcription of upstream genes; therefore, this type of interaction results in a one-way dependence of gene expression . Nonetheless, producing polycistronic RNAs can slow down the transcription of upstream genes due to the longer time needed for the production of polycistronic RNAs compared to monocistronic ones. The downstream genes of Hox clusters are controlled by the transcription factors encoded by the upstream genes, thereby resulting in a spatiotemporally well-controlled stepwise activation of these genes. According to the Waterfall model, the same pattern of gene expression can be achieved through the operation of TINs.The Seesaw model of the TIN hypothesis proposes a mutually interdependent mode for regulating the expression of neighboring genes. The seesaw mechanism is based on the head-on collision of RNAP molecules during the transcription of two or more overlapping genes positioned in convergent or divergent orientations relative to each other.Divergently overlapping genes. Divergently oriented genes can often overlap and generate transcripts with 5′ overlapping regions , one between the E/L genes (ul14–ul15), and four between an E and an L gene , while there is no overlap between the E genes . Apparently, the transcriptional machineries of the opposing genes must collide at the overlapping region unless their expressions are temporally well separated. According to the Seesaw model, TI at the overlapping region plays a pivotal role in the temporal separation of the expression of overlapping genes. The underlying mechanism is based on either the dislocation of RNAP molecules, which results in the inhibition of the assembly of transcription pre-initiation complex and/or the premature termination of the transcription. This process provides an automatism through which a gene with a high expression rate suppresses the expression of the opposing gene(s) with lower activity. The alteration of the transcriptional direction in a locus is initiated by transcription factors (through a change in their composition and/or activity).as yeast , C. eleg elegans , and PRV elegans . ConvergConvergent overlap between a protein-coding gene and an RNA gene. The overlaps between protein-coding genes and RNA genes have been described in several cellular organisms and viruses (Hox cluster play an important role in the regulation of gene expression (ep0)-LAT and the ie180-AST transcript pairs serve as examples for this type of transcriptional overlap in PRV. Additionally, the LLT overlaps both ep0 and ie180 mRNAs . The HSV LAT has been shown to encode miRNAs , indicating that the transcription rates of these molecules were interrelated. The LAT and AST transcripts of PRV are considered non-coding. Intriguingly, however, the complementary DNA strands contain long ORFs, which had led to the speculation of their potential in encoding antisense proteins. However, we showed that these ORFs were generated by the selective accumulation of G and C bases in the silent codon positions of these genes, thus eliminating stop codons from the fourth antisense reading frame on the complementary DNA strand  being composed of two convergently oriented tandem gene clusters. The Seesaw model proposes the existence of a mechanism that allows an extensive transcriptional read-through across the oppositely oriented genes, which is the result of the inefficient termination of transcription at the terminal poly(A) signals of the nested gene clusters. This mechanism is supposedly under the control of the ICP27 protein, resulting in the generation of long transcripts containing sequences that are complementary to the minus DNA strand of a gene and antisense sequences transcribed from the plus strand of the opposing gene. While most of the genes within the tandem gene cluster belong to the same kinetic class, the convergent partners usually belong to different temporal classes . The ul15 gene has to produce a long transcript encompassing the other two genes, which are removed during mRNA processing by splicing. The short Us region appears to be differently organized than the long Ul region of the PRV genome: all the Us genes are tandemly oriented. However, the us1 gene located on the inverted repeats is transcribed in an opposite direction . The Us genes are transcribed in an E/L kinetics , while the us1 (icp22) appears to be an L gene in PRV  site of the mRNA or by removing the antisense transcript by RNA splicing. classes . A specie in PRV  and an Ie in PRV . It is pFigure 6). However, real GMs can be built up somewhat differently: (1) the first step of the transcription cascade of PRV is the production of IE180 mRNA, which does not need de novo viral protein synthesis. The transcription of this transactivator is regulated, among others, by the RNA genes AST and LLT through a seesaw mechanism , and by its own protein product  Next, several E genes are induced by the IE180 transactivator in association with specific cellular factors. At this stage of viral infection, the upstream E genes inhibit both their tandem gene partners through a waterfall mechanism and the convergent L genes through a seesaw mechanism. Furthermore, these upstream E genes also inhibit the expression of the divergently oriented genes belonging to different kinetic (sub)classes. The ie180 and the ep0 genes encode the two key transcriptional activators of PRV. Therefore, these genes and also their antisense partners require a complex regulation, which could explain why LAT, LLT, and AST are controlled by their own promoters. This is in contrast to the rest of the antisense RNAs, which, according to the Seesaw model, are controlled by the promoter of opposite genes and produced via a transcriptional read-through mechanism. (3) In the next step, the ICP27 protein and/or other factors recognize the poly(A) signal of the upstream E genes, thereby allowing the transcription of downstream E genes from their own promoters. (4) Subsequently, gene expression swaps from the E to L stage (capsizing the arms of the seesaw), in which the ICP27 protein may play a critical role by efficiently recognizing the poly(A) signals at the 3′ co-terminals leading to a decreased convergent read-through from the E genes. Furthermore, poly(A) signals at the 3′ co-terminal of the late genes may not be recognized by the RNAP molecule, causing a transcriptional read-through from the L genes toward the E genes. In other words, the poly(A) signals might be differentially recognized by ICP27 and/or other factors, thereby dictating a temporal order in the succession of the ON/OFF switching of gene expression. Other viral proteins, such as VHS and EP0, may also play roles in the kinetic shift, presumably through modulating the expression and/or activity of the IE180 transactivator  overlap at their TATA boxes. In our earlier publication, we categorized the ul20 gene as E/L, and the other three genes as E genes. It is assumed that these genes are expressed in a slightly different kinetics, which is, at least partly, regulated by competition between overlapping promoters for the transcriptional initiation factors. The mechanisms influencing the preferential binding of transcription factors to the two regions of these promoters are currently unknown.Bidirectional promoters can also function in the regulation of the temporal orderliness of gene expression. Two of the divergent gene pairs of PRV . An extensive transcriptional read-through between the Hox genes has been reported in various species  signal, which is occasionally overlooked by the RNAP enzyme. Eukaryotic genes do not necessarily form convergent read-through overlaps to achieve a similar regulatory effect. Instead, they can also overlap as a result of alternative polyadenylation, which is recognized as a major post-transcriptional regulatory mechanism in eukaryotes. Differential polyadenylation depends on a variety of factors, including cell type , embryonic stage, environment and disease  are regulated by antisense transcription from RNA genes (LAT and ASP). Additionally, at the moment, it is not known whether CUTs are also expressed in viruses.A major difference between viruses and cellular organisms is that the interacting partner of a protein-coding gene in a cellular organism, and especially in a eukaryote, appears to be more frequently an RNA gene, rather than another protein-coding gene as in a virus. The reason for this could be that viruses contain extremely compact and efficient genomes, since they have to utilize every detail of the DNA. In PRV, only the two most important transactivator genes  models and compete with each other in the promoter-competition-based model. In the collision-based models, the genes with higher initial expression rates and/or transcriptional read-through efficiencies suppress the transcription of genes with lower initial expression and/or efficiency, thereby further reducing the transcription of the latter genes close to the zero level. The Waterfall model predicts a one-way inhibitory effect (from upstream to downstream genes) among tandem genes, while the Seesaw model envisages a reciprocal downregulatory effect on the transcription of convergently oriented genes. The latter system results in an inverse synchronization of transcription between convergent genes through a self-adjusting automatism, thereby greatly simplifying the coordination of gene expression. The Extended Seesaw model proposes a complex regulatory pattern between genes forming both tandem and convergent clusters in a genomic locus. In the latter case, the direction of transcription can be repeatedly altered. In this study, we focused on the operation of TINs in alpha-herpesviruses. The question is whether the large-scale organization of the genomes of cellular organisms resembles that of the herpesviruses. The number of true convergent overlaps between protein-coding genes is low in the genomes of various cellular organisms, which appears to imply a peripheral significance of transcriptional collision in controlling genome-wide transcription. However, convergent orientation of gene clusters is common in these organisms, which potentially allow transcriptional read-through and alternative transcriptional termination mechanisms. Moreover, the intrinsic bidirectionality of the eukaryotic promoters provides an additional means for neighboring genes to exert a mutual effect on each other’s expression.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.


The framework for playing quantum games in an Einstein-Podolsky-Rosen (EPR) type setting is investigated using the mathematical formalism of geometric algebra (GA). The main advantage of this framework is that the players' strategy sets remain identical to the ones in the classical mixed-strategy version of the game, and hence the quantum game becomes a proper extension of the classical game, avoiding a criticism of other quantum game frameworks. We produce a general solution for two-player games, and as examples, we analyze the games of Prisoners' Dilemma and Stag Hunt in the EPR setting. The use of GA allows a quantum-mechanical analysis without the use of complex numbers or the Dirac Bra-ket notation, and hence is more accessible to the non-physicist. Although its origins can be traced to earlier works The general idea in the quantization scheme proposed by Eisert et al This approach to constructing quantum games was subsequently criticized As a way to counter the criticism in real coordinates, it thus permits a visualization that is simply not available in the standard approach using matrices over the field of complex numbers. Pauli matrices are isomorphic to the quaternions, and hence represent rotations of particle states. This fact paves the way to describe general unitary transformations on qubits, in a simplified algebraic form, as rotors that bring noticeable simplifications and geometrical clarifications. We apply constraints on the parameters of EPR type arrangements that ensure a faithful embedding of the mixed-strategy version of the original classical game within the corresponding quantum game. In particular, we show how using GA we can determine new NE in quantum games of Stag Hunt and Prisoners' Dilemma played in the EPR type setting.The usefulness of applying the formalism of geometric algebra (GA) We have the following payoff matricesThe EPR setting assumes that players Alice and Bob are spatially-separated participants, who are located at the two arms of the EPR system. In a run, each player receives one half of a two-particle system emitted by the same source. We associate Alice's strategies Geometric algebra (GA) Given a linear vector space We denote by In order to express quantum states in GA we use the one-to-one mapping It can then be shown using the Schmidt decomposition of a general two qubit state To simulate the process of measurement in GA, we form a separable state Employing Eq. (9), we firstly calculateWe allow each player the classical probabilistic choice between their two chosen measurement directions for their Stern-Gerlach detectors. The two players, Alice and Bob choose their first measurement direction with probability We now find the optimal solutions by calculating the Nash equilibrium (NE), that is, the expected response assuming rational self interest. To find the NE we simply requireTo embed the classical game, we require at zero entanglement, not only the same pair of strategies being a NE but also to have the bilinear structure of the classical payoff relations. At a NE of Referring to Eq. (15), we then have the conditionsSo, in summary, for both cases we have that the two measurement directions are This then reduces the equation governing the NE in Eq. (26) toWe also have the probabilities for each state Here we explore the above results for the games of Prisoners' Dilemma and Stag Hunt. The quantum versions of these games are discussed in Refs. The game of Prisoners' Dilemma (PD) With reference to Eq. (23), we thus have Consider a particular example of PD by taking The game of Stag Hunt (SH) The SH game can be defined by the conditions The EPR type setting for playing a quantum version of a two-player two-strategy game is explored using the formalism of Clifford geometric algebra (GA), used for the representation of the quantum states, and the calculation of observables. We find that analyzing quantum games using GA comes with some clear benefits, for instance, improved perception of the quantum mechanical situation involved and particularly an improved geometrical visualization of quantum operations. To obtain equivalent results using the familiar algebra with Pauli matrices would be possible but obscures intuition. We also find that an improved geometrical visualization becomes helpful in significantly simplifying quantum calculations, for example unitary transformations on a single qubit become simply rotations of a vector as displayed on the Bloch sphere, and two qubits can be modeled in a real We find that by using an EPR type setting we produce a faithful embedding of symmetric mixed-strategy versions of classical two-player two-strategy games into its quantum version, and that GA provides a simplified formalism over the field of reals for describing quantum states and measurements.For a general two-player two-strategy game, we find the governing equation for a strategy pair forming a NE and the associated payoff relations. We find that at zero entanglement the quantum game returns the same pair(s) of NE as the classical mixed-strategy game, while the payoff relations in the quantum game reduce themselves to their bilinear form corresponding to a mixed-strategy classical game. We find that, within our GA based analysis, even though the requirement to properly embed a classical game puts constraints on the possible quantum states allowing this, we still have a degree of freedom, available with the entanglement angle Analysis of quantum PD game in this paper can be compared with the results developed for this game in Ref. The EPR setting represents a simplified quantum game framework retaining classical strategies, but allowing quantum mechanical features such as entanglement to be employed in classical games. A more general scheme can be described allowing full use of unitary operations by each player, which is a useful framework when contact is not essential with a corresponding classical game. An even more general framework than quantum mechanics can be described, based on the properties of non-factorizable joint probabilities These three results are useful when calculating measurement outcomes in an EPR experiment, with a measurement direction


Automated perimetry has become the mainstream for assessment of functional glaucomatous loss and progressive damage. Recent improvements with the Swedish interactive thresholding algorithm (SITA) strategy and the guided progression analysis (GPA) have further settled standard achromatic perimetry (SAP) as the preferred method for diagnosis and follow-up of functional loss. Although SAP is still considered the gold standard, function-specific perimetry may offer advantages for early diagnosis. Frequency doubling technology (FDT) and short-wavelength automated perimetry (SWAP) have been shown to be helpful, especially when SAP is normal and there is a suspicion of glaucoma. Studies using rarebit perimetry have also shown promising results. Studies have observed that each test identifies a different subset of eyes, and combining the tests may improve sensitivity. Nevertheless, the more sophisticated analyses do not reduce the importance of a correct interpretation of the test results. Automated perimetry has become the mainstream for assessment of functional glaucomatous loss and progressive damage. For the past several years, several different perimeters and test strategies have been developed, but none has had more studies and has been more used than standard white-on-white perimetry with the Humphrey Field Analyzer . Recent improvements with the Swedish interactive thresholding algorithm (SITA) strategy and the guided progression analysis (GPA) have further settled it as the preferred method for diagnosis and follow-up of glaucomatous functional loss. However, the more sophisticated analysis does not reduce the importance of a correct interpretation of the test results. In every exam, it is crucial to check the reliability indices. One can only rely on the results of a visual field exam if the patient was able to follow the instructions and correctly perform the test. Even though the analysis is fully automated, the exam still relies upon the cooperation of the patient. There are three reliability indices reported on the upper left of every exam . The ratOutside Normal Limits, Borderline and Within Normal Limits. In addition, the GHT may report General Reduction of Sensitivity or Abnormally High Sensitivity, for diffuse effects. Lastly, we see at the bottom right of the printout two global indices used to summarize the information from the total deviation and the pattern deviation plots [The glaucoma hemifield test (GHT) is an additional analysis based on the asymmetry in severity observed in glaucoma between both hemifields . It repron plots . The meaon plots  However,As visual field testing is a subjective examination, variable responses may be obtained each time the test is performed or even during the same test. This variability has been the biggest drawback of visual field assessment, as it may greatly confound interpretation of the test. Fluctuation varies among patients and among sectors in the same visual field, and usually increases with severity of disease. Any abnormalities in a visual field test should be confirmed in subsequent tests.Irreversible visual field defects are the final common feature of glaucomatous damage to the retinal ganglion cells (RGC), and for many years, functional evaluation of these cells relied solely on white-on-white standard automated perimetry (SAP). However, although SAP remains the most commonly performed method of visual field assessment in glaucoma, histological and clinical studies have shown that in many cases visual field defects on SAP are detectable only when a substantial number of ganglion cells have been lost.4 SeveralThe FDT perimetry determines the contrast sensitivity for detecting the frequency doubling stimulus. The frequency doubling illusion phenomenon was thought to be mediated by a subset of magnocellular RGCs. However,13The SWAP, on the other hand, isolates the short-wavelength-sensitive pathway and differs from conventional perimetry for its narrow-band blue-light stimulus and the yellow background illumination. The software can be installed on HFA perimeters and, similar to SAP, in its most current version uses SITA strategy that reduces the overall duration of the test. A statis1923Rarebit perimetry (or microdot perimetry) is a new technique that uses a regular computer LCD monitor to present very small stimuli  on a dark background. The patient has to indicate for each presentation whether one or two dots were observed. Rarebit perimetry was not designed to isolate any specific type of RGC and there is no hill of vision. Instead of measuring thresholds for light sensitivity, it uses the microdots to evaluate the density of coverage within the central 30° of the visual field. Defects are seen as clusters of micro defects in the integrity of the system – as micro holes in a flat surface. Such micro defects aggregate in increased numbers within established visual field defects, and the deeper the defect, the larger the density of the micro holes. Rarebit visual field defects show spatial distributions similar to those of SAP, but defects containing sloping borders tend to appear larger in rarebit perimetry. It is a very recent technology but some studies have shown promising results.–29Few studies have compared the performance of the different visual function-specific tests in the same population, and most have used the previous generations of these instruments. It is inMost frequently, progression is identified as deepening of a preexisting scotoma, along with enlargement of the defect. Diffuse sensitivity loss may also represent glaucoma progression, although it is usually accompanied by new localized defects or worsening of previous defects. Progressive diffuse loss that is isolated should always raise the suspicion of cataract progression or other media opacities. Before confirming the existence of a new or progressive visual field defect, it is important to demonstrate that the defect is repeatable on subsequent visual fields. This is one of the most important aspects of evaluating visual field progression in glaucoma. A few scattered points showing significant change are rather common in non-progressing patients. Variable responses that do not reflect further damage will vary in location and pattern in consecutive tests, whereas a true defect reflecting loss of RGC should repeat itself. Data from several clinical trials have suggested that change needs to be repeatable, with a defect of the same type in the same general location, in three consecutive exams before progression can be confirmed.–34There are two main approaches to analyze progression. The first is to compare the current examination with an older one (the baseline). If the results are significantly worse on the follow-up examination, it indicates progression. This is called event-based analysis as it looks for defects (events) on the current examination that were not present before. In the second approach, instead of comparing only a few tests, one looks for progressive change by analyzing all the tests available in a specific period of time. This is called trend-based analysis as change is observed as a trend in the values plotted over time, and significant deterioration can be assessed by observing the slope or decline of the regression line. Besides evaluating whether progression has occurred, trend analysis also allows the estimation of the rate of progression. It is well known that some patients decline faster than others, and estimating each individual’s rate of progression is helpful in evaluating response to therapy and determining how aggressive treatment should be.Possible Progression. If the same three or more points have significant change detected and repeated in three consecutive follow-up tests, the GPA software will flag the last exam as Likely Progression.Different analytical tools have been developed to assist clinicians in identifying visual field progression. The most general and simple way used the MD index plotted against time. Any significant decline was indicative of progressive deterioration. However, even though deterioration on the MD may represent glaucomatous progression, it may also represent progressing cataract or other media opacities. Conversely, cataract surgery in a glaucomatous patient may mask progression and even lead to improvement in MD values. Another analytical tool incorporated on previous versions of the Humphrey perimeters was the glaucoma change probability (GCP), which performed individual comparisons of each point at the follow-up examination with a set of baseline fields. Even though it performed a more individualized analysis of the sectors in the visual field, it was based on the total deviation plot and was still affected by diffuse media opacities such as cataract. The GPA software was developed to overcome most of these limitations. Both the GCP and the GPA are event-based analyses, but the GPA has several potential advantages when compared to the GCP. The GPA is based on the pattern deviation plots, as opposed to the total deviation plots used by the GCP. In addition, the GPA runs on SITA tests, but also accepts Full-threshold tests (the GCP does not), which is convenient for patients whose follow-up started with full-threshold perimetry. Detection of new or progressing visual field defects is performed by comparison to the baseline; therefore, it is critical to have reliable baseline exams. The software automatically selects the first two available exams as the baseline tests. However, one can easily override this selection to a more suitable time-point  or to avoid initial learning effects (which could reduce the sensitivity to detect progression). The GPA software then compares each follow-up test to the average of the baseline tests. It identifies points that show change greater than the expected variability (at the 95% significance level), as determined by previous studies with stable glaucoma patients. If significant change is detected in at least three points, and repeated in the same points for two consecutive follow-up tests, then the GPA software will flag the last exam as The most recent version of the HFA also provides the visual field index (VFI) and VFI progression plot. The VFI is a newly developed index that is proposed to better evaluate the rate of progression with SAP. The aim of this analysis is mainly to provide valuable information on the rate of deterioration. The VFI is calculated as the percentage of normal visual field, after adjustment for age. Therefore, a VFI of 100% represents a completely normal visual field, while a VFI of 0% represents a perimetrically blind visual field. The VFI is shown on the GPA printout both as a percentage value for each individual exam and as a trend analysis, plotted against age . While t


The aim of this study was to compare through surface electromyographic (sEMG) recordings of the maximum voluntary contraction (MVC) on dry land and in water by manual muscle test (MMT).Sixteen healthy right-handed subjects  participated in measurement of muscle activation of the right shoulder. The selected muscles were the cervical erector spinae, trapezius, pectoralis, anterior deltoid, middle deltoid, infraspinatus and latissimus dorsi. The MVC test conditions were random with respect to the order on the land/in water.For each muscle, the MVC test was performed and measured through sEMG to determine differences in muscle activation in both conditions. For all muscles except the latissimus dorsi, no significant differences were observed between land and water MVC scores (p = 0.063–0.679) and precision (%Diff = 7–10%) were observed between MVC conditions in the muscles trapezius, anterior deltoid and middle deltoid.If the procedure for data collection is optimal, under MMT conditions it appears that comparable MVC sEMG values were achieved on land and in water and the integrity of the EMG recordings were maintained during wáter immersion. Electromyography (EMG) has emerged very recently in the area of aquatic physiotherapy (APT) . This coThe surface electromyography (sEMG) has been an important tool in swimming in order to determine the muscle activity in propulsion -7. HowevTherefore, in this study a MVC test of the shoulder muscles is performed both in water and dry land, to determine the degree of comparison, integrity, validity and reliability of sEMG signal in both environments, considering what Silvers & Dolny said on the importance of using sEMG waterproofing procedures .We studied the musculature of the right shoulder of 16 skilled and healthy subjects (8 men and 8 women) upon acceptance of voluntary informed consent. They had a mean age of 26.06 ± 4.48 years. All subjects were interviewed using the questionnaire International Physical Activity Questionnaire  and measAdhesive surface electrodes (5mm diameter) were used on all subjects after skin cleaning with a 90% alcohol swab, application of an adhesive spray (TensoSpray™) and protection by a waterproof adhesive tape  . This waThe MVC test was used to verify the integrity and comparability of the sEMG signal on dry land/in water. This test was completed in accordance with the recommendations of the Perotto  MMT testDuring the MVC test, sEMG data was recorded at a sampling frequency of 1000 Hz. A low pass filter was applied with a bandwidth of 20 Hz, an attenuation of 60dB and a maximum frequency of 400 Hz.Data was analysed with SPSS version 15 for Windows. Following the intervention phase, we continued the inferential analysis between variables, according to type and normality. For the analysis of the variables, we performed the nonparametric Kolgomorov-Smirnov test and used Wilcoxon or Student’s t-test for the normality of the variables. Finally, the ICC and the Cronbach's alpha tests were used to calculate correlation between both measurements. The statistical significance level was set at 0.05.As can be seen in Table There is a body of evidence from underwater sEMG recording that demonstrates reduced signal amplitudes and decreased sEMG/force ratios for maximal isometric muscle contractions compared with measurements in air that would question the results of the present study . Some stThe present study did not attempt to elucidate the neurophysiological factors that have been suggested to explain the differences in sEMG recordings between land and water. Instead, we focused on reducing the errors in the procedure for the sEMG recordings so that land and water MVC tests could be compared more accurately. These minimal differences were attributed to the sealing of the electrodes ,19 and tTo evaluate the reproducibility of the peak was used in the MVC test for each muscle in both conditions ,14,22-24Also observed in the muscles pectoralis, the cervical erector spinae and infraspinatus muscles produce more electrical potential in the aquatic environment on land. This occurs because of the complexity of the MVC test in two environments as different as water and land, because the thrust of the participant and the examiner’s resistance (MMT) is clearly influenced depending on the environment. Although initial tests (pilot study) performed where we found that the waterproofing were absolute and applied low-pass filters to prevent water-noise inferences; we can say that repeating the same procedure for the maximum thrust of the participant and offer the same resistance to the participant in both environments, it is very difficult and complex task.Finally, the correlations according to intra-class correlation coefficient (ICC) and alpha Cronbach test between the two environments are not very linear relationships in the pectoralis, anterior deltoid and middle deltoid, as the values are very close to zero. Others muscle  have a higher degree of positive relationship between them and values away from zero. In general, we say that the relationships between measurements in both environments and the application of the procedure by MVC test measured with sEMG are very weak because the sample is heterogeneous, participants possessed very different anthropometric values and mediation in water (buoyancy) is more complex than on land. The application of randomization tests on dry land/in water interventions had no effect -20,22-24The current study has several limitations. Participants were provided verbal encouragement throughout testing. To facilitate a MVC test, participants were positioned in identical anatomical conditions on land and in water. MMT was used for each MVC test, which may have introduced variation in resistance provided to the limbs. We recognize that the use of external resistance, such as firmly secured mobilization straps, may have facilitated reliability for generating a MVC. Water temperature could have affected the sEMG recordings in this study. However, water temperature was approximately 29–30ºC, reducing the likelihood that temperature distorted the sEMG signal . VeneziaTherefore, the degree of immersion and variety of muscles tested still appear to be factors that may partially account for the disparate results of previous research.We know that the reproducibility of a well-described procedure in the same environment is complicated by numerous factors endogenous and exogenous to the participant/researcher in each of the environments. When comparing the same procedure on land and in water, endogenous and exogenous changes to the participant/researcher are much higher, so it is very difficult to reproduce the procedure exactly as in water/on land. However, following the methodological rigor taken in this study and the accompanying data analysis, we can say that the use of the test protocols described for MVC test, the optimal application of MMT test in water/on land, the maximum force of participant at the MVC test and the waterproofing, which does not allow the entry of water between the electrode/skin, all appear to make the values of the MVC test for sEMG more comparable with that on dry land/in water.sEMG: surface electromyographic; MVC: Maximum voluntary contraction; MMT: Manual muscle test; APT: Aquatic physiotherapy; μV: Microvolts.The authors declare that they have no competing interests.AIC-V participated in the design of the study and performed the statistical analysis and to drafted the manuscript. RC-L collected the data and helped to draft the manuscript. Both authors read and approved the final manuscript.PhD candidate at University of Malaga and Lecturer of Physiotherapy of Faculty of Health Sciences at Osuna Extension University.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2052-1847/5/28/prepub


The current study investigated the functional connectivity of the primary sensory system with resting state fMRI and applied such knowledge into the design of the neural architecture of autonomous humanoid robots. Correlation and Granger causality analyses were utilized to reveal the functional connectivity patterns. Dissociation was within the primary sensory system, in that the olfactory cortex and the somatosensory cortex were strongly connected to the amygdala whereas the visual cortex and the auditory cortex were strongly connected with the frontal cortex. The posterior cingulate cortex (PCC) and the anterior cingulate cortex (ACC) were found to maintain constant communication with the primary sensory system, the frontal cortex, and the amygdala. Such neural architecture inspired the design of dissociated emergent-response system and fine-processing system in autonomous humanoid robots, with separate processing units and another consolidation center to coordinate the two systems. Such design can help autonomous robots to detect and respond quickly to danger, so as to maintain their sustainability and independence. In the research community on human level intelligence , 2, therin vivo the architecture of the neural network in human brain via analysis on the functional connectivity between different brain regions [Functional MRI (fMRI) has allowed us to reveal  regions , 11. It  regions –15. Such regions –18. TherRegions of interest (ROIs) were selected among the primary sensory regions, the amygdala, the prefrontal cortex, the posterior cingulate cortex (PCC), and the ACC. The amygdala is a well-known processing center for fear and relevant response , 20, whe2, flip angle = 90°, matrix 64 × 64). Corresponding structural MRI was also acquired on the same scanner. Totally 200 volumes of 28 contiguous axial slices at 5 mm thickness (without gaps) covering the whole brain were acquired from each subject (subjects were instructed to remain awake with their eyes closed). The original voxel size was 3.75 mm × 3.75 mm × 5 mm. The following preprocessing was conducted on the data of each subject with AFNI [http://www.nitrc.org/).A seven-minute resting state fMRI data were acquired from 29 subjects on a GE 3.0 T Signa Excite Gemse MRI system  with an EPI sequence  between the time series of each pair of ROIs in each individual subject were calculated: The ROIs were outlined on the high-resolution structural MRI of each subject: the left and the right sides of the primary visual cortex , the primary auditory cortex (BA41), the primary motor cortex , the primary somatosensory cortex , the olfactory cortex, the prefrontal cortex, the ACC, and the PCC with reference to the template provided in AFNI. The average time series were extracted from each ROI on each subject. The Pearson correlation coefficients  reflecting the average correlations normalized by the deviation among all subjects (average divided by standard deviation), as plotted in Figures Z matrix to 1 by hand. The significant z-scores between each pair of ROIs were plotted upon the graphs in Figures P < .005.X can help predict Y better than knowing the past of Y alone; if so, a statistical interpretation can be made that X “Granger causes” Y. The Granger causality of two variables can be implemented based on two linear regression functions. A restricted model for prediction of Y by its own past can be expressed as follows:Granger causality analysis is an approach for determining whether one time series is effective in predicting another. Unlike regression analysis which reflects correlations only, Granger causality analysis allows for interpretation on the causality of two time series. The mathematical basis for Granger causality analysis was well explained in previous studies , 31. Them is the time lag considered for estimation, and the unrestricted model for prediction of Y by the past of X can be expressed as follows:F test can be conducted to test whether the residual of the unrestricted model (Y(t)=α2Y, giving rise to The mathematical basis for multivariate estimation is much more complicated. The current study utilized the implementation via multiple vector autoregressive (MVAR) modeling with the assistance of the Granger Causal Connectivity Analysis Matlab Toolbox  and the During waking rest, significant correlations between the left and the right correspondence of a functional brain region robustly appeared significantly among all subjects , 34. DisDissociation was also found among the correlations of the ROIs in the primary sensory system and the other regions. The ACC was found to be negatively correlated with the visual cortex, the motor cortex, and the somatosensory cortex but positively correlated with the frontal cortex and the PCC. The PCC was negatively correlated with the motor cortex and the somatosensory cortex but positively correlated with the olfactory cortex, and the auditory cortex. The amygdala was positively correlated with the olfactory cortex, the auditory cortex, and the somatosensory cortex. The frontal cortex was positively correlated with the auditory cortex, but negatively correlated with the visual cortex. The frontal cortex was positively correlated with the PCC and the ACC, whereas the amygdala was negatively correlated with the PCC and the ACC . The information flow in the neural network under investigation was revealed through the Granger causality analysis , in whicThe connectivity and causality patterns found above are inspiring for the architecture of an autonomous humanoid robot. Dissociation between an emergent-response system and a fine-processing system is proposed. The emergent-response system is consisted of specialized sensory units (such as the olfactory system in human) and an emergent-response center . The fine-processing system consists of a fine-processing center  and corresponding sensory inputs . There is constant communication between them. There are also integration centers that integrate the information from the two systems and consolidate possible confliction between them . The current study explored the functional connectivity of the primary sensory system and their connectivity with the amygdala, the frontal cortex, the PCC, and the ACC. Dissociation was found in the connectivity patterns. The amygdala was found to be strongly connected with the olfactory cortex and the somatosensory cortex, compared to the frontal cortex which was strongly connected to the visual cortex and auditory cortex. The PCC and the ACC were found to play important roles in bridging the frontal cortex and the amygdala as well as the primary sensory system. The dissociation indicates the existence of two dissociated primary sensory systems. There may exist two systems in the neural network of the human brain; one is an emergent-response system, which monitors the outside world, detects danger, and responds very quickly without complicated computation, and the other is a fine-processing system, which processes sensory input information in fine detail and responds based on slow and thorough computation. The olfactory cortex and the somatosensory cortex might serve for the emergent-response system by maintaining bidirectional communication with the amygdala whereas the visual and the auditory cortices might belong to the fine-processing system by maintaining strong connectivity with the frontal cortex. Such dissociation is possible to have evolutionary necessity. Under circumstances when survival is threatened, emergent-response should be taken (such as flee) and fine-processing would be unnecessary. Olfaction and somatosense are very important for animals to avert rotten or poisonous food and physical harm; thus, it is reasonable for them to have a strong connection with the amygdala. On the other hand, the visual cortex has been found to be the major information input for human cognition , 36, andThe dissociation between the emergent-response system and the fine-processing system, with the amygdala and frontal cortex serving as processing centers for the above-mentioned representative primary sensory regions, is inspiring for designing similar dissociated systems in autonomous humanoid robots. Current robots usually rely on one single sensory input, usually vision; this is not sufficient for responding quickly to danger from the outside world. Integration of multiple sensory input, with some being primarily for detecting danger, could be crucial for the survival of autonomous robots. For example, an auditory unit and a tactile sensory unit can be designed to sense the approaching of other objects when they are out of the visual field . Accompanying processing units should also be designed to enable the robots to react quickly when suspected danger is close. Besides designing independent fast-processing units to detect and respond to danger, it is also necessary to include processing units to integrate the multisensory input information and to consolidate the competition between the fast processing units and fine-processing units, to ensure the efficiency of the neural architecture of autonomous robots. In the human brain, the PCC and the ACC play such a role in the integration of sensory input and resolving the confliction between the two systems. The PCC has been found to be a region that is constantly active during the resting state, and it is also a representative region of the default mode network, a network that has higher activity during resting state than during cognitive tasks, and was hypothesized to be monitoring the outside world and getting ready to respond . The PCCThe functional connectivity analysis in resting state fMRI allows neuroscientists to study the complex neural network of human brain in large scale –49, maki


In The Netherlands, helmet therapy is a commonly used treatment in infants with skull deformation . However, evidence of the effectiveness of this treatment remains lacking. The HEADS study (HElmet therapy Assessment in Deformed Skulls) aims to determine the effects and costs of helmet therapy compared to no helmet therapy in infants with moderate to severe skull deformation.Pragmatic randomised controlled trial (RCT) nested in a cohort study. The cohort study included infants with a positional preference and/or skull deformation at two to four months (first assessment). At 5 months of age, all children were assessed again and infants meeting the criteria for helmet therapy were asked to participate in the RCT. Participants were randomly allocated to either helmet therapy or no helmet therapy. Parents of eligible infants that do not agree with enrolment in the RCT were invited to stay enrolled for follow up in a non-randomisedrandomised controlled trial (nRCT); they were then free to make the decision to start helmet therapy or not. Follow-up assessments took place at 8, 12 and 24 months of age. The main outcome will be head shape at 24 months that is measured using plagiocephalometry. Secondary outcomes will be satisfaction of parents and professionals with the appearance of the child, parental concerns about the future, anxiety level and satisfaction with the treatment, motor development and quality of life of the infant. Finally, compliance and costs will also be determined.HEADS will be the first study presenting data from an RCT on the effectiveness of helmet therapy. Outcomes will be important for affected children and their parents, health care professionals and future treatment policies. Our findings are likely to influence the reimbursement policies of health insurance companies.e.g. do participants in an RCT represent the eligible target population and do outcomes of the RCT differ from outcomes found in the nRCT?Besides these health outcomes, we will be able to address several methodological questions, ISRCTN18473161. Infants have malleable and fast-growing cranial bones, and are therefore at risk of developing skull deformation if their head often remains in the same position. When a child turns its head toward one side most of the time, this is defined as positional preference . Skull dSkull deformation is generally considered a purely cosmetic disorder. Yet parents worry that the deformation might be permanent and might influence the child’s attractiveness with the risk of, for example, being teased . Some stThe prevalence of skull deformation can be up to 21.5% in infants younger than 6 months, but decreases within the first years of life -10. A loPrevention or treatment of positional preference and skull deformation include parental counselling, counter-positioning and physical therapy ,18. ChilBoth helmet therapy and no helmet therapy  are standard approaches in The Netherlands. To compare the effectiveness of these two approaches a pragmatic RCT study design is required . PragmatSince the condition of interest changes over time and the decision-making is time-dependent, the RCT needs to be nested in a cohort study -32. The Additionally, nesting the RCT in a prospective cohort study makes it possible to present information on the representativeness of the RCT population, by comparing this population with non-participants ,34. FurtThe main goal of the Helmet Therapy Assessment in Deformed Skulls (HEADS) study is to investigate the effects and costs of six months of helmet therapy compared to no helmet therapy in children with moderate to severe skull deformation. This article describes how this study is designed and reports the recruitment scheme so far. We provide a description of the statistical analysis plan to be used after data collection is completed and conclude with general recommendations on study design.The HEADS study is a two-armed pragmatic RCT nested in a cohort study Figure . The intEthics approval for the study was given on the 8th January 2009 (ref: NL24352.044.08) by the Medical Ethics Committee of the Medisch Spectrum Twente hospital in Enschede, The Netherlands.Participants were recruited (April 2009 to present) and measured by specially trained paediatric physical therapists (HEADS PPTs) in the Eastern part of the Netherlands .In The Netherlands, all infants are screened in the first months of life for positional preference and skull deformation at well-baby clinics. Youth Health Care professionals working at well-baby clinics in the region where the study is carried out have been informed about this study, reminded to look for this condition and asked to refer cases to HEADS PPTs.There are 96 HEADS PPTs involved in the study, working in 73 physical therapist practices. They all received three instruction sessions from the researchers of the HEADS study, including theory lessons on positional preference and skull deformation, a refresher course about plagiocephalometry (PCM) assessment and training in recruiting patients for RCTs.Based on their experience and performance in the HEADS study, six HEADS PPTs were selected to perform the assessments at T24 (T24-HEADS PPTs) and received an extra instruction session.Children could be treated with helmet therapy at ProReva (Zwolle), Deventer Hospital/LIVIT (Deventer) and Slingeland Hospital/Roessingh Rehabilitation Technique (Doetinchem). At the start of the HEADS study, these were the only institutions providing helmet therapy within the region in which the project is carried out, and therefore they were asked to collaborate in the RCT. Parents of children in the nRCT could also choose institutions outside of this region or newer institutes that provide helmet therapy within the region.Children aged two to four months with a positional preference and/or skull deformation are eligible for the cohort study. Premature children , children with congenital muscular torticollis, craniosynostosis and/or dysmorphic features are all excluded.Children aged 5 months with a moderate to severe skull deformation, measured by PCM are eligible for the RCT. PCM is a reliable, valid, non-invasive and easy-to-use method for measuring the shape of the skull ,36. To dAt T5, children meeting RCT eligibility criteria can still enrol in the study (late-enrolment).Children eligible for the RCT, but whose parents declined participation, are invited to participate in the nRCT for follow-up. Children with PCM outcomes above the upper thresholds of the inclusion criteria for the RCT are also asked to participate in the nRCT.Figure A computer-generated blocked randomisation plan with blocks of eight participants is used to allocate treatment in the RCT. After a HEADS PPT enrols a child for the RCT, he or she informs the researcher (RMW) who contacts the parents. Both parents and researcher are unaware of allocation until the parents have signed the informed consent form and confirmed participation. The researcher performs the allocation and informs the parents about group allocation. The child’s HEADS PPT, general practitioner and Youth Health Care professional are also informed about the allocation afterwards.Blinding of parents and professionals to allocation is not possible during the intervention period, including the T8 and T12 assessment. To ensure unbiased long-term outcomes, the T24 assessments are blinded. These assessments are carried out by T24-HEADS PPTs, who are unfamiliar with the history of the infants they are measuring. Furthermore, we instruct parents in the invitation letter and a poster at the assessment location, not to mention group allocation to the assessor.Helmet therapy: parents of participants allocated to the helmet therapy group were asked to make an appointment at one of the three collaborating institutes for helmet therapy. First a (paediatric) physician was consulted to confirm diagnosis and exclude contraindications. Subsequently, the orthotist provided care as usual; he constructed the custom-made helmet, supplied information about introducing the helmet to the infant, regular wearing instructions and instructions about cleaning of the helmet and general care. The helmet has to be worn for at least 23 hours per day from six to twelve months of age.No helmet therapy: Parents of participants allocated to the no helmet therapy group were asked not to start any treatment for the skull deformation of their child. In this group, recovery of deformation of the head was awaited by allowing spontaneous growth of the skull.In the nRCT, parents were able to select a treatment for their child, that is, either helmet therapy or no helmet therapy. The choice was recorded afterwards when the child was twelve months old (T12).The cohort study started with a baseline measurement at two to four months of age (T0). A follow-up measurement was performed in all children at 5 months of age (T5).In the RCT, assessments took place at the age of 8 months (T8), 12 months (T12) and 24 months (T24) .Through the parental questionnaire at T0 and the parental questionnaire for late-enrolment at T5, information about background characteristics, medical characteristics and other possible prognostic factors were collected.The primary outcome is the transverse shape of the skull at 24 months, measured with PCM. The severity of deformational plagiocephaly was determined using the ODDI, and ear deviation (ED) was calculated to determine ear misalignment. The severity of deformational brachycephaly was determined by the CPI. A continuous outcome variable (change in score from pre- to post-test) as well as a dichotomous outcome variable will be used for analysis. The dichotomous variable distinguishes full recovery from no full recovery with a cut-off for full recovery of ODDI < 104% and CPI < 90%.Secondary outcomes are 1) satisfaction of the parents and HEADS PPT with skull shape, face and body ; 2) psychomotor development  ; 3) motoThe questionnaire at T12 assessed whether parents were compliant with the therapy to which their child was assigned. Also recorded, was whether parents switched groups, and if they did, the age this happened and the reason for it. The helmet providers also collected start and end dates of helmet therapy given to infants in the RCT. Furthermore, helmets in the RCT of the HEADS study are equipped with a logging device (LoD). The LoD measures the number of hours a helmet is worn per week (therapy compliance) and will be used to determine a dose–response relationship. The LoD was attached to the helmet and data were sent to the researcher after the invention period.In both groups, parents were asked at T12 whether they provided extra care to treat the skull deformation of their child, such as the use of positioning devices, performing exercises with their child or applying various additional therapies.Cost data were collected alongside the effectiveness study. Both medical costs and indirect costs incurred by parents because of diagnostic work-up and treatment were recorded. Indirect costs were collected with the help of a diary completed by parents during the intervention period. Costs are being determined for both the RCT and the nRCT.The required sample size for the HEADS RCT, based on a significance level of 5%, power of 90% and a difference in mean improvement of at least 4 ODDI-points (SD 6 ODDI-points) was calculated as 72 infants (36 in each arm). Assuming a maximum estimated loss-to-follow up of 25%, we needed to include 96 children in the RCT.In 2008, a preliminary study was performed into the feasibility of an RCT on helmet therapy for skull deformation. Of the parents of 61 children with a skull deformation, 39% agreed to participate in a study as described in the patient information and verbally clarified. In the light of this information, the size of the current study region was chosen and the inclusion period was estimated.Data analyses will be performed using SPSS 18.0. A statistical significance level of 0.05 will be used and missing values will be imputed with multiple imputation .Data analysis will start with descriptive statistics of baseline demographic and clinical characteristics of the total population at T0. At T5, this will be repeated for the clinical characteristics.t-test or the chi square test. The representativeness of the RCT population will be determined by comparing baseline demographic and clinical characteristics of the RCT population with those of the total eligible population at T5. Both the change score (continuous variable) and the success of recovery (dichotomous variable) will be compared between groups on an intention-to-treat basis. After analysis of covariance (ANCOVA), both multiple regression analyses (change score) and logistic regression analyses (success of recovery) will be carried out with predictor variables to control for confounders. Finally, a per-protocol analysis will be performed.At T5, characteristics of the RCT population will be described. In a subsequent analysis, the intervention and control group will be compared with respect to prognostic factors using the independent samples Baseline characteristics and applied therapies will be described for participants in the nRCT and compared between children treated with a helmet and children whose parents chose not to start helmet therapy. Similarly to the RCT, both the continuous and the dichotomous variables will be compared between groups on an intention-to-treat basis. After univariate analyses, both multivariate and logistic regression analyses will be carried out adding predictor variables.Baseline characteristics will be compared between the RCT and the nRCT. To study differences in the continuous as well as the dichotomous variable between the RCT and the nRCT, both a multiple linear regression analysis and a logistic regression analysis will be carried out, with the interaction factor of study (RCT or nRCT) × group (helmet or no helmet).The HEADS trial is the first study to present an RCT on the long-term effects of helmet therapy compared to no helmet therapy in infants with moderate to severe skull deformation. The HEADS study started as a cohort study for infants aged two to four months, and continued as an RCT after the first follow-up assessment at the age of five months. In parallel with the RCT, a non-randomised controlled trial (nRCT) was carried out. This extensive cohort study will provide excellent opportunities to study the determinants of skull deformation. Outcomes of the RCT and the nRCT will provide objective information about treatment options for the parents of affected children. With this information, an informed decision can be made whether to start helmet therapy or not. Additionally, outcomes from the cost-effectiveness study are expected to influence future treatment and reimbursement policies.Recruitment in RCTs is often a challenge and it is common that trials fail to reach their target sample size . In the However, most parents refusing participation in the RCT are willing to enrol in the nRCT. Figure Another methodological advantage of the present study design is its ability to better evaluate the representativeness of the RCT study population. Usually, RCTs have homogeneous yet very selective populations to maximize the likelihood of detecting significant differences. The cohort study of the HEADS study represents a broad population. Due to the nested study design, it is possible to determine the external validity of the RCT, by testing whether the RCT population is representative of the broad, eligible population at T5. The same can be determined for the nRCT population. Furthermore, we can study whether participants in the RCT are comparable to the nRCT participants by comparing the study outcomes and baseline characteristics in both studies.Finally, as the decision for helmet therapy in the nRCT group was made by parents themselves, this will allow us to investigate the relationship between real-world decisions and treatment outcomes. This provides more information on the usefulness of data from non-randomised compared to randomised studies, which is relevant in comparative effectiveness research. Furthermore simultaneous analysis of data from an RCT and nRCT can strongly contribute to the generalizability of the study outcomes and the development of clinical practice guidelines as compared to single RCTs .Final results of the HEADS study are expected in 2013.The trial is ongoing.ANCOVA, analysis of covariance; BSID, Bayley Scales of Infant Development; CPI, cranio proportional index; ED, ear deviation; ITQOL-SF47, Infant Toddler Quality of Life Questionnaire; LoD, logging device; nRCT, non-randomised controlled trial; ODDI, oblique diameter difference index; PCM, plagiocephalometry; PPT, paediatric physical therapist; RCT, randomized controlled trial.The authors declare that they have no competing interests.RMW is responsible for the day-to-day management of the HEADS study, collected the data and wrote the main article. MMB and LAV designed the study and were responsible for the grant application. MMB is the principal grant holder and has overall responsibility for the conduct of the trial. CGMC contributed to both the design of study and the grant application with respect to the statistical analyses. MJI contributed to the design of the study, grant application and writing the article. All authors read and approved the final manuscript.